Table of contents :

Web resources :
• Common enzymes used in MoBio, cloning, PCR and LCR
• ABI 373 Loading Guide
• ABI 377 Loading Guide
• Media recipes
• Molecular reagent list
• Plate Pouring
• Detection of desired sequence
• engineered intrasterically regulated enzyme : while the ssDNA (reporter strand) is unpaired, the inhibitor sits in the cereus neutral protease's active site, preventing the semi-synthetic inhibitor-DNA-enzyme (IDE) construct  from functioning. If the desired sequence is present in the sample, however, it binds the reporter strand, releasing the allosteric inhibitor. With its hands untied, the enzyme activates the fluorescent substrate, announcing the DNA's presence like a stranded mariner setting off flares. The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.


• molecular beacons are stem-loop–forming DNA molecules tagged at one end with a fluorescent dye and at the other with a fluorescent quencher, with a target-specific sequence in between. In the absence of target DNA, the beacon adopts a closed, hairpin-like structure that keeps the dye and quencher in close proximity, dousing fluorescence. When it binds its target, however, the probe unfolds, separating the two ends and producing a measurable light emission. An electrochemical variant involves a stem-loop–forming piece of DNA tethered to a gold electrode to which an electroactive compound called ferrocene is attached. In its closed configuration, this DNA's hairpin structure keeps the iron-containing ferrocene near the electrode surface, where it can undergo a measurable electron transfer with the electrode by electron tunneling : the efficiency of electron tunneling diminishes exponentially with increasing distance. By separating the reporter from the electrode surface, DNA hybridization events reduce the current. Like molecular beacons and unlike existing electrochemical detection methods, Plaxco's approach is reagentless—that is, no chemicals need to be added following hybridization for detection, according to the paper. But unlike fluorescent approaches, electrochemical methods require no expensive hardware such as light sources, optics, and photodetectors. The authors report detecting target DNA at as low as 10 pM concentration, which is pretty much state of the art. Plaxco said that under ideal laboratory conditions, optical systems can detect target at concentrations as low as 10 fM. But in the real world such systems typically report in the nM to pM range, in line with this system. The authors report recovering about 80% of the original signal strength after washing, suggesting that the system is reusable. They attribute the 20% loss to the instability of ferrocene in aqueous solutions at high temperature
• Nucleic acid purification

Web resources :
• DNA purification (plasmids, cosmids and p1) using Diatomaceous Earth
• Introduction
• protocol for Mini, Midi and Maxi preps (GenoMed Kit)
• pick 1 well-defined bacterial colony with a previously fire-sterilized wood-prickle and put the prickle into a flask containing 50 mL of Luria's broth and 50 mL of ampicillin. Put the flask in a shaker o/n
• harvesting bacterial cells : E.coli cells are transferred into a Falcon 50 and pelleted by centrifugation :
• Mini : 4,000 rpm for 4 minutes
• Midi : 4,000 rpm for 10 minutes
• Maxi : 4,000 rpm for 10 minutes
Remove all traces of medium carefully. Wash the pellet with TBS 1x
• cell resuspending : add solution E1 (Mini : 0.4 mL, Midi : 4,0 mL; Maxi : 10.0 mL) to the pellet and resuspend the cells until the suspension is homogenous. When RNase is used to prepare plasmid-DNA, redissolve RNase in 1 mL of solution E1 by gently mixing, then transfer RNase solution completely into bottle E1 (stick supplied label "Contains RNase/store at 4°C" on bottle E1)
• place solution into autoclaved centrifuge tubes
• cell lysis : add solution E2 (Mini : 0p.4 mL; Midi : 4.0 mL; Maxi : 10.0 mL) and mix gently, but thoroughly, until the lysate appears to be homogenous. Do not vortex. Incubate at room temperature for 5 minutes
• neutralization : add solution E3 (Mini : 0.4 mL; Midi : 4.0 mL; Maxi : 10.0 mL) and mix immediately by inverting the tube 5 times. Do not vortex! Centrifuge the mixture at 20°C and > 15,000x g (11,0000 rpm) for 10 minutes
• column equilibration : columns are equilibrated before the cleared lysate is prepared by applying solution E4 (Mini : 2 mL; Midi : 10 mL; Maxi : 30 mL). The flow of solution starts automatically. Allow the column to empty by gravity flow. Do not force out remaining solution.
• column loading : put the supernatant into Falcon 50. Apply supernatant from step 5 to equilibrated column. Allow the lysate to run by gravity flow
• column washing : wash the column with solution E5 (Mini) twice (Mini : 2 x 2.5 mL; Midi : 2 x 10.0 mL) or once (Maxi : 15.0 mL). Allow the column for each wash to empty by gravity flow.
• plasmid elution : elute the DNA with solution E6 (Mini : 0.9 mL; Midi : 5.0 mL; Maxi : 15.0 mL) into tubes placed down the columns. Do not force out remaining solution
• plasmid precipitation : precipitate the DNA adding in each tube 0.7 volumes of isopropanol (Mini : 0.63 mL; Midi : 3.5 mL; Maxi : 10. 5 mL) to a final volume of 5 + 3.5 mL = 8.5 mL, then mix and redistribute each tube into 6 Eppendorf (each Eppendorf willl contain 1.4 mL). Centrifuge at 4°C and > 15,000 x g (11,000 rpm) for 30 minutes. Remove the surnatant with a P200. Wash the plasmid DNA with 150 mL of 70% ethanol and recentrifuge 5 minutes at 14,000 rpm and 4°C. Air dry the pellet for 10 minutes at 37°C, and redissolve the DNA in 200 mL of H₂O (begin to redissolve the pellet in the first of the 6 Eppendorf, then transfer the whole volume into the second one and redissolve, and so on. At the end you'll have all the DNA in the last Eppendorf).
• Maxi-preps and all media, solutions
• Midi-preps
• Mini-preps
• inoculum train : for many microbial fermentation processes, it can have a substantial impact on process performance in terms of productivity, profitability, and process control. In general, it is understood that a well-characterized and flexible inoculum train is essential for future scale-up and implementation of the process in a pilot plant or manufacturing setting. A fermentation process utilizing E. coli DH5 for the production of plasmid DNA carrying the HIV gag gene for use as a vaccine is currently under development in our laboratory. As part of the development effort, we evaluated inoculum train schemes that incorporate 1, 2, or 3 stages. In addition,the effect of inoculum viable-cell concentrations was investigated, either thawed or actively growing, over a wide range (from 2.5 x 10⁴ to 1.0 x 10⁸ viable cells/mL or approximately 0.001% to 4% of final working volume). The various inoculum trains were evaluated in terms of final plasmid yield, process time, reproducibility, robustness, and feasibility at large scale. The results of these studies show that final plasmid yield remained in the desired range, despite the number of stages or inoculation viable-cell concentrations comprising the inoculum train. On the basis of these observations and because it established a large database, the first part of these investigations supports an exceptional flexibility in the design of scalable inoculum trains for this DNA vaccine process. This work also highlighted that a slightly higher level of process reproducibility, as measured by the time for the culture to reach mid-exponential growth, was observed when using actively growing versus frozen cells. It also demonstrated the existence of a viable-cell concentration threshold for the 1-stage process, since we observed that inoculation of the production stage with very low amounts of viable cells from a frozen source could lead to increased process sensitivity to external factors such as variation in the quality of the raw materials used in the medium formulation. However,despite this slight disadvantage, a 1-stage inoculum train was a viable option in many situations, especially if the inoculation viable-cell concentration was kept above 4.8 x 10⁶ viable cells/mL. Because it leads to a reduction in process steps and eliminates some capital investments (i.e., inoculum fermenter), when feasible a one-stage process configuration will positively impact process economics^ref
• plasmid DNA purification :
• current good manufacturing practice (cGMP) conditions for obtaining clinical-grade plasmid DNA to be used in gene therapy and DNA vaccination. The purification of pharmaceutical-grade plasmid DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of recombinant protein pharmaceuticals^{ref1, ref2, ref3}.
• enrichment : production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing^ref.
• 3 topoforms :
• supercoiled (sc) or covalently closed circular (ccc) pDNA gives te most efficient transfection and expression rate in eukaryotic cells
• open circular (oc) pDNA
• linear (l) pDNA
• dimers (concatenamers, catenanes) reduce the plasmid quality and should be minimized in the final drug product. Correlation between Escherichia coli genotypes and quality of pDNA is controversial^ref
CGE analysis of plasmid DNA. The analysis allows the identification and quantification of individual topology variants of plasmid vector DNA and is the base of in-process-control and batch release control in plasmid manufacturing. (a) Selection of different possible plasmid structures and (b) electropherogram (EPG) of a plasmid DNA sample with different topology variants.


• plasmid DNA strands can be cleaved during purification, storage, intravenous administration and cell trafficking, leading to relaxed and denatured forms^ref. SC, OC, and L pDNA were injected at 2,500, 500, 333, and 250 mg doses. SC pDNA was detectable in the bloodstream only after a 2,500 mg dose, and had a clearance of 390(+/-50) ml/min and Vd of 81(+/-8) ml. The pharmacokinetics of OC pDNA exhibited non-linear characteristics with clearance ranging from 8.3(+/-0.8) to 1.3(+/-0.2) ml/min and a Vd of 39(+/-19) ml. L pDNA was cleared at 7.6(+/-2.3) ml/min and had a Vd of 37(+/-17) ml. AUC analysis revealed that 60(+/-10) % of the SC was converted to the OC form, and nearly complete conversion of the OC pDNA to L pDNA. Clearance of SC pDNA was decreased after liposome complexation to 87(+/-30) ml/min. However the clearance of OC and L pDNA was increased relative to naked pDNA at an equivalent dose to 37(+/-9) ml/min and 95(+/-37) ml/min respectively. SC pDNA is rapidly metabolized and cleared from the circulation. OC pDNA displays non-linear pharmacokinetics. Linear pDNA exhibits first order kinetics. Liposome complexation protects the SC topoform, but the complexes are more rapidly cleared than the naked pDNA^ref. pDNA degradation is considered to be a unidirectional process, with supercoiled degrading to open circular and then to the linear topoform. The calculated kinetic parameters suggested that supercoiled pDNA degrades in rat plasma with a half-life of 1.2 minutes, open circular pDNA degrades with a half-life of 21 minutes, and linear pDNA degrades with a half-life of 11 minutes. Complexation of pDNA with liposomes resulted in a portion of the supercoiled plasmid remaining detectable through 5.5 hours^ref. This degradation is mainly due to the activity of nucleases of the production host (E.coli) and of the receptor cells (present in serum and in some intracellular compartments such as lysosomes). Indeed, nake DNA exposed to cell lysates and serum nucleases is rapidly degraded^ref. It is known that certain base sequences (hot spots) can cause variations on the supercoiled helix, thus originating different DNA conformations. For instance, slipped mispaired DNA regions can occur if direct repeats sequences are present, cruciforms occur at adjacent inverted repeats and regions of unwounded DNA, can arise at AT rich regions. All these rearranged DNA sequences constitute potential initation sites for plasmid degradation during purification, storage, transfection and trafficking, ultimately leading to less efficient vectors. The hot spots found are located within the bovine growth hormone polyadenylation signal (BGH-polyA) (bases 3880-4104) and the replication origin (bases 5371-6044). An in silico search revealed that these are G and AT righ regions, respectively. These types of sequences can potentially give rise to secondaray structures in plasmid DNA and originate single stranded regions. It is known that these secondary structures play a biologically relevant role by facilitating the binding of specific proteins to polyadenylation signals and replication origins^ref. The SV40 polyA sequence, but not a synthetic polyadenylation signal that is based on the highly efficient polyA signal of the rabbit b-globin gene^ref, is less prone to degradation (Ribeiro et al, 1st DNA Vaccines Conference, 2002).
• medium compounds of animal origin and antibiotics for plasmid selection are problematic and have therefore to be avoided. The type and mixture of complex carbon and nitrogen sources (e.g. yeast extract, peptones) was shown to improve both the yield and the percentage of supercoiled DNA. Fed-batch fermentations tend to a degradation of the pDNA with proceeding fermentation duration, so that the optimum harvesting point is about 27 hours. A volumetric yield up to 400 mg pDNA per L culture broth is achieved by high-cell-density-cultivation in fed-batch mode
• alkaline cell lysis for extraction of pDNA from biomass avoiding shear forces
• 3 column chromatographic processes :
• hydrophobic interaction chromatography (HIC) delivers 95% supercoiled pDNA. By using matrices derivatised with mildly hydrophobic ligands (e.g. polypropylene  glycol) it is possible to separate supercoiled and relaxed plasmid DNA from the impurities on the basis of differences in surface hydrophobicity. HIC takes advantage of the higher hydrophobicity of single-stranded nucleic acids that show a high exposure of the hydrophobic aromatic bases when compared with double stranded nucleic acids. In double stranded plasmid molecules the hydrophobic bases are packed and shielded inside the helix and thus hydrophobic interaction with the HIC support is minimal. Endotoxins (e.g. LPS) interact even more strongly with the HIC media via the lipide moiety. [Prazeres DMF, Diogo MM, and Queiroz, JA, Purification of plasmid DNA by hydrophobic interaction chromatography, International Patent, International Application Number PCT/PT01/00012, International Publication Number WO 02/04027 A1, filed July 6, 2001]
• anion exchange chromatography (AIEC) : the core of the downstream process if a core monolithic support (methacrylate copolymer convective interaction media (CIM^®), BIA Separations, Ljubljana, Slovenia) providing an oustanding capacity for plasmids (10 mg/ml) and a fast processing time^{ref1, ref2, ref3}. Instrumentation : a binary gradient HPLC system. Separation device : CIM DEAE disk (diameter : 16 mm, thickness : 3 mm; active bed volume : 0.34 ml). Sample : 0.5 mg/ml of plasmid DNA (pCMVb) in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA. Injection volume : 10 mL. Mobile phase : buffer A (20 mM Tris-HCl, pH 7.4); uffer B (buffer A + 1 M NaCl). Conditions : gradient elution from 73-100% buffer B in 4.5 min; flow rate : 3 ml/min; detection : UV at 260 nm.


• size esclusion chromatography (SEC) results in a pDNA bulk > 99% purity and a pDNA homogeneity > 98%
• [protein-mediated dual functional affinity adsorption of plasmid DNA. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ± 0.0013, 0.0095 ± 0.0016, and 0.0080 ± 0.0006 mg, respectively, to 50 L of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 ± 0.0043, 0.0219 ± 0.0035, and 0.0190 ± 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein^ref]
• single histidine-chromatography step to purify sc pDNA from other isoforms and Escherichia coli impurities present in a lysate clarified. The histidine-agarose support combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine-ligand. The 6 kb DNA vaccine backbone pVAX1-LacZ was used as a model target. Following loading at high salt (2.3 M AS), the different species were eluted by a series of reverse salt step gradients (2.0, 1.5, 0 M AS). Open circular pDNA and genomic DNA (gDNA) eluted at 2.3 M, sc pDNA was isolated as a single peak at 2.0 M and RNA eluted at 1.5 M AS and lower. The underlying mechanism is thought to involve not only hydrophobic interactions between the support and pDNA molecules, but also non-specific biorecognition of nucleic acid bases by the histidine ligand. Control analysis showed that the isolated sc pDNA conforms to specifications in terms of gDNA (3.4 ng/mug pDNA), endotoxins (0.02 EU/mug pDNA), RNA and proteins (undetectable) and pDNA homogeneity (~100 % sc). Furthermore, the transfection efficiency of CHO cells (50%) was significantly higher when compared with the efficiency (25%) of a pDNA control. This study confirms the possibility of use a single histidine-agarose chromatography step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate^ref
• quality control :
• DNA concentration is determined with UV-absorption (260 nm)
• general purity is determined with UV-scan (220-320 nm)
• homogeneity (ccc content ; the topologic plasmid forms and restriction fragments) is determined by high performance capillary gel electrophoresis (CGE) (HPCE) due to its high resolution and accuracy. Restriction mapping is also done by using efficient lab-on-a-chip based technologies (e.g. Agilent Bioanalyzer 2100)
• anion exchange HPLC is a rapid, robust and sensitive method for the determination of the percentage of the different pDNA forms and their quantification.
• Production of ccc-supercoiled DNA in ccc-Pilot Grade quality. (a) AGE of a standard DNA sample (lanes 1, 2) and ccc-Pilot-Grade DNA sample (lanes 3, 4). (b) CGE analysis of the ccc-Pilot-Grade DNA sample identifies > 98% of ccc DNA :

The structural homogeneity of plasmid DNA is usually determined by AGE. Different bands in AGE of a plasmid sample may be assigned to different plasmid forms. The assignment of bands to the different topologies, however, is not easy since the electrophoretic mobility of plasmids of different shape changes with the electrophoretic operating conditions. In addition, the quantification of forms based on the signal intensity of stained bands in AGE may not be reliable due to nonlinear responses. Adequate equipment is required in order to obtain reproducible results. It is well known that typically only one band, the ccc form, is observed when only a small amount of a plasmid sample is applied to an agarose gel. Standard AGE usually reveals 2 prominent bands: the ccc form and another slower migrating form, commonly thought to be the oc form. It has been demonstrated that this is not always the case, since the oc form co-migrates with ccc dimers. Capillary gel electrophoresis allows the identification and quantification of all prominent forms mentioned. CGE is performed using thin (100 mm) coated capillaries with a length of 40-60 cm filled with a liquid polymer, e.g. solution of hydroxypropylmethylcellulose. Electrophoretic separation takes place by applying a high voltage (5-30 kV) between both ends of the capillary dipped in buffer reservoirs. Special intercalating dyes, like YOYO, YO-PRO or TOTO, enable on-line detection with high-resolution of the different plasmid forms by laser-induced fluorescence (LIF). The automated system offers high reproducibility, reliable quantification and short analysis time. In contrast to AGE, quantification of plasmid forms by using CGE is possible over a wide range of linearity and needs only a small amount of plasmid DNA (50 ng)
• host-related impurities (endotoxins, genomic DNA, RNA and proteins) have to be specified and minimized. Lab simple commercial small-scale kits deliver small quantities of pDNA (mg-mg) with a purity of approximately 50%.
• purity (visible) is determined by visual inspection
• purity (genomic DNA (gDNA)) is determined by agarose gel (visual), hybridization techniques (Southern blot technique) based on chemical, enzymatic or fluorescence principles (e.g. DIG, Biotin-Systems, Fluos), and quantitative PCR. While in the past contaminations of plasmid DNA with gDNA were in the range of 5-10%, novel purification technologies allow a reduction to <1% of gDNA. The most critical step is the alklaine lysis, where the gDNA as well as the plasmid DNA are denatured by a pH shift to alkaline buffer conditions. A subsequent neutralization step allows the reannealing of plasmid DNA within a short time. The gDNA, however, does not reanneal completely to a DNA double strand again. Therefore, the major part of gDNA double strand again. Therefore, the major part of gDNA will be a component of the flaky material which is generated by the alkaline lysis. It mainly consists of potassium dodecyl sulfate, insoluble proteins, cell debris, LPS and DNA. Chromosomal DNA is extremely shear-sensitive, which can easily result in DNA fragmentation. Some gDNA fragments are large enough to migrate in one distinct band in AGE. Smaller fragments can be detected as an undefined smear by overloading the agarose gel. More sensitive assays like Southern blot  hybridization can demonstrate this - depending on the hybridization and washing conditions applied. The most sensitive assay is a kinetic PCR method using a TaqMan probe to quantify gDNA contaminations. A dot-blot hybridization method utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe to detect residual host cell DNA in purified DNA vaccines. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine^ref.
• purity (RNA) : quantification is important since plasmid DNA is purified without using RNase. It is typically monitored by other blotting techniques and different AGE protocols (visual), fluorescence dyes like Ribogreen after digestion of the plasmid DNA with DNase or after AGE. A novel technique is the determination of RNA by quantitative RT-PCR
• purity (proteins) is typically monitored with colorimetric techniques like the Bradford test or the bicinechonine acid (BCA)
• purity (LPS) is monitored with the specific and sensitive kinetic Limulus amebocyte lysate (LAL) test
• purity (microrganisms) is determined with the bioburden test and the sterility test (sterile required)
• identity (vector structure) : restriction fragment length conforms to reference in AGE (1-3 enzymes). The length of the restriction fragments can be estimated by comparison with a linear DNA size marker (e.g. 1-kb ladder). The determined fragments have to conform to the calculated fragments or to a reference DNA with respect to identity. 4 different enzymes with a minimum 2 restriction sites should be used.
• identity (sequence) : sequencing (double-strand) (identical required)
International Conference on Harmonization (ICH) harmonized tripartite guideline Q1A (2R), Stability testing of new drug substances and products, 6 February 2003
U.S. FDA Guideline (December 1996), Points to consider on plasmid DNA vaccines for preventive infectious disease indications. FDA, Rockville, MD, USA
U.S. FDA, CBER Guideline (May 1998), Guidance for industry - guidance for human somatic cell therapy and gene therapy. FDA, Rockville, MD, USA [.pdf]
EMEA Guideline (1999), Note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products. CPMP/BWP/3088/99
Urthaler, J., Schlegl R, Paril, H., Tarmann, C., Gross, N., Salzbrunn, J and Necina, R (2001), Performance of hydrophobic interaction chromatography for isolation of pDNA from E.coli cell lysates, Proceedings of Prep 2001 Symposium, Washington, 2001, 66
Companies manufacturing pharmaceutical grade plasmid DNA (pDNA)
Bibliography : Schleef M (ed.). Plasmids for therapy and vaccination, Wiley-VCH:Weinheim, 2001
• MacConnell MP-96 and MP-24 purification method
• (1) bacteria are lysed directly in the culture media using enzymes and reagents which break open the bacteria, degrade RNA, and dissolve proteins and bacterial membranes. These reagents are contained in foam pads of the Applicator Comb and Lysis Tablets which are placed into the wells of the disposable sample cassette.
• (2) DNA and other solubilized molecules are resolved in separate lanes of agarose gel which has been precast into the cassette. The resolution process is accomplished through a microprocessor-controlled power supply and electrophoresis rig which is automatically filled by the instrument. Plasmid DNA is separated from other lysed E. coli components by its unique mobility in agarose during programmed electrophoresis. During the separation process, plasmid DNA ranging in size from 2-20 kb is electrophoresed until 2 kb supercoiled plasmids run to the end, but not off the separation gel.
• (3) electroelution of the resolved DNA is accomplished by the microchamber block which is slid into place by rotating cams activated by the instrument's computer. The microchamber block contains a high cut-off ultra filtration membrane at one of its openings. During electroelution, DNA continuously collects as a film on the inner vertical surface of the ultra filtration membrane until the draining step occurs when the buffer runs out of the microchamber block.
• (4) purified DNA then loses its attraction to the membrane and falls downward into a 20-25 µl droplet of buffer at the bottom corner of the microchamber block window, where it is easily recovered using a disposable tip pipettor.

• DNA extraction
• from peripheral or bone marrow blood samples in EDTA
• standard (for :
• IgH rearrangement (CDR3)
• IgH rearrangement (V_H families)
• BCL-1 rearrangement/J_H
• BCL-2 rearrangement/J_H
• TcR rearrangement)
• transfer a vacu-tainer (3.5 mL) of sample in a 15 mL Falcon probe
• fill the probe with lysis buffer (sucrose 54.5 g, Tris HCl 1M pH 7.4 5 mL, MgCl₂ 1M 5 mL, Triton X-100 5 mL; store at 4°C)
• mix slightly for 1-2 minutes
• centrigufate at 4,000 rpm for 10 minutes
• remove supranatant
• resuspend pellet in 10 mL of physiological buffer (NaCl 2.19 g + disodium EDTA 3.65 g; store at room temperature)
• centrifugate at 4,000 rpm for 10 minutes to remove supranatant
• resuspend pellet in 1.5 mL of buffer A (Tris HCl pH 8.0 5 mL; disodium EDTA 0.58 g; store at 4°C)
• add 50 mL of SDS 10% and 50 mL of proteinase K 20 mg/mL
• incubate overnight at 37°C or at 65°C for 1 hr
• add 0.5 mL of a saturated solution of NaCl
• mix strongly for 15 seconds
• centrifugate at 2,800 rpm for 15 minutes
• transfer the supranatant in another test-tube
• add a same volume of isopropanol
• mix the test-tube to precipitate DNA
• collect DNA with a dispensable bacteriological loop 10 mL
• immersion in 70% ethanol
• air drying and resuspension in appropriate volume of TE buffer (Tris HCl 1M pH 8.0 1 mL; disodium EDTA 0.037 g; H₂O 99 mL)
• by column chromatography (for chimerism studies)
• 200 mL whole blood or pellet + 200 mL PBS
• add 20 mL of Qiagen proteinase K
• mix, then add 200 mL of AL buffer
• vortex for 15 seconds
• incubate 10 minutes at 56°C
• quick run up to 3,900 rpm
• add 200 mL ethanol 100%
• vortex for 15 seconds
• quick run
• fix the column in the appropriate test-tubes and transfer the content of the Eppendorf vial into the column
• centrifugate at 8,000 rpm for 1 minute
• remove the filtered component and place into a new appropriate test-tube
• add 500 mL AW1 buffer to the column
• centrifugate at 8,000 rpm for 1 minute
• remove the filtered component and add 500 mL AW2 buffer in the column
• centrifugate for 3 minutes at 12,000 rpm
• remove the filtered component
• centrifugate for 3 minutes at 12,000 rpm
• throw away the appropriate test-tube and place the column in a sterile Eppendorf vial
• add 100 mL AE buffer
• incubate at room temperature for 2-3 minutes and centrifugate for 1 minute at 8,000 rpm
• throw away the column and preserve the Eppendorf vial with DNA in solution
• RNA extraction
• purification of RNA from cells and tissues by acid phenol-guanidinium thiocyanate-chloroform extraction (Chomczynski P, Sacchi N, 1987) : cells are homogenized in guanidinium thiocyanate and the RNA is purified from the lysate by extraction with phenol-chloroform at reduced pH. This method allows many samples to be processed simultaneously and quickly. The yield of total RNA depends on the tissue or cell source and is generally in the range of 4-7 mg per ml starting tissue or 5-10 mg per 10⁶ cells. All reagents used in this protocol must be prepared with diethyl pyrocarbonate (DEPC)-treated H₂O^ref. Procedure :
• processing of cells or tissue samples :
• 1 | prepare cells or tissue samples for isolation of RNA as appropriate for the material under study. Consult the table below for the amounts of solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate x 2H2O, 0.5% (wt/vol) sodium lauryl sarcosinate, 0.1 M 2-mercaptoethanol) required for different types or samples.
• 100 mg  of tissue : 3 ml
• 75-ml (T-75) flask of cells : 3 ml
• 60-mm plate of cells : 1 ml
• 90-mm plate of cells : 2 ml
• for tissues :
• (i) : isolate the desired tissues by dissection and place them immediately in liquid nitrogen.
• (ii) : transfer 100 mg of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tissue using a pestle. Keep the tissue frozen by the addition of liquid nitrogen.
• (iii) : transfer the powderized tissue to a polypropylene snap-cap tube containing 3 ml of solution D and homogenize at 15-25°C for 15-30 s with a Polytron homogenizer (Kinematica)
• for mammalian cell growth in suspension :
• (i) : harvest the cells by centrifucation at 200-1,900 g at 15-25°C for 5-10 min. Resuspend the cells in 1-2 ml of sterile ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄).
• (ii) harvest the cells again by centrifugation, remove the PBS completely by aspiration and add 2 ml of solution D per 10⁶ cells
• (iii) homogenize the cells with a Polytron homogenizer at 15-25°C for 15-30 s
• for mammalian cells grown in monolayers :
• (i) remove the medium and rinse the cells once with 5-10 ml of sterile ice-cold PBS
• (ii) remove PBS and lyse the cells in 2 ml of solution D per 90-mm culture dish (1 ml per 60-mm dish). Transfer the lysates to a polypropylene snap-cap tube
• (iii) homogenize the lysates with a Polytron homogenizer at 15-25°C for 15-30 s
• 2 | Trasnfer the homogenate to a fresh tube and sequentially add 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of phenol and 0.2 ml of 49:1 (wt/vol) chloroform/isoamyl alcohol per milliliter of solution D. After addition of each reagent, cap the tube and mix the contents thoroughly by inversion.
• 3 | vortex the homogenate vigorously for 10 s. Incubate the tube for 15 min on ice
• 4 | centrifuge the tube at 10,000g at 4°C for 20 min and then transfer the upper, aqueous phase containing the extracted RNA to a fresh tube
• purification of RNA :
• 5 | add an equal volume of isopropanol to the extracted RNA. Mix the solution well and allow the RNA to precipitate at -20°C for at least 1 h
• 6 | collect the precipitated RNA by centrifugation at 10,000g at 4°C for 30 min
• 7 | carefully decant the isopropanol and dissolve the RNA pellet in 0.3 ml of solution D for every 1 ml of this solution used in Step 1
• 8 | transfer the solution to a microcentrifuge tube, vortex it well and precipitate the RNA with 1 volume of isopropanol at -20°C for 1 hr or more
• 9 | collect the precipitated RNA by centrifugation at maximum speed at 4°C for 10 min in a microcentrifuge. Wash the pellet twice with 75% ethanol, centrifuge again and remove any remaining ethanol with a disposable pipette tip. Allow the pellet to air dry for a few minutes before dissolving it in 50-100 ml of DEPC-treated H₂O and storing at -70°C
• 10 | estimate the concentration of the RNA by measuring the absorbance at 260 nm of an aliquot of the final preparation
• from peripheral or bone marrow blood samples in EDTA (for
• BCR/ABL
• quantitative BCR/ABL
• PML/RARa
• CBFb/MYH11
• AML1/ETO
• FIP1L1/PDGFRA
• FLT-3 mutations
• usually 4 tubes are prepared with 3 mL Lymphoprep each
• 2 vacutainers are put into a separate tube, then filled with NaCl solution
• add the double of diluted blood sample to each Lymphoprep vial (pipette gently with inclined tip along the wall of the tube to prevent turbolence and rupture of the surface tension in Lymphoprep)
• centrifuge 25' at 1,900 rpm or 15' at 3,100 rpm, at 20°C
• collect the lymphocyte/monocyte cloud from the 2 tubes into another tube and fill with NaCl
• centrifuge 5' at 1,900 rpm (washing)
• remove the plasma by fastly inclining the tube, then add 1 mL NaCl
• move the content into an Eppendorf vial
• centrifuge 5' at 98,00 rpm
• remove the plasma over the pellet
• store the pellet at -80°C
• abbreviated protocol for RNA isolation
• homogeneization :
• 1 mL TRI Reagent per 50-100 mg tissue, 10 cm2 culture dish, or 5-10 x 106 cells,
• store homogenate for 5-15 minutes
• RNA extraction
• add 0.2 mL chloroform, mix vigorously
• store sample 2-15' at RT
• centrifuge 12,000 g for 15' (4°C)
• RNA precipitation
• transfer aqueous phase into a new tube
• add 0.5 mL isopropanol mix
• store for 5-10' RT
• centrifuge 12,000 g for 15' (4-20°C)
• RNA wash
• mix RNA pellet with 1 mL 75% ethanol
• centrifuge 7,500 g for 15' (4-20°C)
• solubilization
• air dry the RNA pellet for 5-10'
• dissolve in 50-200 mL of formazol
• 0.5% SDS, or water by pipetting and incubating at 55-60°C for 10'
• DNA purification, plasmid mini-preps, diatomaceous earth
• DNA purification, bacterial-genomic, triton extraction method
• DNA purification, bacterial-genomic via Qiagen columns
• DNA purification, bacterial genomic, specifically Gram positive bacteria
• DNA isolation from low melting-temperature agarose
• Low copy plasmid purification using Qiagen Midi columns
• DNA/RNA precipitation
• Plasmid "Miniprep" isolation
• Single stranded template from pBluescript phagemid
• DNA detection
• DNA isolation from low melting-temperature agarose
• Metaphase chromosome preparation
• DNA preparation by cryostom tissue dissection
• RNA Preparation
• Solublization of RNA in formamide
• RNAse protection assay
• LiCl RNA preparation
• Mini-Prep procedure for E. coli plasmid purification
• Chromosome banding : for cytogenetics analysis, whole peripheral blood is cultured at 37°C in the presence of tetradecanoyl phorbol 12-myristate 13-acetate (TPA) (0.05 µg/mL) for 3 to 5 days. Cells are treated with colcemid (0.1 µg/mL) for 60 minutes at 37°C before harvesting. After hypotonic treatment (KCL 0.075 mol/L for 10 minutes at 37°C), cells are resuspended in fixative (methanol: glacial acetic acid 3:1). Standard cytogenetic preparations are made, G banded, and karyotyped according to the International System for Cytogenetic Nomenclature (1995).

Web resources :
• DAPI chromosome identification
• Image analysis
• Image capture
• RNA sequencing
• mapping RNA with nuclease S1 : preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe. At the end of the reaction period, nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by either autoradiography or Southern hybridization. The method can be used to quantify RNAs, to map the positions of introns and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates^{ref1, ref2, ref3, ref4}.
• preparation of the DNA probe
• 1. Prepare a polyacrylamide minigel of the desired concentration containing 8 M urea (for example, Bio-Rad Mini-Protean).
• 2. While the gel is polymerizing, mix the following reagents:
• unlabeled oligonucleotide : 10 pmol (1 ml)
• [g³²-P]ATP (3,000 Ci/mmol, 10 mCi/ml) : 10 ml
• 10X polynucleotide kinase buffer : 2 ml
•  water : 6 ml
•  polynucleotide kinase : 10 units (1 ml)
 Incubate the reaction mixture at 37 °C for 45 min and then at 95 °C for 3 min to inactivate the polynucleotide kinase. The oligonucleotide used to prime probe synthesis should be 20-25 nucleotides in length and complementary to the RNA strand to be analyzed. It should hybridize to the template DNA 250?500 nucleotides 3' of the position to be cleaved by the chosen restriction enzyme. Store oligonucleotides in aliquots at -20 °C.
• 3. Add to the kinase reaction:
• single-strand DNA template : 2 ml (2 mg)
• 10X annealing buffer : 4 ml
• water : 14 ml
Incubate the reaction mixture at 65 °C for 10 min and then allow it to cool to 15-25 °C. Use standard procedures to prepare single-stranded DNA from a recombinant bacteriophage M13 carrying the insert DNA strand in the same sense as the test RNA.
• 4. To the reaction mix from Step 3, add 4 ml of deoxynucleoside triphosphate (dNTP) mixture (20 mM) and 1 ml (10 units) of the Klenow fragment of Escherichia coli DNA polymerase I. Incubate the reaction mixture at 15-25 °C for 15 min and then inactivate the DNA polymerase by incubation at 65 °C for 3 min.
• 5. Adjust the ionic composition and pH of the reaction mixture to suit the restriction enzyme, add 20 units of enzyme and incubate the reaction mixture at the appropriate temperature for 2 h. Recover the DNA probe by ethanol precipitation.
• 6. Dissolve the DNA in 20 ml of gel-loading buffer, load onto the minigel and run the gel until the bromophenol blue reaches the bottom of the gel (200 mA for 30 min).
• 7. Expose the gel to X-ray film. Realign the gel on the glass plate with the film, identify the radiolabeled band and excise it with a scalpel.
• 8. Transfer the gel fragment to a fresh sterile microcentrifuge tube, add just enough gel elution buffer to cover the fragment (250-500 ml) and elute the labeled DNA at 15-25 °C overnight. Store at -70 °C.
• hybridization of RNA and DNA
• 9. Transfer 0.5-150-g aliquots of RNA (test and standard) into separate sterile microcentrifuge tubes. Add an excess of uniformly labeled single-stranded DNA probe to each tube. To prepare the RNA standard, synthesize it in vitro by transcription of the appropriate strand of a recombinant plasmid containing the DNA sequences of interest and a bacteriophage promoter. Prepare test RNA (poly(A)⁺ or total RNA) using any standard isolation method.
• 10. Recover the RNA and DNA by ethanol precipitation and dissolve the nucleic acid pellet in 30 l of hybridization buffer in a microcentrifuge tube.
• 11. Close the lid of the tube tightly and incubate the hybridization reaction in a water bath set at 85 °C for 10 min to denature the nucleic acids.
• 12. Quickly transfer the tube to a water bath set at the desired hybridization temperature (usually 65 °C). Hybridize the DNA and RNA at the chosen temperature for 12-16 h.
• 13. Keeping the body of the tube submerged, open the lid of the hybridization tube. Rapidly add 300 l of ice-cold nuclease S1 digestion buffer and immediately remove the tube from the water bath. Vortex gently to mix and then transfer the tube to a water bath set at the temperature appropriate for digestion with nuclease S1, depending upon the objective^ref. Incubate 1-2 h, depending on the degree of digestion desired.
• 14. Chill the reaction to 0 °C, add 80 ml of nuclease S1 stop mixture and vortex the tube to mix. Extract the reaction once with 1:1 (vol/vol) phenol/chloroform and recover the products by ethanol precipitation.
• 15. Dissolve the pellet in 4 ml of Tris-EDTA (TE) buffer (pH 7.6). Add 6 ml of gel-loading buffer and separate the DNA by electrophoresis through a polyacrylamide-8 M urea gel.
• 16. Transfer the gel on the glass plate to a tray containing an excess of 10% trichloroacetic acid (TCA). Gently rock or rotate the tray at 15-25 °C for 10 min, pour off the 10% TCA solution and replace with an excess of 1% TCA. Gently rock or rotate the tray at 15-25 °C for 5 min.
• 17. Pour off the 1% TCA solution and briefly rinse the fixed gel with distilled, deionized water. Lift the glass plate together with the gel out of the tray and place them on a flat bench top. Remove excess water with paper towels or Kimwipes.
• 18. Cut a piece of Whatman 3MM filter paper (or equivalent) that is 1 cm larger than the gel on all sides. Transfer the gel to the filter paper by laying the paper on top of the gel and inverting the glass plate.
• 19. Remove the plate and dry the gel on a gel dryer at 60 °C for 1.0-1.5 h.
• 20. Obtain an autoradiographic image of the dried gel. Scan the image by densitometry or phosphorimaging or excise the fragments from the gel and count them by liquid scintillation spectroscopy.
• DNA sequencing

Web resources :
• companies offering equipment, reagents and software for DNA sequencing : Accelrys, Agencourt Bioscience, Applied Biosystems, Beckman Coulter, Biotage, Bio S&T, Bio-Rad, Cambrex, CBS Scientific Company, Inc., DNASTAR, Inc., GE Healthcare, 454 Life Sciences, Genamics, Geospiza, Gene Codes Corporation, GeneFlow, Ltd., Invitrogen, Mekentosj, MiraiBio, Mobious Genomics, Molecular Cloning Laboratories, MWG Biotech, Nanofluidics, Network Biosystems, Nucleics, Parallabs, PerkinElmer, Polymorphic DNA Technologies Inc., Retrogen, Inc., Roche Applied Sciences, Rockland Immunochemicals, Inc., Solexa, Stratagene, Technelysium, Textco, TimeLogic, TriStar Technology Group, USB, US Genomics,

VisiGen Biotechnologies
• Pyrosequencing
• Nanopore sequencing
• Sequencing gel solutions
• Sequencing gels: wedge gel performance without pouring wedge gels
• Linear amp seqI
• Linear amp seqII
• Linear amp seqIII
• Linear amp seqIV Licor
• Terminator reaction clean-up
• Sequencing through stop regions of M13 or pUC subclones, and PCR products
• Direct automated sequencing of bacterial genomic DNA
• AGTC 96 well mini-prep
• Sequenase 1 dye-termination
• Sequenase II ALF primers
• Sequenase V2.0 licor
• Nucleic acid hybridizations
• Hybridization
• in situ hybridization (ISH)
• fluorescent ISH (FISH)
• chromosome painting
• Octavian Henegariu's Multicolor FISH Page: color changing karyotyping (CCK), custom fluorescent nucleotide synthesis, and improved cytogenetic slide preparation protocols
• DNA-FISH
• RNA-FISH
In situ hybridization (ISH) is used to visualize defined nucleic acid sequences in cellular preparations by hybridization of complementary probe sequences. Probe sequences can be labeled with isotopes, but nonisotopic ISH is used increasingly as it is considerably faster, usually has greater signal resolution, and provides many options to simultaneously visualize different targets by combining various detection methods. The most popular protocols use fluorescence detection, as described here. These protocols have many applications, from basic gene mapping and diagnosis of chromosomal aberrations^{ref1, ref2, ref3} to detailed studies of cellular structure and function, such as the painting of chromosomes in three-dimensionally preserved nuclei^{ref1, ref2}. This protocol describes FISH of biotin- or digoxigenin-labeled probes to denatured metaphase chromosomes and interphase nuclei. The hybridized probes are detected and visualized using fluorochrome-conjugated reagents. Procedure
• preparation of probes
• 1| For probes labeled with biotin or digoxigenin, prepare the probe cocktail: in a microcentrifuge tube combine 1020 ng of probe DNA and 1-3 mg of salmon sperm DNA. Lyophilize the DNA in a Savant Speed Vac or in a heating block, resuspend in 6-7 ml of deionized formamide and mix well (at 15-25 °C for at least 30 min).
• 2| For probes targeting a chromosome-specific repeat cluster of DNA, add hybridization buffer (final concentrations: 2X SSC, 10% dextran sulfate, 25 mM sodium phosphate) to the probe cocktail for a total volume of 10 ml. Mix well at 15-25 °C for 30 min.
When the probe DNA contains interspersed repetitive sequences (IRSs), chromosomal in situ suppression hybridization^{ref1, ref2} is performed to prevent binding of labeled IRSs within the probe to sequences other than the targeted locus. Competitor DNA is added to the probe mix, and a preannealing step is performed. The competitor DNA can be total genomic DNA (for example, human placental DNA) or the Cot-1 fraction of DNA.
• denaturation of probe and specimen
• 3| Denature the double-stranded probe mixture at 75 °C for 5 min in a water bath or in a heating block.
• 4| For repetitive probes where no Cot-1 DNA has been added, place the probe mixture directly on ice and go to Step 5.
• For a probe DNA that contains IRS sequences and has Cot-1 DNA in the mixture, incubate the probe mixture at 37 °C for 20 to 30 min as a preannealing step.
• 5| Denature fixed chromosome specimens by immersing the slides in denaturation buffer (70% formamide, 2 SSC (pH 7.0)) at 70 °C for 2 min. Use a Coplin jar in a 70 °C water bath, but do not place more than four slides in a Coplin jar at any one time.
• 6| Dehydrate slides through an ice-cold ethanol series of 70%, 90% and 100% for 3 min each and then air-dry.
• 7| Apply 10 ml of the denatured probe onto a 18X 18-mm glass coverslip (prewarmed to 37 °C) and lower the slide onto the probe mix to avoid trapping air bubbles between coverslip and slide. Apply rubber cement to seal around the coverslip.
• hybridization
• 8| Place the slides with samples to be hybridized in a humidified chamber in a hot-air oven at 37 °C or 42 °C for 1?18 h, as appropriate for the specimen and probe.
• 9| Remove rubber cement from slides with forceps and carefully remove coverslips by washing with 2 SSC.
• 10| Place the slides in wash buffer A (50% formamide, 2X SSC (pH 7), preheated to 42 °C) into a shaking water bath at 42 °C. Replace the buffer every 5 min for 15 min.
• 11| Place slides in wash buffer B (0.1-1X SSC (depending on stringency required), pH 7.0, preheated to 60 °C) into a shaking water bath at 60 °C. Replace the buffer every 5 min for 15 min.
• 12| Exchange wash buffer B for 4X SSC at 15-25 °C.
• detection
• 13| Depending on which hapten was used to label the probe and whether amplification of the signal is needed, select the appropriate detection system (for example, avidin/streptavidin if the probe was labeled with biotin or anti-digoxigenin antibody if the probe was labeled with digoxigenin).
• 14| Place 50-200 ml of blocking solution (4% bovine serum albumin (BSA) or dried milk powder, 4X SSC) on the hybridized sample, cover the area with a coverslip and incubate at 15-25 °C for 20 min.
• 15| Add 50 ml of detecting solution (1-20 mg/ml in blocking buffer with 1% BSA or other blocking compound) onto a 24 x 40-mm glass coverslip.
• 16| Lower the slide over the coverslip to present the hybridized area to the detecting solution. Incubate in a humidified chamber at 15?25 °C for 1 h or at 37 °C for 30 min.
• 17| Wash slides in wash buffer C (2X SSC, 0.1% Tween 20; counterstaining dyes such as propidium iodide, DAPI and Hoechst can be included to stain chromosomes) 3 times at 42 °C for 5 min in a shaking water bath.
• 18| Mount the slides with the appropriate mounting medium, and view the samples using fluorescence microscopy. The slides can be stored for a few days to many months in the dark at -20 °C or -70 °C
Web resources :
• In situ hybridization
• comparative genomic hybridization (CGH)

Web resources :
• Spectral karyotyping (SKY) and CGH database and protocols at NCBI
• Comparative Genomic Hybridization at Dartmouth
• CGH
• Southern blot technique : a technique for transferring DNA fragments separated by agarose gel electrophoresis to a nitrocellulose filter, on which specific fragments can then be detected by their hybridization to probes, which were labeled radioactively in the original technique but are now often labeled using nonradioactive methods. Procedure^{ref1, ref2} :
• Digestion and separation of the DNA
• 1| Digest an appropriate amount of genomic DNA with one or more restriction enzymes.
• 2| Measure the concentrations of the digested DNAs and transfer the desired amount of each digest to a fresh microcentrifuge tube. Add 0.15 volume of 6X sucrose gel-loading buffer.
• 3| Separate the fragments of DNA by electrophoresis through an agarose gel (for example, a 0.7% gel cast in 0.5X TBE or 1X TAE buffer). Maintain a low voltage through the gel (< 1 V/cm).
• 4| After electrophoresis is complete, stain the gel with ethidium bromide (10 mg/ml) or SYBR Gold (Molecular Probes) and photograph the gel. Place a transparent ruler alongside the gel to assess in the photograph the distance that any band of DNA has migrated.
• Preparation of the gel and membrane for transfer
• 5| Transfer the gel to a glass baking dish. Use a razor blade to trim away unused areas of the gel, and cut a small triangular piece from the bottom left-hand corner to simplify orientation.
• 6| Denature the DNA by soaking in a denaturing solution as follows.
• For transfer to uncharged membranes (nitrocellulose or nylon):
• (i) Soak the gel for 45 min at 15–25 °C in 10 gel volumes of denaturation solution (1.5 M NaCl, 0.5 M NaOH) with constant gentle agitation.
• (ii) Rinse the gel briefly in deionized H₂O, and then neutralize it by soaking for 30 min at 15–25 °C in 10 gel volumes of neutralization buffer (1 M Tris, pH 4.0, 1.5 mM NaCl) with constant gentle agitation. Change the neutralization buffer and continue soaking for a further 15 min.
• For transfer to charged nylon membranes:
• (i) Soak the gel for 15 min at 15–25 °C in several volumes of alkaline transfer buffer (0.4 M NaOH, 1 M NaCl) with constant gentle agitation.
• (ii) Change the solution and continue soaking for 20 min.
• 7| Cut a piece of nylon or nitrocellulose membrane and two sheets of thick blotting paper 1 mm larger than the gel in each dimension. Cut a corner from the membrane to match the gel (step 5).
• 8| Float the membrane on the surface of a dish of deionized H2O until it wets completely, then immerse in the appropriate transfer buffer for at least 5 min.
Use neutral transfer buffer (10 SSC or 10 SSPE for uncharged membranes. Use alkaline transfer buffer (0.4 M NaOH with 1 M NaCl) for charged nylon membranes.
• Assembly of transfer setup and transfer of DNA
• 9| Place a piece of thick blotting paper on a sheet of Plexiglas or a glass plate to form a support that is longer and wider than the gel, draping the ends of the blotting paper over the edges of the plate. Place the support inside a large baking dish on top of four neoprene stoppers to elevate it.
• 10| Fill the dish with the appropriate transfer buffer (see step 8) until the level of the liquid reaches almost to the top of the support. When the blotting paper on the top of the support is thoroughly wet, smooth out all air bubbles with a glass rod or pipette.
• 11| Remove the gel from the solution in which it was placed in step 6 and invert it so that its underside is now uppermost. Place the inverted gel on the support, centered on the wet blotting paper, and ensure there are no air bubbles between the blotting paper and the gel.
• 12| Surround, but do not cover, the gel with Saran Wrap or Parafilm.
• 13| Wet the top of the gel with the appropriate transfer buffer (step 8). Place the wet membrane on top of the gel, aligning the cut corners. One edge of the membrane should extend just over the edge of the line of slots at the top of the gel.
• 14| Wet the two pieces of thick blotting paper in the appropriate transfer buffer and place them on top of the wet membrane. Smooth away air bubbles with a pipette.
• 15| Cut or fold a stack of paper towels (5–8 cm high) just smaller than the blotting papers and place on the blotting papers. Put a glass plate on top of the stack and weigh it down with a 400-g weight.
• 16| Allow the transfer of DNA to proceed for 8–24 h, replacing the towels as they become wet.
• 17| Remove the papers above the gel. Turn the gel and the attached membrane over and lay them, gel side up, on a dry sheet of blotting paper. Mark the positions of the gel slots on the membrane.
• 18| Peel the gel from the membrane. Discard the gel or stain it to gauge the success of transfer.
• Fixation of DNA to the membrane
• 19| Soak the uncharged membrane in one of the following solutions as appropriate:
• For neutral transfer: soak the gel in 6X SSC for 5 min at 15–25 °C and proceed to step 20.
• For alkaline transfer: soak the gel in 0.5 M Tris-Cl (pH 7.2) with 1 M NaCl for 15 min at 15–25 °C and proceed to step 21. Alkaline transfer results in covalent attachment of DNA to positively charged nylon membranes.
• 20| Immobilize the DNA that has been transferred to uncharged membranes.
• To fix by baking in a vacuum oven:
• (i) Drain excess fluid from the membrane and dry it on a paper towel for at least 30 min at 15–25 °C.
• (ii) Sandwich the membrane between two sheets of dry blotting paper and bake for 30 min to 2 h at 80°C in a vacuum oven.
• To fix by baking in a microwave oven:
• (i) Place the damp membrane on a dry piece of blotting paper.
• (ii) Heat the membrane for 2–3 min at full power in a microwave oven (750–900 W).
• To cross-link by UV irradiation:
• (i)Place the damp membrane on a dry piece of blotting paper.
• (ii) Irradiate at 254 nm to cross-link the DNA to the membrane.
• 21| Proceed directly to hybridization of immobilized DNA with the appropriate probe(s).
Membranes not used immediately in hybridization reactions should be thoroughly dried, wrapped loosely in aluminum foil or blotting paper, and stored at 15–25 °C, preferably under vacuum.
Web resources :
• Alkaline Southern blotting
• Southern blot-chemiluminescent detection, using Lumigen or Lumiphos (BM "Genius" system)
• Southern blot-chemiluminescent detection, using CSPD (BM "Genius" system)
• "Northern" blot technique : a technique analogous to Southern blot technique, but performed on fragments of RNA instead of DNA; a nylon membrane is often substituted for the nitrocellulose filter. Northern blotting and hybridization are used to study gene expression by detecting RNA species of interest, and to identify alternate RNA splicing patterns. It is analogous to Southern blotting, which is used to analyze DNA. This protocol describes the transfer of RNA from agarose gels to nylon membranes and the fixation of the RNA to the membrane^{ref1, ref2, ref3, ref4, ref5}.

Procedure :
• partial hydrolysis of RNA sample :
• 1| (optional) partially hydrolyze the RNA sample, fractionated through agarose, by soaking the gel in the appropriate soaking solution as described below.
• for transfer to uncharged nylon membranes:
• rinse the gel with DEPC-treated H₂O and soak for 20 min in 5 gel volumes of 0.05 N NaOH.
• transfer the gel into ten gel volumes of 20X SSC for 40 min and then proceed directly to Step 2.
• for transfer to charged nylon membranes:
• rinse the gel with DEPC-treated H₂O; soak for 20 min in 5 gel volumes of 0.01 N NaOH/3 M NaCl.
• proceed directly to Step 2.
• 2| move the gel to a glass baking dish and trim away unused areas of the gel. Cut a small triangular piece from the bottom left-hand corner to simplify orientation.
• assembly of the transfer setup and transfer of RNA
• 3| place a piece of thick blotting paper on a sheet of Plexiglas or a glass plate to form a support that is longer and wider than the gel. Drape the ends of the blotting paper over the edges of the plate. Place the assembly inside a large baking dish with the support elevated on four neoprene stoppers.
• 4| fill the dish almost to the level of the support with the appropriate RNA transfer buffer (0.01 N NaOH, 3 M NaCl for positively charged membranes, and 20X SSC for uncharged membranes) until the level of the liquid reaches almost to the top of the support. When the blotting paper on the top of the support is thoroughly wet, smooth out all air bubbles with a glass rod or pipet.
• 5| cut a piece of the appropriate nylon membrane 1 mm larger than the gel in both dimensions and float it on the surface of a dish of deionized H₂O until it wets completely. Then immerse the membrane in 10X SSC for at least 5 min. Cut a corner from the membrane to match the gel (Step 2)
• 6| remove the gel from the solution in which it was placed in Step 1 and place it face down on the wet blotting paper. Surround, but do not cover, the gel with Saran wrap or Parafilm.
• 7| wet the top of the gel with the appropriate transfer buffer (see Step 4) and place the wet membrane on top of the gel, aligning the cut corners. One edge of the membrane should extend just beyond the line of wells at the top of the gel.
• 8| wet two pieces of thick blotting paper (cut to exactly the same size as the gel) in the appropriate transfer buffer and place them on top of the membrane. Smooth out any air bubbles with a glass rod or pipet.
• 9| prepare a stack of paper towels (5–8 cm high), just smaller than the blotting papers, and place them on the blotting papers. Put a glass plate on top of the stack and weigh it down with a 400-g weight.
• 10| allow the transfer of RNA to proceed for no more than 4 h in neutral buffer and 1 h in alkaline buffer.
• 11| dismantle the capillary transfer system. Mark the positions of the slots on the membrane. Transfer the membrane to a glass tray containing 6> SSC and agitate very slowly for 5 min.
• 12| drain excess fluid from the membrane and dry it, RNA side upward, on a sheet of blotting paper for a few minutes.
• fixation of RNA and staining of the membrane
• 13| depending on the type of transfer and membrane used, stain and/or fix as follows:
• positively charged nylon membrane, alkaline transfer. Stain with methylene blue (see below).
• uncharged or positively charged nylon, neutral transfer. Fix by UV irradiation and then stain with methylene blue (see below).
• uncharged or positively charged nylon, neutral transfer. Stain with methylene blue and then fix by baking the membrane or heating in a microwave oven (see below)
• stain with methylene blue.
• soak the damp membrane in methylene blue solution (0.02% (wt/vol) in 0.3 M sodium acetate (pH 5.5)) until the rRNAs become visible (3–5 min).
• photograph the membrane under visible light with a yellow filter.
• destain the membrane by washing in 0.2X SSC, 1% (wt/vol) SDS for 15 min.
• to fix by baking:
• place the dry membrane between two pieces of blotting paper.
• bake for 2 h under vacuum at 80 °C.
• to fix in a microwave oven:
• place the damp membrane on a piece of blotting paper.
• heat for 2–3 min at full power in a microwave oven (750–900 W).
• to cross-link by UV irradiation:
• place the damp, unstained membrane on a piece of blotting paper.
• irradiate at 254 nm for 1 min 45 s at 1.5 J/cm².
Membranes not used immediately in hybridization reactions should be thoroughly dried, wrapped loosely in aluminum foil or blotting paper and stored at 15–25 °C, preferably under vacuum.
Web resources :
• Northern blots
• The "Neverfail" Northern Blot Hybridization
• single cell gel electrophoresis (SCGE) / comet assay : a sensitive microscopic gel electrophoresis method capable of measuring DNA strand breaks in individual cells. The original form of this assay was described first by Östling et al. An alkaline comet assay has been introduced by Singh et al. A number of variations of this assay greatly increase the flexibility and utility of this technique for detecting various manifestations of DNA damage (e.g., single- and double-strand breaks, oxidative induced base damage, and DNA-DNA/DNA-protein crosslinking) and DNA repair in virtually any eukaryotic cell. It has been used widely in genetic toxicology, radiation biology and medical and environmental research for the detection of single-strand breaks, double-strand breaks and alkali-labile sites in DNA and can be employed also to visualize DNA degradation due to necrosis or apoptosis. The assay essentially involves embedding of eukaryotic cells in low-melting agarose gel on a slide, followed by cell lysis and electrophoresis. Fragmented DNA is visualized after appropriate staining as a tail projecting from the original cell that resembles the tail of a comet (laddering pattern). Visual display of the detected comet shape and calculation of comet head radius, tail length, and integral intensity of fluoresence can provide information about the nature and degree of DNA fragmentation^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}
• conformation-sensitive gel electrophoresis (CSGE) : a method that requires no special equipment or preparation of the DNA. It detects conformational differences between heteroduplexes and homoduplexes, which are detected in differences in electrophoretic migration that are accentuated by the use of a mild denaturing agent^{ref1, ref2}.
• 2-D DNA gel electrophoresis (2-DDGE) : HMW-DNA fragments restricted with a rare-cutter restriction enzyme are separated by PFGE. After staining and photography the lane is cut from the gel and incubated in a solution containing another restriction enzyme. Subsequently, the gel-strip and placed on top of a new gel for electrophoretic separation in the second dimension^ref.
• denaturing gradient gel electrophoresis (DGGE) : resolves partially denatured double-stranded DNA in precisely defined conditions of temperature and denaturant concentration. Different alleles may denature to various extents under such conditions, and migrate differently on DGGE acrylamide gels^ref.
• denaturing detergent gradient gel electrophoresis (DDGE) : method based on differential electrophoretic migration of single versus double-stranded DNA, which can easily be detected by commonly used ethidium bromide staining methods. However, it still requires special apparatus for generating detergent gradients and optimization of the experimental conditions^ref.
• field inversion gel electrophoresis (FIGE) : electrophoresis method (comparable to PFGE) for the separation of high molecular weight DNA. [Van Daelen, RA., Jonkers JJ, Zabel P (1989) Preparation of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE). Plant Mol. Biol. 12, 341-342.]
• pulsed field gel electrophoresis (PFGE) : gel system to separate DNA fragments sizing 0.5 to 3 Megabases with an electrical current alternately delivered from 2 angles in timed intervals, which minimizes diffusion of large molecules^ref.
• transverse alternating field electrophoresis (TAFE) : electrodes are positioned on opposite sides of a vertical gel. The DNA moves first toward one electrode and then toward the other, forming a zigzag pattern. The vector of this oscillation is a straight line from the loading well to the base of the gel. Variation in voltage and pulse time allows separation of DNAs ranging in size from 2 Kb to > 6,000 Kb. Procedure^ref :
• separation of DNA fragments by TAFE :
• 1| cast a 1% agarose gel in 1X TAFE gel buffer (20 mM Tris-acetate (pH 8.2), 0.5 ml EDTA) without ethidium bromide and allow the gel to set. Run the gel in the same buffer solution as used for preparing the gel.
• 2| prepare agarose plugs containing the DNA of interest and carry out digestion with restriction enzymes. Prepare and embed the appropriate DNA size standards
• 3| rinse all of the plugs in 10 volumes of Tris-EDTA (pH 8.0) for 30 minutes with 2 changes of the buffer
• 4| embed the digested and rinsed DNA plug in individual wells of the gel. Seal the plugs in the wells with molten 1% agarose in 1X TAFE gel buffer
• 5| place the gel in the TAFE apparatus filled with 1X TAFE gel buffer, cooled to 14°C
• 6| connect the gel apparatus to an appropriate power supply set to deliver 4-s pulses at a constant current of 170-180 mA for 30 minutes. This setting forces the DNA to enter the gel rapidly. After this time period, decrease the current input to 150 mA, change the pulse time to a setting optimum for the size range of DNA to be resolved as described below and continue electrophoresis for 12-18 hrs :

size range (kb)	pulse time (s)	electrophoresis time (h)
5-50	1	10
20-100	3	10
50-250	10	14
100-400	20	14
200-1,000	45	18
200-1,600	70	20

In a Tris-acetate-EDTA buffer,  a pulse time of 15 s will separate DNA fragments in the 50-400-kb size range. This same range of fragments can be separated in the Tris-borate-EDTA buffer using a program of 8-s pulses at 350 mA for 12 h followed by 15-s pulses at 350 mA for an additional 12 h.
• staining and transfer of the separated DNA fragments :
• 7| disconnect the power supply, dismantle the gel apparatus and stain the gel in 1X TAFE gel buffer containing 0.5 mg/ml ethidium bromide or an appropriate dilution of SYBR Gold. Photograph the gel under UV light
• 8| rinse the stained gel twice with water. Then incubate in denaturation solution for 30 min with gentle shaking. Change the denaturation solution and incubate for additional 30 minutes. In general, standard methods of Southern blotting can be used to detect hybridizing gene sequences in TAFE gels. Some investigators find that very large DNA fragments transfer more efficiently after partial hydrolysis of the DNA by acid treatment. When acid treatmens are used in the protocol, it is important to stain the agarose gel after transfer of the DNA to the nylon membrane. A slight residual smear of the DNA should be visible. If no residual DNA is detected, then the acid treatment may have been too harsh (that is, too long or too strong)
• 9 | trasnfer the DNA directly to a nylon membrane by capillary blotting in denaturation solution (1.5 M NaCl, 0.5 M NaOH). Some investigators find that the transfer of larger DNA fragments in enhanced when 6X SSC buffer is used in capillary blotting rather than the more standard 10X SSC solution
• 10| after transfer, fix the DNA to the nylon membrane by baking for 2 h at 80°C, by ultraviolet-light crosslinking, or by heating for 2-3 min at full-power in a microwave oven (750-900 W)
• 11| carry out prehybridization and hybridization with with labeled probes in a formamide-containing buffer (for example, 6X SSC, 5% Denhardt's reagent, 0.5% (wt/vol) SDS, 1 mg/ml poly(A), 100 mg/ml salmon sperm DNA, 50% (vol/vol) formamide). We typically use ³²P-labeled single strand bacteriophage M13 probes at a concentration of 5 x 10⁶ to 6 x 10⁶ c.p.m./ml of hybridization buffer to detect a single-copy gene in a complex mammalian genome. A membrane hybridized for 12-16 h in this fashion is washed in 500 ml of 2X SSC containing 1% (wt/vol) SDS for 15 min at 15-25°C, scrubbed gently with a sponge, washed in 1 l of 0.5X SSC containing 1% (wt/vol) SDS for 2 h at 68°C and then autoradiographed.
• contour-clamped homogeneous electric field electrophoresis (CHEF) for the resolution of DNA fragments > 6,000 Kb^ref
• single stranded confirmational polymorphism (SSCP) : relies on secondary and tertiary structure differences between denatured and rapidly cooled amplified DNA fragments that differ slightly in their DNA sequence. Different SSCP alleles are resolved on non-denaturing acrylamide gels, usually at low temperatures. The ability to resolve alleles depends on the conditions of electrophoresis^{ref1, ref2, ref3}.
• temperature-gradient gel electrophoresis (TGGE) : method based on differential electrophoretic migration of single versus double-stranded DNA, which can easily be detected by commonly used ethidium bromide staining methods. However, it still requires special apparatus for generating temperature gradients and optimization of the experimental conditions^ref
• microfluidics-based gel electrophoresis : 5100 Automated Lab-on-a-Chip Platform (ALP) (source : Agilent Technologies of Palo Alto, Calif) integrates the process of resolving both nucleic acids and proteins – including sample handling, injection, separation, staining, and detection – into a single 5.5-cm² chip that can process 6,000 samples before being replaced. A high-throughput implementation of the technology underlying the company's popular 2100 Bioanalyzer, the 5100 ALP can run up to 4,608 samples overnight, with run times of 1 minute per sample at full capacity (twice as fast as the 2100 system). Unlike the manually loaded 2100, the 5100 ALP uses capillaries to load samples from twelve 96- or 384-well plates. Agilent currently sells compatible kits for quality control and screening of PCR fragments and identification and analysis of proteins. The new system offers improved data quality, which should aid data sharing across laboratories. Gel electrophoresis by its nature is not a very reproducible technique, and quantitative accuracy is poor. The microfluidic lab-on-a-chip systems get very high reproducibility and get good quantitation accuracy with digital data output. Agilent's early-access customers have been pleased with the 5100. Te 2100 can run only 12 samples per chip every 30 to 40 minutes.
• retrotransposon-based insertion polymorphisms (RBIP)^ref
• single nucleotide amplified polymorphisms (SNAP) : PCR based marker
• single nucleotide polymorphisms (SNP) : polymorphism based on a nucleotide substitution. Used as a marker diagnostic for a specific trait. Often mentioned in connection with a technique which allows the specific recognition of the SNP^ref.
• DNA amplification methods :
• polymerase chain reaction (PCR) : a rapid technique for in vitro exponential amplification of specific DNA or RNA sequences, allowing small quantities of short sequences to be analyzed without cloning: oligonucleotide primers are annealed to single-stranded nucleotide sequences, which are copied by a thermostable polymerase; the number of copies is geometrically amplified by repeated cycles of annealing and copying^{ref1, ref2}. Laminar convection current in a Rayleigh-Bénard convection cell (hot water rises in the middle of the chamber and flows towards its edge, where it cools and sinks) can drive PCR 4 times faster than standard techniques. There is no need to use an alternating cycle of heating and cooling. The enzyme copies single DNA strands at the chamber's cool periphery, and these strands then pair up. The convection current carries the double strands to the hot centre, where they are prised apart before being borne back to the edges. It makes 100,000 copies from a single DNA strand in less than half an hour, and could be miniaturized to just 0.1 mm^{ref1, ref2}. Percentage of Taq DNA polymerase usage associated with specific molecular biology techniques :
• classic end-point PCR (57.8%)
• classic end-point reverse transcriptase PCR (14.7%)
• real-time quantitative PCR, RNA or cDNA template (13.0%)
• real-time quantitative PCR, DNA template (8.6%)
• sequencing (5.7%)
• other (0.2%)
• site-selected insertion (SSI) : a PCR-based mutagenesis and tagging technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes^{ref1, ref2}
• site-directed mutagenesis by the singe-tube megaprimer PCR method

The 'megaprimer' method of site-directed mutagenesis uses three oligonucleotide primers and two rounds of polymerase chain reaction (PCR)^ref. One oligonucleotides is mutagenic; the others are forward and reverse primers that lie upstream and downstream from the binding site for the mutagenic oligonucleotide. The mutagenic primer and the nearer of the external primers are used in the first PCR to generate and amplify a mutated fragment of DNA. This amplified fragment—the megaprimer—is used in the second PCR in conjunction with the remaining external primer to amplify a longer region of the template DNA. This protocol is based on a method that uses forward and reverse external primers with significantly different melting temperatures (T_m)^ref.
• generation of the mutant megaprimer
• in a sterile 0.5-ml microcentrifuge tube or amplification tube (on ice), mix the following reagents for the first amplification reaction:
• 10x amplification buffer : 10 mL
• plasmid template DNA : 200-400 pg
• 2.5 mM dNTP solution : 8 ml
• mutagenic primer : 10 pmol
• low-T_m flanking primer : 100 pmol
• thermostable DNA polymerase : 0.5 ml (2.5 units)
• water : to 100 ml
If the 10x amplification buffer supplied by the manufacturer of the thermostable DNA polymerase does not contain MgCl₂, add an appropriate volume of 0.1 M MgCl₂ so that the reaction contains the optimum concentration of divalent ion for the particular DNA polymerase being used. If the thermocycler does not have a heated lid, overlay the PCRs with 1 drop (50 ml) of light mineral oil.
• place the tubes in a thermocycler and amplify the nucleic acids using the denaturation, annealing and polymerization times and temperatures listed in the table below.

cycle number	denaturation	annealing	polymerization
first cycle	4 min at 94°C	1 min s at 42-46°C	1 min at 72°C
24 cycles	40 s at 94°C	1 min s at 42-46°C	1 min at 72°C
last cycle	40 s at 94°C	1 min s at 42-46°C	5 min at 72°C

Times and temperatures may need to be adapted to suit the particular reaction conditions. Polymerization should be carried out for 1 min for every 1,000 bp of length of the target DNA.
• after completion of the first PCR, add to the reaction tube:
• high-T_m flanking primer : 100 pmol
• thermostable DNA polymerase : 0.5 ml (2.5 units)
• 2.5 mM dNTP solution : 3 ml
• mix the reagents gently by pipetting the reaction mixture up and down several times. If necessary, centrifuge the tube briefly to deposit the reagents in the bottom.
• amplification of the mutant templates
• carry out the second amplification reaction, which consists of 25 cycles with the following 2-step temperature profile:
 

cycle number	denaturation	annealing and polymerization
24 cycles	40 s at 94°C	90 s at 72°C
last cycle	40 s at 94°C	5 min at 72°C

• analyze 5% of the second amplification reaction on an agarose or polyacrylamide gel and estimate the concentration of amplified target DNA.
• isolation of the mutant clones
• clone the amplified target into an appropriate vector, transform the construct into competent cells and prepare plasmid DNA from at least six transformants.
• sequence the selected clones to verify the presence of the desired mutation and the absence of any additional mutations in the entire product DNA sequence.
This method yields mutants with an efficiency of 80%; therefore, it is generally necessary to screen only 6 colonies by sequencing to identify the desired clone.
• repeat sequence primed PCR (REP-PCR)
• reverse transcriptase PCR (RT-PCR)

Web resources : Reverse transcriptase PCR (RT-PCR)
Web resources :
• Polymerase chain reaction (PCR) and related methods
• Error rates for thermal resistant DNA polymerases
• PCR and multiplex PCR : guide and troubleshooting
• Cloning PCR-generated DNA fragments
• Core sample PCR: a method to re-PCR unique bands from products of mixed size
• Tc1 deletion PCR tips
• Long PCR protocol
• Degenerate PCR protocol
• ssPCR magnetic beads
• companies offering PCR reagents and instrumentation : Abgene, Active Motif, Agilent Technologies, AlleLogic Biosciences Corp., Ambion, Apogent, Invitrogen, Applied Biosystems, Artus Biotech, Biochain, Biogene, BioHelix Corp. (NEB), Bioneer, Bio-Rad Laboratories, Biosearch Technologies, Biosource, Biotrove, Bio S&T, Brinkmann Instruments, Chemicon International, Cepheid, Corbett Research, DxS Limited, Epicentre, Epoch Biosciences, Eurogentec, Exiqon, Fermentas Inc., Finnzymes, Fluoresentric, Genetic Research Instruments, Genetix, Gene Therapy Systems, Inc., GeneWorks, GenoMechanix, GenScript, Hy-labs, Idaho Technology Inc., Integrated DNA Technologies, Lark Technologies, LGC, Lucigen, Maxim Biotech Inc., MJ Research (Bio-Rad), Midwest Scientific, Molecular Probes (Invitrogen), MWG Biotech, New England BioLabs, Novagen (EMD Biosciences), PGC Scientifics, PerkinElmer, Premier Biosoft International, PrimerDesign Ltd, Promega, Qiagen, Roche Applied Science, Seegene, Serologicals Corporation, Sigma-Aldrich, Sigma-Genosys, Stratagene, SuperArray Bioscience Corp., Synthegen, Takara Mirus Bio, Thermo Electron Corporation, Techne, USB Corp.,
• real-time PCR combines the amplification of a DNA sequence with the detection of the amplified products during each reaction cycle—in other words, in real time. In comparison to conventional PCR, it can be used to detect a much wider range, over 10⁷ fold, of starting template concentration. It is also less time-consuming, as it does not require analysis of the end products by gel electrophoresis, and can provide a quantitative result. It is no surprise that the technique, first described in the mid-1990s, has quickly grown in popularity. Researchers use it to measure gene expression and copy number, calculate viral titers, and carry out single-nucleotide polymorphism analysis, to mention just a few applications. In particular, real-time reverse transcription (RT)-PCR has become the method of choice to rapidly and quantitatively examine the expression of specific genes. Scientists can readily detect as little as a twofold change in the expression of a target gene in response to different treatments in hundreds of samples per day. Because of the prominence of real-time PCR, companies are flooding the market with newer, improved reagents for every step of the process, from sample preparation to reverse transcription to amplification—all of which promise to make the process even faster, more efficient and more reliable. In addition, the methods and instruments used to measure the amplification products continue to become more sophisticated. As a result, researchers wanting to use real-time PCR are faced with a staggering choice of options. PCR was developed in 1983, a discovery that earned Kary Mullis the Nobel Prize in Chemistry ten years later. The technique, now a staple of every molecular biology lab, uses the thermostable Taq DNA polymerase to extend short single-stranded synthetic primers using the target DNA or cDNA as a template during repeated cycles of heat denaturation, primer annealing and primer extension. With each cycle the amount of template DNA is doubled until one of the reagents becomes limiting, and the reaction reaches a plateau. At the completion of the reaction, amplification products are analyzed by size fractionation using gel electrophoresis. Real-time PCR follows the same course, except that the products are detected as they are made, during the exponential phase of the reaction rather than at the end. Detection reagents now on the market are based on probes and dyes that produce a fluorescent signal each time a double-stranded product is made. The more copies of nucleic acid present at the start of the reaction, the fewer amplification cycles are required to make sufficient product to detect by fluorescence imaging. The cycle in which a significant increase in fluorescence above the threshold is measured—referred to as the CT value—can therefore be used to calculate the quantity of DNA in the sample. To carry out real-time PCR, researchers have to choose not only what primers to design (see Box 2) but also what detection chemistry to use. In many cases these decisions will be influenced, if not determined, by the thermocycler (see Box 3) that they have access to and the instrument's chemistry and dye compatibilities. There are many popular chemistries for real-time PCR. One class uses different fluorescent dyes incorporated in short oligonucleotide probes specific for the amplified target. The second class consists of dyes that bind double-stranded DNA and become fluorescent; the most commonly used of these is SYBR Green I, sold by many companies that provide PCR reagents. As the amount of PCR product increases, more SYBR Green I dye binds to DNA, resulting in a steady increase in fluorescence. The technique is inexpensive and generic, as it requires the same detection reagent for each template to be tested. But detection with dyes like SYBR Green I is less specific than probe-based detection methods. For example, if primers bind to each other, the dye will bind to these so-called primer dimers and generate a signal. In addition, SYBR Green I cannot be used in multiplexed assays—in which several distinct targets are included in a single tube or well—because it will not distinguish among different sequences. Despite these drawbacks, SYBR Green I can be used to quantify the amount of template in a sample if the PCR is fully optimized. But many researchers prefer to use this dye to optimize PCR and check that the primers are working well, before ordering a probe-based assay. A variety of probes specific for the amplified target (or amplicon) can be used in real-time PCR. By far the favorite, especially among scientists who have just started using the technique, are TaqMan probes. Each TaqMan probe consists of a single-stranded oligonucleotide that is complementary to a sequence within the target template. It has a fluorescent dye at its 5' end, whose signal is stifled by a quencher moiety at the 3' end. Soon after the TaqMan probe hybridizes to one of the strands on the template, it is digested by the exonuclease activity of the Taq DNA polymerase as it extends the amplification primers. Chopping up the probe releases the fluorescent dye from the quencher, resulting in an irreversible increase in the fluorescence signal. Applied Biosystems Group (ABI) currently offers "over 200,000 TaqMan Gene Expression Assays for human genes and their variants," says Kelly McDonald, senior scientist at ABI. An advance in the technology has been the addition of a DNA minor groove-binding (MGB) moiety at the 3' end of the TaqMan probes, increasing their stability and specificity. This modification allows the probes to be shorter—about 13-20 base pairs (bp) long—and thus "cheaper to make and more flexible to move where you want within the gene," says McDonald. Another company, Epoch, produces its own brand of MGB probes called MGB Eclipse. ABI has exploited the high specificity and sensitivity of the TaqMan assays to develop a method to specifically amplify and quantify microRNAs (miRNAs), which are short endogenous RNAs believed to play primary roles in gene regulation^ref. ABI's new line of assays uses special looped primers for reverse transcription of the miRNAs; the resulting cDNAs are then amplified by real-time PCR. These assays can be used to quantify miRNA levels and discriminate between mature miRNAs and their precursors. "We currently have close to 200 validated assays for human miRNAs. They can quantitate miRNAs with a broad range of expression levels, starting from as little as 1 ng of total RNA," says McDonald. "The assay can be done directly on whole cells or cell lysates to save time and eliminate the risk of losing the miRNAs during the isolation process." ABI plans to expand its product portfolio to include similar assays for the detection of short interfering RNAs (siRNAs)—RNA molecules used to knock down the expression of specific genes^ref. "These assays can be used to validate whether a siRNA is efficiently transfected into cells, as well as monitor the molecule's lifespan," says McDonald. ABI plans to introduce its preliminary miRNA assays through an early access program this spring. Exiqon developed a line of probes incorporating locked nucleic acids (LNAs) that can be integrated into different types of probes, including TaqMan and molecular beacons, to increase the stability of the probe-target duplex. LNA-containing probes can be even shorter than MGB probes and offer greater flexibility in design. Exiqon has developed a ProbeLibrary of 90 prevalidated real-time detection probes substituted with LNAs that can recognize 98% of transcripts cataloged in the RefSeq database at the National Center for Biotechnology Information. The reason these probes can recognize such a high number of transcripts is that each probe is only 8?9 nucleotides long and can therefore detect more than one transcript. Assays specific for a particular target can be designed by using the right primer-probe combination. Real-time PCR detection using molecular beacons. (Courtesy of Salvatore Marras, Public Health Research Institute.)


To help with this task, Exiqon has launched a free online software product to design primers for a particular transcript, usually spanning the exon-exon splice junctions, and then identify one or more LNA probes that will detect the amplification product. "A big issue in real-time PCR is the time spent on assay design," says Peter Roberts, sales manager for Exiqon. "A typical design will use up most of the morning for a researcher. Our software does it in seconds." By combining individual ProbeLibrary probes and target-specific PCR primers selected using the software, Exiqon's Assay Design Center can be used to create > 644,000 distinct assays for the human transcriptome. "It is the only commercially available system that provides specific kits for real-time PCR for model organisms such as Arabidopsis, Drosophila and C. elegans, in addition to the usual human, mouse and rat," says Roberts. Several chemistries developed after the TaqMan probes flaunt a hairpin loop structure. Molecular beacons^ref, first developed at the Public Health Research Institute (PHRI) in Newark, New Jersey, consist of a single-stranded loop complementary to the target template and a double-stranded stem, about six bases in length, with a fluorophore at one end and a quencher at the other end. When the probe is in a hairpin configuration, the fluorophore and quencher are in close proximity. But the probes are designed in such a way that they will bind to the amplicon at a specified temperature—because the probe-target duplex is thermodynamically more stable than the hairpin structure. Upon binding, the stem comes apart and the fluorophore and quencher are separated, giving off fluorescence. "These probes are very flexible and can be used for any existing PCR application and machine," says Salvatore Marras, one of the developers of this technology. Because the stem structure does not participate in the hybridization reaction itself, its length can be adjusted to add specificity to the probe. "Once you have the desired target you can optimize the primers and hybridization temperature to create the most optimal thermal cycle conditions for the PCR, and then adjust the structure of the molecular beacons so that they will only hybridize to the amplicon at that temperature," says Marras. Unlike with TaqMan probes, the fluorescence produced at each amplification cycle is reversible, as the probe is not destroyed, resulting in a lower overall background. You can use six or seven different molecular beacons in the same tube because of the low background," says Marras, whose group has also designed assays that can identify 15 different targets in one tube. "In one assay a particular target will be detected as a combination of two fluorophores. If you use a machine that can detect six colors, you could use 15 pairs of molecular beacons—each pair specific for one target but labeled with a combination of two different fluorophores—in the same tube and look for different signatures," says Marras. Marras has also used molecular beacons synthesized using 2'-O-methyl RNA, which are nuclease resistant and have higher affinity for RNA, to measure the formation of RNA transcripts in a test tube^ref. "We wanted to study inhibitors of RNA polymerase, many of which are antibacterial drugs currently on the market," says Marras. "We designed molecular beacons to bind to transcripts that are formed when an RNA polymerase transcribes from an artificial DNA template. The system allows us to follow transcription in real time. We could potentially screen through thousands of inhibitors and look not only at the level of inhibition but also at the kinetics." Another chemistry that uses hairpin structure is that of Scorpions, first developed by David Whitcombe at DxS^ref. Unlike beacons, Scorpions are PCR primer and probe in one. They consist of a primer that has a 5' stem-loop tail with a fluorophore and a quencher in close proximity. This 'tail' will hybridize to the extension product of the primer, thereby releasing a fluorescent signal. "For TaqMan and molecular beacons, the target and probe have to collide to have a signal, but with Scorpions you have everything on the same molecule," says Stephen Little, DxS CEO. As a result, Scorpions have a fast reaction mechanism. "A probe cannot bind to its target if the target amplicon hybridizes to itself, for example if it contains repetitive sequences. But this is not a problem with Scorpions because they hybridize so quickly after the amplicon is produced," says Little. "They are well suited for problem sequences." Promega has developed a new chemistry that lies between the nonspecific detection using SYBR Green I and the probe-based assay. The soon-to-be-released Plexor system uses certain modified nucleotides that are recognized by DNA polymerase but do not form duplexes with normal, unmodified nucleotides. In a Plexor reaction, one oligoprimer for the PCR is tagged at the 5' end with the modified nucleotide and a fluorophore. The reaction is then carried out in the presence of the four natural nucleotides and a modified nucleotide (complementary to the one at the 5' end of the primer) that has a quencher moiety attached. As the PCR proceeds and the target is amplified, the quencher winds up base-paired to the fluorophore. Thus, the Plexor system measures a decrease in fluorescence as the reaction proceeds. "But the quantification is calculated exactly the same as with TaqMan," says Gary Madsen of Promega. Overview of the core technology in Plexor Systems for qPCR and qRT-PCR. (Courtesy of Promega.)

The technology is similar to detection with SYBR Green I in its simplicity, but allows multiplexing. To help researchers configure single- or multiplex assays, the Promega website will have a Plexor Primer Design tool that will recommend the primer sets, including fluorophores, to be ordered for one or several target genes. One advantage of multiplexing is that it allows researchers to run the target and internal control in the same well. "Right now most researchers will do one assay for the target and one for the control," says Madsen. "Plexor allows you to do things faster and more accurately." The company plans to launch the product in May 2005. Regardless of the detection method used, researchers calculate the quantity of a target gene or transcript in a sample from the CT value obtained during the PCR. The calculation can be done in several ways, either by using a standard curve of CT values obtained from a serially diluted standard solution (absolute quantification) or by comparing the difference in the CT values of two samples (relative quantification). As the name implies, absolute quantification results in an actual number of DNA molecules in a sample, but many experts caution that such claims should be treated with a dose of skepticism. "I always tell customers I quantify relative to the standard I am using. There is really no absolute quantification," says Gregory L. Shipley, director of the Quantitative Genomics Core Laboratory at the University of Texas Health Science Center in Houston, who provides real-time PCR as a service to academic labs. But for most basic research applications, absolute quantification may not be a necessity. "Usually you only want to have a relative measure. For example, you want to see a gene go up relative to another," says Shipley. Real-time PCR is a relatively easy technique to learn, and as new, cheaper instruments and off-the-shelf kits come on the market, many labs are embracing it as a valuable tool in their research. But the apparent accessibility of the technique may be somewhat misleading. "The trick is not the PCR or detection. With most probes you can detect as few as 10 to 1 copies of target DNA and about 100?200 copies for RT-PCR," says Shipley. "But the problem is sample purification, the method used for the reverse transcription, the choice of internal controls and standards." The deluge of products and kits that researchers have at their hands can make it difficult to compare the results of one experiment to another. As a result, discussions among experts in the field often center on the issue of standardized protocols and controls to use in real-time PCR experiments. Another property of PCR that can complicate quantification is that it is an exponential rather than a linear reaction. This means that a change in amplification efficiency between samples translates into a huge difference in results when the samples are amplified through dozens of doublings. The problem is particularly important when the reaction uses very little template DNA. "When you use small starting samples you can run up against real problems," says Lawrence Wangh of Brandeis University, who developed a new method for real-time quantitative analysis of RNA called linear-after-the-exponential (LATE)-PCR5. "When using real-time PCR to quantitate low levels of starting materials, one of the problems researchers can encounter is more scatter among replicates. This is because the more cycles you need to get a fluorescent signal, the more opportunity to get an error," says Wangh. "Another problem is that you can get mispriming, leading to changes in reaction efficiency. If the amplification reaction does not start in the first cycle, it can result in one CT shift." Wangh's method uses primers at two different concentrations—a limiting concentration and an excess one—so that when the limiting primer is used up, the reaction proceeds in a linear fashion. A key feature of the technique is that the melting temperature of the limiting primer is equal to or greater than that of the excess primer. "This means that we can shift the annealing temperature up. This makes the reaction more stringent and cleaner, without affecting efficiency. This leads to less scatter among replicates and a stronger signal," says Wangh. LATE-PCR is just one example of the developments in the PCR field. As methods to quantify gene expression by real-time PCR become more sophisticated and more products and reagents become available to researchers, real-time PCR is poised to become a prominent tool for scientific research in the genomics information era.
• In the beginning, for those wanting to do PCR, there was Taq. Nowadays thermostable DNA polymerases come in different flavors, each with its own unique capabilities. Researchers also benefit form a variety of kits for amplification, reverse transcription and sample preparation. Some master mixes are optimized for fast reactions, whereas others contain proprietary reagents that prevent mispriming. Many of the commercially available Taq polymerases are special blends, such as Fermentas' high-fidelity PCR enzyme mix. Stratagene markets the Pfu polymerase, which has a lower error rate than Taq thanks to its proofreading capacity. Takara Mirus Bio, on the other hand, sells Bca BEST, a DNA polymerase with strand-displacing and template-switching activities that can be used to perform reverse transcription and DNA amplification in a single tube. So-called hot-start polymerases are variations of the naturally occurring enzyme that become active only at high temperatures (typically 95 °C). This property reduces the chances of mispriming—that is, the polymerase extending primers bound to complementary or partially complementary sequences on nontarget DNAs while the reaction is being set up. "Products created during setup decrease the efficiency of PCR," says Joseph Donnenhoffer of Roche. The company's FastStart Taq polymerase has a one-base deletion that renders the enzyme inactive at room temperature. The polymerase is sold as part of the LightCycler real-time PCR kits. Sigma Aldrich sells a Taq polymerase that is supplied inactivated by an antibody, which is then released by heat. In addition to making the amplification step more effective, companies are also developing methods to make sample isolation more straightforward and less error-prone. Cepheid's GeneXpert kit integrates sample preparation with real-time PCR. "You can load the sample directly into a cartridge, which is then inserted into a GeneXpert module. It is the technology currently used by the US post office for monitoring the presence of biothreat agents in air samples," says Bill McMillan. Ambion sells a Cells to Signal kit that produces a sample ready for PCR in 5 minutes. "It is the first of its kind," says Christy Ogrean, associate product manager. You can skip the RNA isolation and do the reverse transcription in a crude cell lysate so that there is no loss of template. It can be used for cultured cells and primary cells alike."
• Many companies provide internet-based tools to help researchers design PCR primers and probes. But a new service provided by the National Cancer Institute will do the work for you. The Quantitative PCR Primer Database profiles more than 3,000 published primers and probes that can be used to quantify human and mouse mRNA by RT?PCR. Users can search the database by gene, species or type of assay (TaqMan, SYBR Green I or RT-PCR) to find the primer's location, sequence and original reference. Similarly, the RTPrimerDB database provided by Ghent University in Belgium contains lists of real-time PCR primers and probes for SYBR Green I, TaqMan, hybridization and molecular beacon assays.
• Many thermocyclers currently on the market are real-time instruments—in other words, they monitor the accumulation of PCR product in real time and automatically analyze the data. They vary in sample capacity—from high-throughput instruments to handhelds—fluorescent dye flexibility, speed and of course price. Roche's LightCycler—a popular machine among scientists—can run a PCR in 16?30 minutes. Instead of using plastic tubes and plates, it uses glass capillaries. The machine was originally set up for use with Roche's own brand of hybridization probes, but it will accommodate other chemistries, such as molecular beacons and Scorpions. Cepheid's Smartcycler is also a very fast machine. One of its unique features is that it contains up to six processing blocks, each with 16 independently programmable modules that perform four-color real-time PCR. The blocks can be linked together for high-throughput applications or kept separate so that multiple users can simultaneously carry out individual experiments, each with a distinct protocol. "It is not a batch machine. A user can run six or seven samples, and while those are running start another independent reaction," says Bill McMillan. ABI recently released its 9800 Fast Thermal cycle, a high-speed version of its 7900 thermocycler for high-throughput applications. "It has the most features and the highest capability," says Andi Felton. "It can use the fast block, regular 96- and 384-well plates, and the low-density array block." In January of this year, ABI also released a high-speed version of its 7500 platform for medium- to low-throughput applications. The two instruments differ in the types of fluorophores they can support. Other popular machines for real-time PCR include Bio-Rad's iCycler IQ, a 96-well machine that can detect four dyes, and Stratagene's Mx3000P, a lower-cost personal thermocycler.
• long-distance PCR^ref
• long and accurate PCR (LA PCR) : in 1992, investigators working on mixing and fusing various domains of different DNA polymerases showed that, when combined with the robust reliability of Taq DNA polymerase, these enzymes produced longer amplicons. Long and accurate polymerase chain reaction (LA PCR) refers to the production of amplified product longer than 3 kilobases (kb) with high fidelity. Long PCR mixtures typically yield PCR products with some tenfold fewer mutations^ref than those observed in products resulting from conventional PCR. The mixture of DNA polymerases typically included either Taq or Klentaq1 (which have no 3'-exonuclease proofreading activity) as the major component and, as the minor component, an archaebacterial DNA polymerase (with proofreading activity) such as Deep Vent, Vent or Pfu^ref. Among other factors that improve LA PCR are the enzyme deoxyuridine triphosphatase (dUTPase)^ref, which prevents the incorporation of dUTP, the deaminated form of deoxycytosine triphosphate (dCTP) into DNA, and the chemical betaine. Although introduced for amplification of high G-C content targets^ref, betaine, when included at surprisingly high concentrations, usually helps to promote long PCR up to at least 20 kb. Since the introduction of mixtures of DNA polymerases, most PCRs of any length have improved in reliability and in yield of product. The following protocol is based on the use of ten various primer pairs for the amplification of a genomic template DNA of 5 kb in a reaction volume of 50 ml^{ref1, ref2}.
• assembly of the reaction :
• 1| Combine the following components of the master mix on ice and mix thoroughly:
• 10X KLA reaction buffer (pH 9.2) [500 mM Tris base, 160 mM (NH₄)₂SO₄, 25 mM MgCl₂, 1% Tween 20. Note, the pH is 9.2 (the optimal for long PCR) without adjustment; some vectors may require pH 7.2. 10-40 dNTP mix] : 60 ml
• 10-40 dNTP mix [10 mM each dNTP, 40 mM MgCl₂; dissolve the dry form of each dNTP in water to 100 mM stock solutions and store at -80 °C] 6 ml
• 5 M betaine 156 ml
• H₂O to make 12 50-ml reactions 345 ml
• KlentaqLA (use 1 ml if the target is < 2 kb in length) 3 ml
• 2| Remove 48 ml to serve as the no-template control reaction and add 2 ml of primer pair 1.
• 3| Add 5.5 ml of genomic DNA at 10 ng/ml to the master mix (Step 1) and mix thoroughly.
• 4| Distribute 48 ml to each of ten reaction tubes.
• 5| To each of the ten reaction tubes, add 1 ml of each primer (or 2 ml of each primer pair), at 10 pmol/ml to the corresponding reaction. There is no need to mix, as this will occur with convection during the cycling.
• amplification :
• 6| Program the thermal cycler as follows. To minimize melt time, ensure that the cycler remains at temperature for no more than 5–10 s for the heat step of each cycle.

cycle number	denaturation	annealing	polymerization
1	1-2 min at 93°C
2-31 (see step 7)	50 s at 92°C	1 min at 50-58°C	10 min at 63°C

The initial melting temperature of 93 °C should be used only for genomic DNA. For other templates, the use of lower temperatures (80 °C) is advised to avoid depurination. The precise annealing temperature is determined by the melting temperature of the primer.
• 7| Carry out amplification, using the following table to estimate reasonable cycle numbers, depending on the type of template used.

template DNA	PCR cycle number
plasmid	18-22
bacterial	25-30
mammalian	35-40

• inverse polymerase chain reaction (IPCR) : a technique to amplify genomic DNA flanking the insertion site of a transposon or T-DNA construct. The obtained flanking genomic DNA can be used as RFLP-probe to determine the map position of the insertion site of the contruct^ref.
• long-distance inverse polymerase chain reaction (LDI-PCR)^ref
• liposome-PCR assay for the ultrasensitive detection of biological toxins : uses liposomes with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as a detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time PCR. Assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods^ref
• hybridization chain reaction (HCR) : binding of DNA to a substrate can accomplish the roles of recognition and signal amplification without any external inputs. HCR came about not from a desire to compete with PCR but as a byproduct of engineering DNA hairpins that could be used as fuel packets to power DNA mechanical machines. By eliminating the machines from the design scenario, they found that the hairpins could be triggered to undergo a chain reaction. The key to HCR in its simplest form is the storage of potential energy in two hairpin species. When a single-stranded DNA initiator is added to this previously stable mixture, it opens a hairpin of one species, exposing a new single-stranded region that opens a hairpin of the other species. This process, in turn, exposes a single-stranded region identical to the original initiator. The resulting chain reaction leads to the formation of a nicked double helix that grows until the hairpin supply is exhausted. Detection of the resulting products does not require any specialized detection equipment. All you need is a gel apparatus, which can be found in any wet lab. We are also trying to make a nanogold-based colorimetric assay that will enable detection by eye alone. For more diverse biosensing applications, DNA and RNA aptamers selected to bind specific molecules hold promise for the development of HCR triggers that will initiate the chain reaction only in the presence of the target molecule. The authors have used this aptamer trigger concept to specifically discriminate ATP from GTP. If we succeed in developing a general aptamer triggering mechanism, then HCR amplification could be incorporated in sensors for a wide range of small molecules. Unlike PCR, which provides exponential amplification, the current form of HCR provides linear amplification. We are now developing nonlinear versions of HCR that provide quadratic, cubic or exponential growth after being triggered by the initiator. However, false positives become a much bigger problem, as spurious initiation events are also amplified nonlinearly. Successful development of an exponential HCR amplification system would increase the sensitivity to target molecules at very low concentrations. This would open up the possibility of attaining a PCR-like level of sensitivity for a variety of small molecules without the need for any expensive equipment or reagents^ref


• ligase chain reaction (LCR) : technique to detect a single-base substitution in a known sequence. The ligase enzyme links 2 adjacent oligo's which hybridize agains the target sequence. In case of a mismatch the ligation of the adjacent oligo's will fail. Ligated oligo's resemble the target sequence and are available as target sequence for unligated oligo's in subsequent cycles resulting in an exponential amplification of ligated oligos^ref.
• ligation-mediated PCR (LM-PCR) analysis of dsDNA breaks (DSBs)^ref
• nucleic acid sequence based amplification (NASBA) : this method has the unique ability to amplify a specific RNA sequence in the presence of homologous DNA sequences, under iso-thermal (=no PCR) conditions. NASBA employs reverse transcriptase and primers to generate a double stranded cDNA template with a T7 promoter. T7 RNA polymerase ten initiates transcription and generates multiple copies of RNA, which re-enters the cycle to generate cDNA. Sooknanan R., and L.T. Malek (1995) NASBA a detection and amplification system uniquely suited for RNA. Bio/Technology 13:563-564.
• rolling circle amplification (RCA) : a DNA amplification method (alternatives to PCR or NASBA). A probe is hybridised to the target sequence to form an open circle, because the ends are inversely complementary to the target sequence. With ligase the circular probe can be closed and will surround the target sequence as a padlock. A single primer homologous to the non-complementary part of the circle is extended by phage f 29 DNA polymerase. Copies of the target sequence roll from the circle in a long sequence of tandem repeats^ref
• arbitrary primed marker :
• arbitrary primed polymerase chain reaction (AP-PCR) : DNA amplification using a single random primer of 16-20 bases. Products are often analysed on a polyacrylamide gel and detected by autoradiography^ref
• amplification fragment length polymorphism (AFLP) / DNA amplification fingerprinting (DAF) : refers to DNA amplification using a single random primer of 8-10 bases. DAF products are analysed on polyacrylamide gels and detected by silver staining^ref
• DNA finger printing (DFP) : general name for multi-locus techniques used for identification of individuals or mapping.
• inter-simple sequence repeat amplification (ISSR) / SSR-anchored PCR : PCR-based multi-locus marker system using oligonucleotide primers homologous to SSR sequences (such as (GATA)n ). To avoid stutterings these primers can be anchored to unique genomic sequences flanking the repeat. Amplification products are only obtained in case SSRs in opposite orientation are found within a PCR-able distance, with a flanking sequences matching the oligo's. 3'-anchoring give better results than 5'-anchoring. Repeat polymorphisms within the SSR do not influence the chance for ISSR polymorphisms. Kantety R.V., X. Zeng, J.L. Bennetzen and B. Zehr (1995) Assessment of genetic diversity in dent and popcorn (Zea mays L.) inbred lines using Inter-Simple Sequence Repeat (ISSR) amplification. Molecular Breeding 1:365-373.
• multiple arbitrary amplicon profiling (MAAP) : basically similar to DAF or AFLP, but using multiple endonuclease digestions of template DNA or amplification products, significant increase of polymorphisms in fingerprints are generated^ref.
• oligonucleotide polymorphism (OP) : PCR based markers using two 18-mer oligonucleotide primers used for tagging specific loci or alleles (see ASO). Primer design is based on known cDNA sequences. Beckmann J.S. (1988) REVIEW Oligonucleotide polymorphisms: A new tool for genomic genetics. Bio/Technology 6:161-164.
• random amplified multiple polymorphisms (RAMP)^ref
• random amplified polymorphic DNA (RAPD) : a PCR product that is obtained from genomic DNA using a single or a combination of typically 10-mer oligonucleotides. Alleles are visualized by the fragments that are amplified, separated on agarose gels and stained with EtBr. RAPDs show dominant inheritance. Variation is based on the position and orientation of primer-annealing sites and the interval they span^ref.

Web resources : Usenet bionet.molbio.rapd
• single primer amplification reactions (SPAR) : PCR based marker. SSRs like (GATA)4 are used to prime PCR on genomic DNA. The stretches of unique DNA in between the SSR are reproducibly amplified. Tetra-nucleotide repeats perform better than di- or tri-nucleotide repeats. Gupta M., Y.-S. Chyi, J. Romero-Severson and J.L. Owen (1994) Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theor. Appl. Genet 89:998-1006.
• non-arbitrary primed marker :
• amplified fragment length polymorphism (AFLP) : PCR based marker. Not based on arbitrairy priming of oligo's, but amplification of specificly selected restriction fragments (see SRFA). Zabeau M. and P. Vos (1992) European Patent Application. Publication no. 0 543 858 A1.
• allele specific associated primers (ASAP) : similar to ASO or AS-PCR. The sequence of the deca-mer oligo is derived from normal RAPD, which generated an absence/presence polymorphism. These absence/presence polymorphisms do not require electrophoretic separation of the sample. Presence of an amplification product is detected by measuring fluorescence of Ethidium-Bromide stained DNA. Gu W.K., N.F. Weeden, J. Yu and Wallace DH (1995) Large-scale, cost-effective screening of PCR products in marker-assited selection applications. Theor. Appl. Genet. 91:465-470.
• allele specific polymerase chain reaction (AS-PCR) : refers to amplification of specific alleles, or DNA sequence variants, at the same locus. Specificity is archieved by designing one or both PCR primers so that they partially overlap the site of sequence difference between the amplified alleles (ASO)^ref.
• allele specific oligo (ASO) : a special kind of oligo for a PCR reaction. The sequence of the oligo is designed in such a way to allow/inhibit hybridisation at the spot where the mutant (resistant) allele differs from the wildtype (suseptible) allele. (Variant of AS-PCR). Beckmann J.S. (1988) REVIEW Oligonucleotide polymorphisms: A new tool for genomic genetics. Bio/Technology 6:161-164.
• cleaved amplified polymorphic sequences (CAPS) : PCR amplified DNA (STS-, EST- or SCAR-product) which is not yet polymorphic is digested with restriction endonucleases to reveal polymorphisms in restriction sites^{ref1, ref2}
• directed amplification of minisatellite-region DNA (DAMD)
• degenerate oligonucleotide primed-PCR (DOP-PCR) : the technique uses 22-mer primers of partially degenerate sequence in a dual annealing temperature protocol and can therefore amplify DNA in a general and non-specific manner^ref.
• expressed sequence tag (EST) : PCR based marker. Highly specific oligos (16-20-mers) are designed by using sequence information of a cDNA. The locus represents a functional gene and is located in an actively transcribed region of the genome.
• inverse sequence-tagged repeats (ISTR) : PCR-based multi-locus marker system using oligonucleotide primers homologous to dispersed high copy sequences, e.g. copia like transposons. Multi-locus polymorphic amplification products are obtained with two primers, designed in a direction outward the element, and allows the amplification of the stretch connecting the elements. Rohde W. (1996) Inverse sequence-tagged repeat (ISTR) analysis, a novel and universal PCR-based technique for genome analysis in the plant and animal kingdom. Journal of Genetics & Breeding 50(3): 249-261.
• repeat sequence primed PCR (REP-PCR)
• sequence characterized amplified regions (SCAR) : PCR based marker. A genomic DNA fragment at a single genetically defined locus that is identified by PCR amplification using a pair of specific (16-24-mer) oligonucleotice primers. Paran I. and R.W. Michelmore (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet 85:985-993
• sequence tagged microsatelite site (STMS) : a PCR-based marker. The DNA sequence of unique DNA flanking microsatellites (GATA)n, (CT)n etc. is used to construct 20-mer oligos. Amplification products spanning the microsatellite show rich allele divergency^ref
• short tandem repeats (STR) : synonymous to SSR detected as (TG)n.(CA)n^{ref1, ref2}
• selective restriction fragment amplification (SRFA) : a procedure taking oligo's that are elongated with selective bases. Because of these bases not all restiction fragments can be amplified in a PCR reaction. Zabeau M. (1992) Gast-College hybride rassen, Vakgroep Plantenveredeling LUW.
• simple sequence length polymorphism (SSLP) : identical to SSR, The jargon is used in (for example) mouse genetics^{ref1, ref2, ref3}
• simple sequence repeats (SSR) : synonymous to STR or micro satellite repeats, in particular the dinucleotide repeats (AC)n (AG)n (AT)n^{ref1, ref2}
• selective amplification of microsatellite polymorphic loci (SAMPL) : based on AFLP. The template is identical to AFLP template, but the rare cutter primer is replaced by a (CA)72(TA)22 or (GT)₇₂(AT)₂. Dean L, Witsenboer H, Kesseli R, Paran I. and R.W. Michelmore (1996) Identification of makers clusters of disease resistance genes in lettuce. Plant Genome IV Poster abstract. Morgante M and Vogel J: Compound microsatellite primers for the detection of genetic polymorphisms. U.S. Patent Appl 08/326456 (1994). Paglia G., M. Morgante (1998) PCR-based multiplex DNA fingerprinting techniques for the analysis of conifer genomes. Molecular Breeding 4:173-177.
• S-SAP
• sequence tagged site (STS) : a PCR-based marker derived from a RFLP-probe, by constructing primers according to the end-sequence of the RFLP-probe^ref. Olsen M., L. Hood, C. Cantor and D. Botstein (1989) A common language for physical mapping of the human genome. Science 245:1434-1435.
• variable number tandem repeat (VNTR) : a genetic locus studied from a Southern blot, probed with a labelled minisatellite repeat. It's used in fingerprinting and forensic studies. It is not similar to a STMS. The core units vary in lenght from 11 to 60 bp^{ref1, ref2}
• synthesis of complementary DNA : the basic strategy for enzymatic conversion of RNA into DNA has changed little since the 1970s; however, there have been great improvements to the efficiency of the overall process. In this method^{ref1, ref2, ref3}, the product of a first-strand synthesis (the cDNA-mRNA hybrid) is used as template for a nick translation reaction. Ribonuclease (RNase) H produces nicks and gaps, creating a series of RNA primers used by Escherichia coli DNA polymerase I during the synthesis of the second-strand DNA. Residual nicks are then repaired by E. coli DNA ligase, and the frayed termini of the double-stranded cDNA are polished by a DNA polymerase. Finally, bacteriophage T4 polynucleotide kinase catalyzes the phosphorylation of the ends of the cDNAs to facilitate cloning.

Procedure :
• synthesis of first-strand cDNA :
• 1| To synthesize first-strand cDNA, mix the following in a sterile microcentrifuge tube on ice:
• poly(A)⁺ RNA (1 mg/ml) : 10 ml
• oligonucleotide primer(s) (1 mg/ml) : 1 ml
• 1 M Tris HCl (pH 8.0 at 37°C) : 2.5 ml
• 1 M KCl : 3.5 ml
• 250 mM MgCl₂ : 2 ml
• dNTP solution (5 mM each) : 10 ml
• 0.1 M DTT : 2 ml
• RNase inhibitor (optional) : 25 units
• water : to 48 ml
Add the manufacturer's recommended amount of Moloney strain murine leukemia virus reverse transcriptase (M-MLV RT) to the reaction and mix well by gentle vortexing.
• 2| Transfer 2.5 l of the reaction to a fresh 0.5-ml microcentrifuge tube, add 0.1 ml [a-³²P]dCTP (400 Ci/mmol, 10 mCi/ml) to the small-scale reaction, and incubate both the large- and small-scale reactions at 37 °C for 1 h.
• 3| Add 1 l of 0.25 M EDTA to the small-scale reaction containing the radioisotope and transfer it to ice. Heat the large-scale reaction to 70 °C for 10 min and transfer it to ice.
• 4| Measure both the total amount of radioactivity and the amount of trichloroacetic acid (TCA)-precipitable radioactivity in 0.5 l of the small-scale reaction. Also, analyze the products of the small-scale reaction by alkaline agarose gel electrophoresis and store the remainder at -20 °C.
• 5| Calculate the amount of first-strand cDNA synthesized as follows: [cpm incorporated x (66 mg - x mg)] / (total cpm) = ml of first strand of cDNA synthesized total cpm
• second-strand synthesis :
• 6| Add the following reagents directly to the large-scale first-strand reaction mixture (Step 3):
• 10 mM MgCl₂ : 70 ml
• 2 M Tris-HCl (pH 7.4) : 5 ml
• 10 mCi/ml [a-³²P]dCTP (400 Ci/mmol) : 10 ml
• 1 M (NH₄)₂SOm : 1.5 ml
• RNase H (1,000 units/ml) : 1 ml
• E.coli DNA polymerase I (10,000 units/ml) : 4.5 ml
Mix the reagents by gently vortexing and centrifuge the reaction mixture briefly in a microcentrifuge to eliminate any bubbles. Incubate the reaction at 16 °C for 2-4 h.
• 7| Add to the reaction 1 ml of 50 mM b-nicotinamide adenine dinucleotide (b-NAD) and 1 l E. coli DNA ligase (1,000-4,000 units/ml). Incubate the reaction at 15-25 °C for 15 min.
• 8| Add 1 ml of a dNTP solution (each at 10 mM) and 2 ml (5 units) of bacteriophage T4 DNA polymerase. Incubate the reaction mixture at 15-25 °C for 15 min. Set aside a small aliquot (3 m) of the reaction for use in Step 11.
• 9| To the remainder of the reaction, add 5 ml of 0.5 M EDTA (pH 8.0). Extract the mixture once with phenol/chloroform and once with chloroform. Precipitate the DNA with ethanol in the presence of 0.3 M sodium acetate (pH 5.2) and dissolve in 90 ml of Tris/EDTA (TE) buffer (pH 7.6).
• 10| To the DNA, add 10 ml 10X T4 polynucleotide kinase buffer and 1 ml T4 polynucleotide kinase (3,000 units/ml), and incubate the reaction at 15?25 °C for 15 min.
• 11| Determine the total amount of radioactivity and the TCA-precipitable counts in 1 l of the small aliquot set aside in Step 8. Calculate the amount of cDNA synthesized in the second-strand reaction, taking into account the amount of the dNTPs already incorporated into the first strand of cDNA: [cpm incorporated in the second-strand reaction x (66 mg - x mg) / total cpm] = mg of second-strand cDNA sequence, where x is the weight of the first strand of cDNA determined in Step 5. The amount of second-strand cDNA synthesized is usually 70-80% of the weight of the first strand.
• 12| Extract the phosphorylated cDNA (from Step 10) with an equal volume of phenol:chloroform (1:1).Separate the unincorporated dNTPs from the cDNA by spin-column chromatography through Sephadex G-50 equilibrated in TE buffer (pH 7.6) containing 10 mM NaCl.
• 13| Precipitate the eluted cDNA with 2 volumes of ethanol in the presence of 0.3 M sodium acetate (pH 5.2). Recover the precipitated DNA by centrifugation at maximum speed at 4 °C for 15 min. Use a handheld minimonitor (Geiger counter) to check that all the radioactivity has been precipitated.
• 14| Wash the pellet with 70% ethanol and resuspend in the desired volume of TE (pH 7.6).
• rapid amplification of cDNA ends (RACE)
• 5'-full RACE (FAQs) : used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target—in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation. Procedure^{ref1, ref2, ref3, ref4, ref5, ref6, ref7} :
• reverse transcription

1| Add 0.001-100 ng of poly(A)⁺ mRNA or 0.01-1 mg of total RNA into each of 3 microcentrifuge tubes. Adjust the volumes to 9 ml with water. Denature the RNA by heating at 75 °C for 5 min, then rapidly chilling on ice. Total RNA extracted from cells with chaotropic agents is generally the template of choice for RT-PCR of mRNAs that are expressed at moderate-to-high abundance. Poly(A)⁺ mRNA is preferred as a template for all forms of RT-PCR when the target mRNA is expressed at low abundance.
2| Set up the following reverse transcription reaction and three controls:

	reaction	control 1	control 2	control 3
denatured RNA (from step 1)	9 ml	-	9 ml	9 ml
5x reverse transcriptase buffer	4 ml	4 ml	4 ml	4 ml
deoxynucleoside triphosphates (dNTPs; 20 mM (pH 8.0))	1 ml	1 ml	1 ml	1 ml
gene-specific antisense primer 1 (10 mM)	4 ml	4 ml	4 ml	-
placental RNAse inhibitor (20 units/ml)	1 ml	1 ml	1 ml	1 ml
reverse transcriptase (100-200 units/ml)	1 ml	1 ml	-	1 ml
water	-	9 ml	1 ml	4 ml

Incubate the reactions at 37 °C for 60 min. The success or failure of 5' RACE is determined here at the beginning of the experiment. If the reverse transcription step works efficiently, the chance of isolating clones that contain the 5'-terminal sequences of the target mRNA is high. On the other hand, no amount of optimization during the later steps of the protocol can compensate for ineffective reverse transcription. Carry these controls through all of the following steps of the protocol.
3| To remove excess primer, dilute each of the reactions to a final volume of 2 ml with water and apply to a Centricon-100 microconcentrator. Centrifuge at 500-1,100g at 4-25 °C for 20 min. Repeat the dilution step and centrifuge again. Transfer the retentates containing cDNA to fresh 0.5-ml microcentrifuge tubes, and reduce the volumes to 10 ml in a rotary vacuum evaporator.
4| To the cDNA in a volume of 10 ml add:
• 5x terminal transferase buffer : 4 ml
• deoxyadenosine triphosphate (dATP; 1 mM) : 4 ml
• terminal transferase (10-25 units) : 2 ml
Incubate the reactions at 37 °C for 15 min, and then inactivate the terminal transferase by heating the reactions at 80 °C for 3 min. Dilute the dA-tailed cDNA to a final volume of 1 ml per reaction with Tris-EDTA buffer (pH 7.6). The tailing reaction can be optimized by setting up mock reactions containing 50 ng of a control DNA fragment, 100-200 nucleotides (nt) in length. After tailing, the size of the fragment should increase by 20-100 nt as measured by electrophoresis through a 1% neutral agarose gel.
• amplification of reverse transcription products

5| In sterile 0.5-ml microcentrifuge tubes, amplification tubes or wells of a sterile microtiter plate, set up a series of PCRs containing the following:
• diluted DNA (from step 4) : 0-20 ml
• 10x amplification buffer : 5 ml
• dNTPs (20 mM) : 5 ml
• (dT)₁₇-adaptor primer (10 mM; 16 pmol) : 1.6 ml
• adaptor-primer (10 mM, 32 pmol) : 3.2 ml
• gene-specific primer-2 (10 ml; 32 pmol) : 3.2 ml
• thermostable DNA polymerase (1-2 units) : 1 ml
• water : to 50 ml
The (dT)₁₇-adaptor primer 5'-GACTCGAGTCGACATCGA(T)₁₇-3' binds to the poly(A)⁺ tract added to the 5' terminus of cDNAs by terminal transferase. The adaptor-primer is 5'-GACTCGAGTCGACATCG-3'.
6| Place the tubes or the microtiter plate in the thermal cycler. Amplify the nucleic acids using the following denaturation, annealing and polymerization times and temperatures:

cycle number	denaturation	annealing	polymerization
1	5 min at 94°C	5 min at 50-58°C	40 min at 72°C
2-31	40 s at 94°C	1 min at 50-58°C	3 min at 72°C
32	40 s at 94°C	1 min at 50-58°C	15 min at 72°C

7| Withdraw a sample (5-10 ml) from each amplification reaction and analyze them by gel electrophoresis. Stain the gel with ethidium bromide or SYBR Gold to visualize the DNA.
A successful amplification reaction should yield a readily visible DNA fragment of the expected size. Note what amount of tailed cDNA generated the largest quantity of amplified 5' termini. The identity of the band can be confirmed by DNA sequencing, Southern hybridization and/or restriction mapping.
8| Extract the nucleic acids with 150 ml of chloroform. Separate the amplified DNA from the residual thermostable DNA polymerase and dNTPs. The DNA can now be ligated to a blunt-ended vector or a T vector, or it can be digested with restriction enzymes and ligated to a vector with compatible termini.
• 5' and 3'-full RACE
• 3'-full RACE (FAQs)
• loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity, and rapidity under isothermal conditions at 65°C. This method employs a DNA polymerase with strand displacement activity and a set of 4 specially designed primers that recognize a total of 6 distinct sequences on the target DNA.
• isothermal DNA amplification kits of lyophilized reagents for field use (e.g. point-of-care or point-of-need molecular analyses of forensic samples or for testing seawater) in genetic diseases and infections like HIV and methicillin-resistant Staphylococcus aureus could appear soon : in a kit developed by New England Biolabs amplification is done at one temperature without doing thermal cycling, and a DNA helicase is used to open the helix, rather than using heat^ref. All you need to do is put a sample on it, and add water and amplify. The method would be useful for techniques such as in situ PCR on tissue sections that are otherwise destroyed. From a technical point of view, genetic testing at home would be possible, in the same way that pregnancy tests are currently available. For a single nucleotide change causing sickle-cell anemia, for example, it would be possible to design probes to amplify the DNA, and then couple that with DNA strips : you spot the finished reactants on a membrane and you will see a color change. This is not the first isothermal DNA amplification system : there are products from Cambio and Amersham. The paper showed a 100-bp fragment when it claimed its strength lay in amplifying longer products than the competition, but initially it looks good. In the clinical application, very long products are not particularly useful : a lot of the time you are looking for specific mutations, and all you want is good-quality stuff for sequencing. Some foresee problems with primer specificity at low temperatures, however.
• cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes has been isolated. The action of 2 catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, has been used to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The 2-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k_obs) of the optimized deoxyribozyme ligase was found to be 1 x 10^-4 min^-1. Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10⁵-fold faster than that generated by a simple DNA template. So far the deoxyribozymes work 100,000 times slower than the natural DNA polymerase. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated^ref


• micrococcal nuclease-Southern blot assay : micrococcal nuclease (MNase) is unique among nucleases in its ability to induce double-strand breaks within nucleosome linker regions, but only single-strand nicks within the nucleosome itself. Because of this property, MNase can be used to determine whether a DNA fragment of interest is within a nucleosome^{ref1, ref2}. In addition, MNase can be used to determine the approximate positions of nucleosomes in a region of DNA, if the nucleosomes are consistently positioned. In brief, cell nuclei are isolated and limiting concentrations of MNase are added to the nuclei, resulting in cleavage at nucleosome linker regions. The genomic DNA is purified and the fragments are separated by agarose gel electrophoresis; the resulting ladder of stained bands corresponds in size to multiples of the nucleosome core plus the linker (200 base pairs (bp)). To determine whether a DNA fragment of interest is within a nucleosome, the genomic DNA is subjected to Southern blot analysis. If a probe derived from the DNA fragment hybridizes to the ladder of nucleosomal bands, the fragment may indeed be assembled into nucleosomes. To determine nucleosome positioning, the purified genomic DNA must be cleaved with a restriction enzyme before gel electrophoresis and Southern blot analysis. Procedure^ref :
• preparing the nuclei
• 1| Pellet 10⁸ cells at 300g at 4 °C for 10 min in a centrifuge. The nuclei should be kept cold throughout the procedure. Manipulations and transport to the centrifuge should be done on ice.
• 2| Discard the supernatant. Wash the cell pellet with 10 ml of ice-cold 1X phosphate-buffered saline. Pellet cells at 300g at 4 °C for 10 min in a centrifuge.
• 3| Resuspend the cell pellet in 5 ml of ice-cold NP-40 lysis buffer. Incubate on ice for 5 min. Pellet the nuclei at 120g at 4 °C for 10 min and remove and discard the supernatant. The nuclei will form a loose pellet. Care should be taken not to disturb the pellet when removing supernatant. Do not vacuum aspirate from this point forward. NP-40 lysis buffer consists of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂, 0.5% Nonidet P-40, 0.15 mM spermine and 0.5 mM spermidine.
• digesting with MNase and purifying genomic DNA
• 4| Wash the nuclei with 2.5 ml of MNase digestion buffer. Pellet the nuclei at 120g at 4 °C for 10 min. Carefully discard the supernatant. MNase digestion buffer consists of 10 mM Tris-HCl (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.15 mM spermine and 0.5 mM spermidine.
• 5| Resuspend the nuclei in 1 ml of MNase digestion buffer containing 1 mM CaCl₂.
• 6| Transfer 100 ml of nuclei to a series of microcentrifuge tubes containing diluted MNase (0.25-75 units). Incubate samples at 15?25 °C for 5 min.
• 7| Add 80 ml of MNase digestion buffer and 20 ml of MNase stop buffer to each sample. Add 3 ml of proteinase K (25 mg/ml) and 10 ml of 20% SDS. Incubate at 37 °C overnight. MNase stop buffer consists of 100 mM EDTA and 10 mM EGTA (pH 7.5).
• 8| Extract samples with 200 ml of phenol/chloroform and gently mix by flicking or rocking the tubes. Centrifuge the samples at maximum speed for 5 min in a microcentrifuge. Carefully transfer each aqueous layer to a new microcentrifuge tube. The genomic DNA may appear as insoluble 'strings'. Slowly remove the aqueous layer with a pipet, including the strings, while minimizing disturbance of the phenol/chloroform interface.
• 9| Extract samples with 200 ml of chloroform and centrifuge samples at maximum speed for 5 min in a microcentrifuge. Carefully transfer each aqueous layer to a new microcentrifuge tube.
• 10| Add 2 ml of heat-treated RNase A (10 mg/ml) to each sample. Incubate at 37 °C for 2 h.
• 11| Repeat Steps 8 and 9.
• 12| Recover the digested, genomic DNA by ethanol precipitation.
• 13| Gently resuspend the pellet in 100 l of water or Tris-EDTA buffer. If necessary, allow the DNA to dissolve at 4 °C overnight.
• digesting with restriction enzymes
• 14| Determine the DNA concentration using a spectrophotometer. Use 5-20 g of DNA for in vitro restriction enzyme digestion.
• 15| Digest DNA in 35 ml total volume at 37 °C overnight. Use an excess of restriction enzyme in its optimal buffer to ensure complete digestion. Several restriction enzyme sites should be tested surrounding the region of interest. Adjacent restriction enzyme sites within the distance of 1 nucleosome (147 bp) are recommended.
• 16| Separate fragments by agarose gel electrophoresis. Prepare a Southern blot to transfer the DNA fragments onto a membrane.
• 17| Prepare a radiolabeled probe of 200 bp, using either nick translation or random priming, complementary to one end of the restriction enzyme site. To ensure that the probe will detect only MNase-digested fragments associated with an in vitro restriction enzyme cleavage, the probe should be limited to the approximate size of one nucleosome and linker (200 bp).
• Southern blotting
• 18| Carry out prehybridization at 65 °C for 2 h (or 42 °C for 2 h if using formamide prehybridization solution).
• 19| Proceed with hybridization at 65 °C overnight (or 42 °C if using formamide buffer).
• 20| Wash the membrane, remove excess liquid by blotting the membrane with Whatman paper, wrap the membrane in plastic wrap, and expose to film or phosphorimager screen.
• mutagenesis :
• biochemical method to make changes in a gene in the test tube via site-directed mutagenesis by the singe-tube megaprimer PCR method and then insert the gene into an organism to determine the properties of its protein. You then have to grow it up and pull the gene out in order to do any further mutation cycles. So, you're constantly putting the gene in to find out its properties and pulling it out to make more changes. It's extremely tedious and time-consuming, and the size of the library of mutant genes is limited by the efficiency with which we can introduce at most one mutant copy of the gene per cell. If 2 different mutant genes end up in the same cell, the effect of the good mutant would be diluted and masked by the not-so-good version.
• treat cells with x-rays or chemicals that generate mutations. The problem there is that if you induce too much mutation, you kill the organisms because you're frying all the genes they need.
• immuno–spin trapping (IST) : the detection of DNA radicals is based on the trapping of radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), forming stable nitrone adducts that are then detected using an anti-DMPO serum. DNA radicals are very reactive species, and because they are paramagnetic they have previously been detected only by electron spin resonance (ESR) with or without spin trapping, which is not available in most bioresearch laboratories. IST combines the simplicity, reliability, specificity and sensitivity of spin trapping with heterogeneous immunoassays for the detection of DNA radicals, and complements existing methods for the measurement of oxidatively generated DNA damage^ref
• SHM produces mutant genes at roughly 1 million times the rate of mutation elsewhere in the genome. Other researchers had demonstrated that SHM could be induced in B cells to repair a single mutation in a non-antibody gene. SHM offers a strategy to evolve nonantibody proteins with desirable properties for which a high-throughput selection or viable single-cell screen can be devised^ref
• targeting induced local lesions in genomes (TILLING) : a reverse genetic, nontransgenic method, to improve a quality trait in a polyploid crop plant. Waxy starches, composed mostly of amylopectin, have unique physiochemical properties. Wheat with only 1 or 2 functional waxy genes (granule-bound starch synthase I, or GBSSI) produces starch with intermediate levels of amylopectin. 246 alleles of the waxy genes were identified by TILLING each homolog in 1,920 allohexaploid and allotetraploid wheat individuals. These alleles encode waxy enzymes ranging in activity from near wild type to null, and they represent more genetic diversity than had been described in the preceding 25 years. A line of bread wheat containing homozygous mutations in two waxy homoeologs created through TILLING and a preexisting deletion of the third waxy homoeolog displays a near-null waxy phenotype. This approach to creating and identifying genetic variation shows potential as a tool for crop improvement^ref.
• Labeling
• nanoparticles : particles with controlled dimensions on the order of nanometres. Examples include :
• colloidal gold
• magnetite particles
• quantum dots (QDs)
• superparamagnetic magnetite nanoparticles : nanometre-sized particles of magnetite (Fe₃O₄), which locally amplify an external magnetic field, but are too small to maintain their own field in the absence of an external field
• colorimetric detection
• biotin-avidin or -streptavidin : in immunochemistry avidin or streptavidin binds diaminobenzidine (DAB) which undergo oxidation and precipitation.
• biotin-XX
• DSB-X biotin
The endogenous biotin induces false-positive signals with the avidin-biotin detection system commonly used for IHC and ISH analyses^ref.
Streptavidin and avidin bind the small molecule biotin with femtomolar affinity^ref. This tight and specific binding has caused the (strept)avidin-biotin system to be used widely for biomolecule labeling, purification, immobilization and patterning. But because streptavidin and avidin are both tetramers, they can tetramerize the biotin conjugates to which they bind. This multimerization can interfere with normal biomolecule function or trafficking, or complicate affinity measurements, limiting the applications of this system. Much effort has been made to develop monomeric (strept)avidin. Disruption of the tetramer interface is straightforward, but is always accompanied by a dramatic decrease in biotin affinity (at least 10⁴-fold in the best case for streptavidin^ref and 10⁸-fold for avidin^{ref1, ref2}) because the biotin binding site lies at the interface between subunits^{ref1, ref2}. An alternative approach to generate monovalent (strept)avidin is to titrate in three equivalents of biotin per tetramer, but this is unsatisfactory because it generates a statistical mixture of mono-, di-, tri- and tetravalent (strept)avidins. Site-specific protein biotinylation and streptavidin labeling has been used to image cellular proteins^ref. This method can be used to specifically tag proteins at the cell surface, requires genetic fusion of the protein of interest to only a 15-amino-acid peptide, provides a stable linkage that does not dissociate over hours and can be used for introduction of a wide range of probes, from organic fluorophores to quantum dots. The streptavidin could cross-link biotinylated cell-surface proteins (such as the AMPA receptor^ref), thus slowing their trafficking and causing unwanted receptor activation^ref. Here we report the development of a monovalent streptavidin with a single femtomolar biotin binding site, and its application to cross-linking–free labeling of biotinylated neuroligin-1 on the surface of living neurons : it retains the affinity, off rate and thermostability of wild-type streptavidin^ref
• enzymes
• horseradish peroxidase (HPO)
• substrates :
• Amplex Red reagent => resorufin
• alkaline phosphatase (AlkP)
• colorimetric detection method for identifying nucleic acid sequences based on the distance-dependent optical properties of gold nanoparticles : nucleic acid diagnostics is dominated by fluorescence-based assays that use complex and expensive enzyme-based target or signal-amplification procedures. Many clinical diagnostic applications will require simpler, inexpensive assays that can be done in a screening mode. In a 'spot-and-read' assay, nucleic acid targets are recognized by DNA-modified gold probes, which undergo a color change that is visually detectable when the solutions are spotted onto an illuminated glass waveguide. This scatter-based method enables detection of zeptomole quantities of nucleic acid targets without target or signal amplification when coupled to an improved hybridization method that facilitates probe-target binding in a homogeneous format. In comparison to a previously reported absorbance-based method, this method increases detection sensitivity by over 4 orders of magnitude^ref.
• bioluminescent molecules

Web resources : the chemistry of bioluminescent reporter assays
• chromophores : groups with characteristic optical absorptions. They usually contain alternating single and double bonds.

fluorophores			labsorption peak(s) (nm)	lemission peak (nm)
Alexa Fluor 350			346	442
Alexa Fluor 430			434	539
Alexa Fluor 488			495	519
Alexa Fluor 532			531	554
Alexa Fluor 546			556	573
Alexa Fluor 555			555	565
Alexa Fluor 568			578	603
Alexa Fluor 594			590	617
Alexa Fluor 610-R-PE			496, 546, 565	630
Alexa Fluor 647			650	668
Alexa Fluor 647-R-PE			496, 546, 565	668
Alexa Fluor 660			663	690
Alexa Fluor 680			679	702
Alexa Fluor 680-R-PE			496, 546, 565	702
Alexa Fluor 700			696	719
Alexa Fluor 700-R-PE			650	723
Alexa Fluor 750			752	779
Alexa Fluor 750-R-PE			650	775
BODIPY FL
BODIPY 564/570
BODIPY 558/568
BODIPY 594
BODIPY TMR-X
Cascade Blue			400	420
CY3
CY5			649 (633 commonly used)	670
CY7			743	767
fluorescein			494	518
fluorescein isothiocyanate (FITC)			495 (488 commonly used)	525
Marina Blue			365	460
Oregon Green 488			496	524
Oregon Green 514
Pacific Blue			410	455
Peridinin chlorophyll protein (PerCP)			488	677
phycobiliproteins / biliproteins are homologous chromoproteins that have covalently bound phycobilin chromophores and constitute the phycobilisomes, the light harvesting complexes of the photosynthetic apparatus in Cyanobacteria (C),  Rhodophyta (R) and Cryptophyta. The phycobilisomes constitute up to 50% of the total protein in Cyanobacteria and their content in PC or PE can be up- or down-regulated by using different light conditions (chromatic adaptation).	phycoerythrin (PE) carries covalently bound phycoerythrobilin (PEB) and phycourobilin (PUB) as chromophoric groups and is composed of 2 subunits, a and b, with Mr of 14000 and 17000, respectively	C-phycoerythrin (C-PE)	565 (488 commonly used)	576
		R-phycoerythrin (R-PE)	496, 546, 565	578
	phycocyanin (PC) carries covalently bound phycocyanobilin (PCB) as chromophoric group and is composed of 2 subunits having a similar Mr of 21000	C-phycocyanin (C-PC)
		R-phycocyanin (R-PC)
	allophycocyanin (APC)		650 (633 commonly used)	660
	phycoerythrocyanin carries covalently bound phycoviolobilin as chromophoric group		565
Proteins : when a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1	green fluorescent protein (GFP) from Aequorea victoria (jellyfish) : 238 amino acids, with a chromophore consisting of the phenolic  side chain of Tyr66 and the imidazolidinone ring formed by cyclization of Ser65-Tyr66-Gly67. The chromophore is located at the center of a cylinder-shaped 3D structure with a Ø = 24 Å and height = 42 Å.  Web resources : GFP applications page	photoactivatable GFP (PA-GFP) is monomeric and gives 100-fold fluorescence contrast, can be applied for protein tracking.	498 (488 commonly used) (blue light)	516 (green light)
	GFP mutants with absorption and emission bands ranging from the violet to the red spectrum of the spectrum	W399X
		enhanced GFP (eGFP / EGFP) : improved brightness after blue-light excitation with respect to wild-type GFP. After irradiaton with l ~ 400 nm, the chromophore undergoes photoconversion to anionic form : as mutant T302H has a very low minor absorbance peak, it is photoinducible at following irradiations with lmajor absorption peak.	lminor absorption peak ~ 397 nm (associated to the neutral phenol form of the chromophore) lmajor absorption peak ~ 488 nm (associated to the anionic phenolate form of the chromophore)
		enhanced cyan fluorescent protein (ECFP) Cerulean (ECFP/S72A/Y145A/H148D) has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activationref.
		enhanced yellow fluorescent protein (EYFP)
	monomeric red fluorescent protein 1 (mRFP1 / DsRed1) derived from Discosoma striata (sea anemone) by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. 3 monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficienciesref.		277, 335, 487, 530 and 558	583
	mPlum was obtained by iterating SHM over 23 rounds of FACS to create monomeric RFPs with increased photostability and far-red emissions (e.g., 649 nm), surpassing the best efforts of structure-based designref1, ref2
	DsRed-E5 is a mutant of the RFP drFP583 that changes its fluorescence from green to red over time, enabling accurate monitoring of the temporal ("fluorescent timer") and spatial pattern
	photoactivatable protein Kaede
	DsRed 'greening' technique
	kindling fluorescent proteins
photoswitchable cyan fluorescent protein (PS-CFP) is a dual-color monomeric protein capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and FRET applicationsref.
tetramethylrhodamine
Texas Red-X			595-596	615-620
binding directly to DNA	7-aminoactinomycin D (7-AAD)		546 (488 commonly used)	650
	propidium iodide (PI)		305 (488 commonly used)	617
	EMA
	Hoeschst 33342
binding directly to RNA	thiazole orange
tandem conjugates of 2 fluorochromes in which the energy is transferred from the first to the second, creating a larger Stoke's shift	PE-CY5		565 (488 commonly used)	670
	PE-CY7		565 (488 commonly used)	767
	PE-TxRed		565 (488 commonly used)	516
	APC-CY7		650 (633 commonly used)	767

Fluorocromes can be conjugated to :
• mAb (fluorobodies) : by inserting diverse antibody binding loops into 4 of the exposed loops at one end of GFP, the natural antibody binding footprint has been mimicked to create robust binding ligands that combine the advantages of antibodies (high affinity and specificity) with those of GFP (intrinsic fluorescence, high stability, expression and solubility). Fluorobodies show affinities as high as antibodies and their intrinsic fluorescence correlates with binding activity, allowing the rapid determination of functionality, concentration and affinity^ref
Other fluorescence applications :
• autofluorescence : the fluorescence from endogenous cell constituents such as NADH, riboflavin and flavin coenzymes, which can contribute to background levels during cell imaging
• total internal reflection : when a light beam strikes the interface of 2 media of different refractive indices at an angle beyond the critical angle, all of the light energy is reflected back into the incident medium. In this situation, an "evanescent field" develops at th interface with an energy that decreases exponentially into the medium with the lower refractive index. This allows selective excitation of fluorophores located within 100 nm of the interface.
• fluorescence correlation spectroscopy : a microspectroscopy technique, in which fluorescence-intensity flucutations in a small focal volume (1 mm³) are measured to enable the number, size and movement of fluorescent molecules to be determined at the single-molecule level.
• fluorescence polarization : the absorption of a fluorophore depends on the polarization angle of the excitation light, and fluorescence emission is also polarized. Fluorescence polarization is used to detect the direction and rotation of molecule movement
• fluorescence quantum yield : the ratio of photons emitted to photons absorbed. The fluorescence brightness of a species is proportional to the product of its molar extinction coefficient and fluorescence quantum yield
• fluoronanogold : fluorescent complexes of small gold clusters that are tagged with an antibody or Fab fragment
• fluorescence lifetime imaging (FLIM)
• fluorescence recovery after photobleaching (FRAP)
• fluorescence loss in photobleaching (FLIP)
• proximity imaging (PRIM)
• fluorescence resonance energy transfer (FRET) / Förster resonance energy transfer (FRET) : : detection method using a combination of fluorecence labels. The first fluorescent molecule is used to absorb the energy, then the energy is transferred to the second florescent molecule. Detection of a light signal from the second dye is diagnostic for the two dyes being in a close proximity. This means that certain molecular reaction took place, which is diagnostic for the presence of a certain allelic state
• template-directed dye-terminator incorporation (TDI) assay : a homogeneous DNA diagnostic method based on FRET^ref
• single-photon FRET : FRET that occurs from a single donor fluorophore to a single distribution of a g-emitting radionuclide tracer in a subject in 2D or 3D
• 3-chromophore FRET (3-FRET) :
• time-resolved fluorescence (TRF) :
• time-resolved FRET (TRET) (also called HTRF™, TruPoint™  and Lance™) :
• dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA^®) :
• fluorescence polarization (FP) :
Web resources :
• Fluorochrome Absorption and Emission Spectra
• Staining Nucleic Acids with Propidium Iodide
• bioluminescence resonance energy transfer (BRET) allows real-time detection of protein-protein interactions in vivo. This technique is based on the nonradiative transfer of energy between a luminescent energy donor (e.g. Renilla luciferase, Rluc) and a fluorescent energy acceptor (e.g., green fluorescent protein (GFP)). This is a system of choice for monitoring both constitutive and regulated intermolecular interactions because of the strict dependence on molecular proximity (<100 Å) between the donor and acceptor molecules for energy transfer^ref.As compared to western blot analysis, the use of BRET presents several advantages:
• because the BRET assay is carried out in living cells, it avoids the possible signal alterations that could result from cell lysis, protein solubilization or any purification steps before western blot analysis
• the ability to follow the evolution of the BRET signal in real time allows one to capture the dynamic nature of the ubiquitination process
• one can monitor changes in ubiquitination levels resulting from specific treatments in one population of cells using BRET, whereas western blot experiments require distinct cellular pools
• because of the ratiometric nature of the BRET technique, a reduction in signal truly represents a decrease in ubiquitination and does not result from the degradation of the ubiquitinated protein target
• DNA labeling by nick translation
• TdT-mediated dUTP nick-end-labeling (TUNEL) : this assay has been designed to detect cells dying of apoptosis. This widely used and highly sensitive assay is based on the fact that nuclear DNA becomes fragmented by endogenous endonucleases during apoptosis and that characteristic, nonrandom DNA fragmentation is common to all cells undergoing this form of programmed cell death . Fragmented DNA is detected easily by agarose gel electrophoresis. Essentially, the TUNEL assay involves labeling of the 3'-hydroxyl DNA ends generated during DNA fragmentation by means of terminal deoxyribonucleotidyl transferase (TdT) and labeled dUTP (fluorescein, biotin, digoxigenin). The DNA fragmentation assay can be used with tissue sections, adherent cells, and cells in suspension. DNA fragmentation can be quantified also if the assay is used in conjunction with flow cytometry. Several reports suggest that some forms of non-apoptotic DNA fragmentation are labeled also by the TUNEL assay and that results, therefore, should not be considered a specific marker of apoptosis. In general it is probably advisable to employ 2 independent methods to confirm that cell death is occurring by apoptosis^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}.
• annexin V apoptosis assay : annexin-V is a member of a highly conserved protein family that bind acidic phospholipids in a calcium-dependent manner. Annexin-V is known also as 35 kDa calelectrin, 35-gamma calcimedin, anchorin C2, placental anticoagulant protein-1 (PAP-1), calphobindin-1, endonexin-2, lipocortin V, vascular anticoagulant-a (VAC-a). The protein has been shown to possess a high affinity for phosphatidylserine. Phosphatidylserine is translocated from the inner side of the plasma membrane to the outer layer when cells undergo death by apoptosis or cell necrosis and serves as one of several signals by which cell destined for death are recognized by phagocytic cells. Cell death by apoptosis and necrosis differ from each other in that cell membranes maintain their integrity during the initial stages of apoptosis , while they become leaky during necrotic cell death . If Annexin-V binds to the cell surface this indicates that cell death is imminent. In order to differentiate apoptosis from necrosis a dye exclusion test with propidium iodide is performed to establish whether membrane integrity has been conserved or whether membranes have become leaky. A combination test measuring Annexin-V binding and dye exclusion thus allows discrimination between intact cells, apoptotic cells, and necrotic cells. The test allows detection of apoptosis at early stages before gross morphological changes have occurred and has been adapted for use with flow cytometry, light and electron microscopy and can be applied to vital and fixed material^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9,ref10}.
• dye exclusion test
• fluorence activated chromosome sorter (FACS)

Assays :
• immunophenotyping : measurement of different cellular antigens, such as cell surface and intracellular receptors, nuclear and mitochondrial proteins.
• directly conjugated antibodies or 2-step systems (avidin-biotin, primary and secondary antibodies).
• simultaneous use of up to 5 fluorescent probes.
• DNA analysis
• cell cycle analysis (percentage of cells in G₀/G₁, S and G₂/M phases).
• S-phase fraction (SPF) : percentage of cells in S-phase
• analysis of aneuploid populations ^ref : DNA index (DI) = ratio of the number of peak channels for the aneuploid G₀/G₁ to that for the diploid
• DNA-index heterogeneity : difference in the DNA index among aneuploidy
• DNA-ploidy heterogeneity
DNA histograms of normal cells are usually dominated by a large, normally-distributed (Gaussian) peak representing the DNA content of diploid G₀/G₁ cells. Often, the DNA content of malignant cells varies from this value, resulting in a second G₀/G₁ peak at a different horizontal (x-axis) location on the DNA histogram. Indeed, the appearance of 2 G₀/G₁ peaks (or more) is an important feature in establishing that DNA-content abnormalities are present^{ref1, ref2}. The ability to visualize a second peak depends on 3 factors:
• population representation : if only a small number of cells with an abnormal amount of DNA are present in the sample, the correspondingly-small abnormal G₀/G₁ peak will be overwhelmed by the much larger peak of the normal G₀/G₁ cells. Conversely, a small number of normal cells also may be unrecognized if overwhelmed by the abnormal cells, possibly tempting the viewer to consider the single peak as representing the normal cells. (The latter situation neglects to consider a shift in G₀/G₁ peak location.)
• magnitude of DNA-content differences : 2 histogram peaks representing cell populations with very minor DNA content differences will appear close together on the resulting histogram and may merge ("fuse") into a single, perhaps broader peak. Greater differences result in increased horizontal separation of the peaks and enables visualization of the abnormality as a bimodal (dual "topped") peak or 2 individual peaks.
• resolution of the technique: the apparent "sharpness" of the DNA histogram peaks can be affected by several factors, including the ability of the preparation method to stain the cells uniformly, as well as the instrument performance during data collection. 2 populations with DNA content differences will be more easily recognized as a bivariate peak or 2 peaks on the DNA histogram if the preparation and instrument performance are optimal. Sub-optimal staining and analysis will result in wider peaks and increased possiblity that the individual peaks fuse fully into one.
• correlation between DNA content and the expression of receptors.
• cell cycle kinetics and BrDU.
• analysis of apoptosis
• decrease in cellular DNA content (sub G₁ method).
• increase in DNA strand breaks (TUNEL method).
• appearance of phosphotidylserine residues on cell surface (annexin V method).
• change in mitochondrial transmembrane potential.
• detection of activated caspases.
• functional assays : several physiological characteristics in cells are closely regulated, and changes can occur in response to stimuli or signals.
• intracellular calcium ion concentration.
• intracellular pH.
• changes in membrane potential.
• special assays
• gene reporter assays allow the quantitation of a transfected gene using fluorescent
• proteins such as EGFP, dsRed2, EYFP and ECFP.
• cell viability.
• cell tracking to determine the location of particular cells in different organs.
• rate of cellular proliferation.
• phagocytosis (microsphere and dye based protocols).
• measurement of short-lived changes in fluorescence.
• concentration of soluble molecules : microspheres, coated with antibodies specific for certain molecules such as ILs, TNF, and IFNs, combined with these same antibodies tagged with a fluorescent marker, can be used to measure the concentration of these important cellular regulators in solution.
• sorting : isolation of cells from a mixture based on any of the above characteristics
• simultaneous collection of cells from 2 subpopulations into test tubes, dishes, or unto slides.
• cloning of single cells into multiwell plates (24, 48, 96 well and custom plates).
• sterile isolations routinely performed.
• isolation of rare populations at rates of up to 10,000 cells/second.
• temperature control of samples.
Web resources :
• ICRF FACS Laboratory: detection of apoptosis using flow cytometry
Bibliography :
• Alice Longobardi Givan. Flow Cytometry: First Principles, 2nd Ed. Wiley-Liss, 2001.
• Current Protocols in Cytometry. Updated quarterly
• Howard Shapiro. Practical Flow Cytometry, 4th Ed. Wiley-Liss, 2003
• Measuring the abundance of mRNA transcripts
• microarrays: complex nucleic-acid samples are investigated by hybridization

Arrays contain tens of thousands of 50- to 150-mm-diameter spots/cm²
• oligonucleotide microarrays : one type is manufactured by Affimetrix (Gene Chip™). It contains hundreds of thousands of ordered, single-stranded synthetic oligonucleotides that are typically 25 bases in length, which have been synthesized in situ on a solid support. Each gene is generally probed by several (> 15) oligonucleotides. DNA and RNA samples are labelled and fragmented before being hybridized to the array. Quantitative estimates of the transcript abundance can be obtained directly by averaging the signal from all the probes that belong to one gene
• spotted DNA microarrays : these usually contain ordered, double-stranded DNA molecules that were created by PCR. They correspond to either genomic or cDNA sequences that have been deposited onto glass slides. Usually there is one probe per gene. For technical reasons, the information that is obtained from these microarrays generally gives the relative concentration (ratio) of a given transcript when 2 conditions are compared. Samples of mRNA from 2 experiments are labelled with different dyes, pooled and hybridized to the microarray by competitive hybridization
• microarray readers : arrays are imaged using LASERs to excite the fluorophores on the chip :
• resolution : 5-10 mm are needed to detect transcripts with a relative abundance of one copy in 500,000, or 3-10 copies per cell
• sensitivity : the presence of at least 2-5 molecules/mm² is necessary for low-level fluorescence detection.
• comparative view using AtlasImage 2.0 software
• green : equal expression of the gene
• red : up-regulation of array 2 versus array 1
• blue : down-regulation of array 2 versus array 1
• gray : one or both arrays below signal threshold (background level)
• upper half of each gene-box : ratio between array 2 and array 1
• lower half of each gene-box : difference of array 2 versus array 1
• array imagers capture a snapshot of the glowing array using a charge-coupled device (CCD) camera, an electronic sensor that detects and records light patterns
• array scanners read the chip point-by-point using a photomultiplier tube (PMT), which then transmits these signals on a point-by-point basis to a computer that assembles the image. The first microarray scanner, built in 1989 by Stephen Fodor and colleagues at Affymax, was a table-sized, home-built affair that included a Zeiss confocal microscope, a laser, and several mirrors. Fodor would go on to head Affymetrix, a Santa Clara, Calif., Affymax spin-off that now holds the lion's share of the microarray market. Unlike some of its competitors, whose glass microscope slide-based arrays can be read in any array reader, Affymetrix's GeneChip microarrays are imaged using a proprietary instrument. Shown here are the major components of the newest version of that device, the GeneChip Scanner 3000.


• the flying objective performs fast horizontal scans; the actual trajectory is an arc with a one-inch radius. The scanner acquires fluorescence data only during the right-toleft scan (left-to-right is the flyback time), and the array is scanned from the top to bottom, as the array inches upwards. The chip transport moves the array by a distance equal to one pixel and then stops, allowing the objective lens to scan one line (arc) of data.
• the dichroic separates excitation and emission beams and allows a small sample of the laser beam to be monitored.
• the system auto-focuses using laser light reflected from the chip’s chrome border.
• the fluorescent signal is measured using a photomultiplier tube (PMT). Photons strike a photoemissive cathode, which emits electrons due to the photoelectric effect. These electrons are accelerated towards a series of electrodes called dynodes, each of which generates additional electrons. This cascading effect creates 10⁷ or more electrons for each photon hitting the first cathode, depending on the number of dynodes and the accelerating voltage. This amplified signal is finally collected at the anode, where it can be measured.
Affymetrix’s microarrays are built in parallel on large wafers and then cut into individual chips, with one wafer yielding anywhere from 49 to 400 chips. Measuring just 1.28 cm on a side, the GeneChip Human Genome U133 Plus 2.0 Array contains 1.3 million distinct sequence features. The chip covers more than 47,000 individual transcripts, each represented by 11 pairs of 25-mer oligonucleotidesDNA features on Affymetrix’s GeneChip microarrays are built using a photolithographic process adapted from the semiconductor industry. The DNA building blocks (called phosphoramidites) are photosensitive, meaning that they become “deprotected,” or competent to participate in a chemical reaction, when exposed to light. Photomasks, bearing windows just microns on a side, deprotect only those regions of the chip that are to couple to the next added nucleotide. In this way, thousands of chemically unique polymers can be assembled in parallel.
The current trend in microarray scanners seems to be for 2 basic types of instrument :
• large capacity systems with multiple slide capability, which appeal to core microarray facilities
• smaller stand-alone units, which appeal to individual researchers
Agilent Technologies of Palo Alto, Calif., claims its laser scanner is the most sensitive in the industry : it's about 3 times as sensitive as any other DNA microarray scanner. The scanner simultaneously conducts 2-color scanning at 10-mm resolution, taking about 8 minutes per slide. It is also the first 48-slide scanning system that can read any combination of 2.5 x 7.6 cm glass slide microarrays. People can use an Agilent array, arrays from any other vendor, or homebrew arrays in the scanner; they're not locked into a closed system. That's in contrast to systems from such vendors as Affymetrix of Santa Clara, Calif., and Applied Biosystems of Foster City, Calif., whose readers accept only their own chips. In addition, upgrades to the scanner's new feature-extraction software means that homebrew array users no longer have to feature-extract using another package. GE Healthcare (formerly Amersham Biosciences)'s Typhoon 9410 Variable Mode Imager became the first large-format gel and blot imager that could also image microarrays. Scanners must also meet the demands of increasingly high-throughput technologies, such as Affymetrix's new 96-well HighThroughputArray (HTA). Axon Instruments, based in Union City, Calif., recently introduced the GenePix Autoloader 4200AL, an add-on to the company's GenePix Professional 4200A microarray scanner. The autoloader loads up to 36 standard microarray slides to automate the processes of scanning and analysis. Axon will also supply scanners to Affymetrix for the company's 96-well HTA technology. Likewise, Alpha Innotech, San Leandro, Calif., recently released its white-light AlphaArray 8000, which uses CCD-detection technology coupled with a broadband excitation source and compatible with a wide variety of fluorophores. The technology provides resolution images down to 4 mm and accommodates standard slides as well as custom formats and microplate based arrays. Tecan Group, based in Maennedorf, Switzerland, has developed the new LS Series of laser scanners can operate up to 4 excitation lasers and 28 emission filters  It allows researchers to scan many different samples automatically, from glass slides to microtiter plates, on the same equipment without having to switch scanners or make extensive adjustments each time the format type or size changes. The introduction of reliable and affordable 3-laser systems such as the GenetixaQuire have allowed the use of a greater range of dyes such as fluorescein and green fluorescent protein.
• samples analyzed by microarrays are generally fluorescently labeled, often using probe families with similar chemical structures but different spectroscopic properties, such as the cyanine (Cy) dyes Cy3 and Cy5. But fluorophores present problems, including photobleaching and spectral overlap (or bleed-through), which can diminish the quality of collected data. Because of these problems, some foresee a move away from fluorescence detection. Some of these systems might not be better technically, but they may be adequate for a particular application. For example, colorimetric detection certainly isn't as quantitative, but not everybody needs quantitation. TeleChem's SpotWare Colorimetric Microarray Scanner allows researchers to use horseradish peroxidase, alkaline phosphatase, gold-silver developers, secondary antibodies, and any other labeling system that produces a pigmented reaction precipitate. According to Martinsky this scanner will read DNA and protein, and costs about $4,000.
• Applied Biosystems' new Microarray Expression Analysis System combines chemiluminescence and fluorescence as a dual method for detecting gene expression in biological samples. Each spot on the arrays contain a 60-mer and a 24-mer oligonucleotide. The former is labeled with digoxigenin for chemiluminescent detection, the latter with a fluorescent dye. In the absence of a chemiluminescent signal, or the presence of a very low signal, each of the approximately 35,000 spots can be precisely located through the fluorescence signal. This allows the system to find each spot and reproducibly map it, and if there is a lack of correlation, the spot is rejected. The Expression Analysis System also includes a CCD camera and high-powered light-emitting diode for its "pseudo-fluorescent" illumination system.
• resonance light scattering (RLS) is another relatively new detection strategy for microarrays. The ultrasensitive signal generation and detection technology, developed by Genicon Sciences, San Diego, (now owned by Invitrogen, Carlsbad, Calif.) can be applied to a wide variety of molecular assay formats and platforms. The company's GeniconRLS System can generate, detect, and image 1- or 2-color gene-expression microarrays using as little as one to 2 mg total RNA for 10-fold increased sensitivity over fluorescence. In addition, it can detect 2-fold differences in gene expression and perform multiple readings without signal decay, the company says.
• Qiagen, based in the Netherlands, has released its HiLight Array Detection System based on the RLS technology
Researchers at Sandia National Laboratories and the UniversityNew of  Mexico, Albuquerque, have developed an alternative strategy, called hyperspectral scanning, which could soon surpass current fluorescence systems. The scanner achieves resolutions between 3 and 30 mm and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each microarray pixel. According to a recent report, this spectral information, when coupled with multivariate data-analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of micro-array experiments^ref. It quantifies every emitting source, including those not associated with the labels, such as those from the buffers used in printing the DNA microarrays. It separates them out at every pixel in the image. Then when coupled with multivariate curve resolution, the relative fluorescence of all fluorescing components can be quantified to separate out the effects of the contaminant and glass emissions from those of the fluorophore, resulting in a more accurate image of the DNA labels.
The microarray field holds great promise, but some of the experimental variance in the data makes it difficult to see the biology. These new detection approaches make the whole industry better and will allow scientists to get more information.
• per-chip genome density
Aim :
• whole proteome microarray
• whole genome microarray :
• single chip : arrays to genotype 100,000 single nucleotide polymorphisms (SNPs) at once have also come to market :
• market leaders Affymetrix of Santa Clara, Calif., and Agilent Technologies, of Palo Alto, Calif., beat Applied Biosystems to the punch, releasing their human whole-genome chips last October. Amersham Biosciences of Chalfont St. Giles, UK, (now part of GE Healthcare) announced its CodeLink Human Whole Genome Bioarray in April, and Taiwan-based Phalanx Biotech Group will begin selling its Human OneArray this fall. NimbleGen Systems of Madison, Wis., has been offering a single microarray, human whole-genome product on a services-only basis since summer 2003
• Affymetrix also is increasing throughput, but in a 96-well format. The company's GeneChip HighThroughputArray (HTA) system, which can process hundreds of samples per day, adapts standard Affymetrix chips to a 96-well microtiter plate, and runs on an automated system built with off-the-shelf robotic components. The technology is licensed to a limited number of companies (currently Johnson & Johnson Pharmaceutical Research & Development of Raritan, NJ, and Aventis Pharmaceuticals of Bridgewater, NJ) to analyze 96 GeneChip Human Genome U133A Arrays. The company plans to release a follow-up product that will contain a whole genome in each microtiter well.
• whole mammalian genomes spread over multiple chips. MWG Biotech of Ebersberg, Germany, released its two-chip Human 40K Array last October
• multiple human genomes on a single chip for parallel sample analyses : Illumina of San Diego launched in January 2 Sentrix BeadChip products. One runs 6 samples against 6 sets of 48,000 human transcripts; the other runs 8 samples against the roughly 22,000 genes in the consensus RefSeq database
• transcriptome : protein-coding genes, which account for just 2% of transcription
• Affymetrix researchers are currently using a specialized GeneChip microarray to "tile" millions of DNA probes representative of the complete human genome. The company recently published data that identifies transcription factor-binding sites across human chromosomes 21 and 22.
• SNP analysis (the most common source of genetic variation) : the human genome is thought to contain over 10 million of them, about one every 300 bases. In April, Affymetrix announced the early-access availability of its GeneChip Mapping 100K Array Set, a 2-chip product slated for summer release. Illumina is also planning to release a 100,000-SNP chip for beta testing by year's end.
A problem inherent in microarray data analysis is the false-discovery rate. Normally in biology, a p value of 0.01 is significant, but in the case of microarray data, it is not. If you look at 40,000 things, you'll be able to find 400 response variables that seem significant just by chance alone. How many replicates is enough? There's the illusion that you need fewer samples because the gene is broken up into several different regions and that means the gene is represented multiple times. Affymetrix of Santa Clara, Calif., sponsored a microarray data analysis "cook-off" between eight software vendors, using a common dataset. Overall, the packages produced largely similar results. VizX Labs in Seattle recently upgraded its easy-to-use GeneSifter.net, a Web-based analysis package. The product includes new features specific for Affymetrix platform users, incorporating a new pathways report, clustering analytics (CLARA), a boxplot option for use in quality control, enhanced dendrograms, additional sorting and searching options, and improved ability to construct gene lists. Ingenuity Systems of Mountain View, Calif., has recently upgraded its Pathways Analysis software. The program is a Web-delivered application that enables biologists to explore therapeutically relevant networks significant to their gene- or protein-expression array datasets. Using the product, a researcher can cross-reference biological networks generated from their datasets with well-known pathways. The software also allows researchers to expand from a single network to visualize other biologically related networks and contains new gene and protein dataset mapping, including two new Affymetrix arrays. InforSense, based in London, recently introduced a new application module for its InforSense 1.9: BioScience package that eases analysis of genomics and proteomics data. According to the company, the upgrade directly integrates sequences, expression data, and ontology-based knowledge for a wide range of analytical applications in genomics and proteomics studies. Example applications include integrative gene-expression analysis and high-throughput gene and protein annotation. Likewise, Iobion Informatics, based in La Jolla, Calif., recently released PathwayAssist 2.5, desktop software for biological pathway visualization and analysis. New features include molecular-network databases for four model organisms (Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana), the capability to map gene-expression data onto KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, and the ability to save pathways in HTML format as interactive, clickable maps. In addition, the new version features integration with GeneSpring and ArrayAssist gene-expression analysis software. The database performance has been increased 5-fold, and the 'Update Pathway' feature instantly extracts new information about a specified protein from PubMed. For thrifty scientists, or even those skilled in programming, the open-source software movement offers analysis tools at no cost.
• Bioconductor, for example, is a collaborative open-development software project started by researchers at the Dana-Farber Cancer Institute, Boston : many of the software tools are general and can be used broadly for the analysis of genomic data, such as serial analysis of gene expression (SAGE), single nucleotide polymorphisms, or sequence data. Bioconductor has been extremely useful because it provides a resource where researchers can put their scripts and methods. It's a way of galvanizing methods so people can have access to other people's source code and they can see whether the method is a valid method or not. And that reduces the need for bioinformaticians to reinvent the wheel : there used to be a tendency of people reinventing the same method, but just calling it something different
• TM4, developed by John Quackenbush's group at The Institute for Genomics Research in Rockville, Md. TM4 consists of 4 major applications : Microarray Data Manager, TIGRSpotfinder, Microarray Data Analysis System, and Multiexperiment Viewer, as well as a Minimal Information About a Microarray Experiment (MIAME)-compliant MySQL database.
MIAME, a standard developed by the Microarray Gene Expression Data Society (MGED), lays out the absolute minimum amount of information researchers should provide to allow experiment reproducibility. Several journals now require researchers to meet MIAME requirements when submitting research for publication. MGED is trying to use standard object models for microarray data as well as data for proteomics, mass spectrometry data, and 2-D gels. The major trend in the last couple of years has been to analyze microarray data relative to genetic processes and pathways. Someone may identify 500 genes that seem interesting in the experiment, but then it is extremely time consuming for the biologist to go through those lists to try to figure out what those genes mean and their relationship to biological processes. Now, with the help of many types of software, people are much better able to process data on the genes of interest and interpret the meaning of their experiment. Still, major errors are often made in the statistics of microarray papers :
• the one mistake that's made in about half of all microarray papers, is when people cluster data. If you have 2 cancer samples, and you find that all the data from cancer one clusters together and all the data from cancer two clusters together, this is interesting because that means that the gene-expression profile and the two samples may be different. But then the researchers make a mistake, he says. Instead of selecting all 40,000 genes, they instead choose a subset of genes that can distinguish the two cancer types. It is a subtle error, but it is one that makes expression patterns appear distinguished when they are not
• another common error is that people will develop prediction models to determine whether they have cancer type one or cancer type two from their gene-expression data, but they'll only have, say, 5-10 samples with which they are working. Correctly predicting four samples out of five gives an accuracy rate of 80%. But with only 5 samples, there's a huge variability in that number. The point is that you look at sample sizes that are so small that the variability of your prediction as to make it meaningless
So researchers need more than just solid statistical packages when working with array data : you need a good experimental design and directed biological questions to guide the analysis of microarray data. Often, there are a lot of transcriptional changes associated with a certain biological condition or disease, but only a few of them play a causal role. The challenge is to isolate these from the mass of bystanders. You need to pull together all available metadata or additional datasets to achieve this.
In January the European Union's Sixth Framework Programme initiated the MolTools project (€ 9 million over 3 years). A consortium of 18 academic and biotech concerns, the project aims to develop more powerful array-based research tools to examine DNA, RNA, and proteins
• cytokine gene expression : choosing the right one for your lab requires weighing sample throughput (how many samples can be tested at once) against gene throughput (how many genes are tested per sample). Arrays and northern blots occupy opposite ends of the throughput spectrum. Northern blots test a few RNA samples (perhaps 10 to 20) for the expression of a single gene, whereas arrays test a single sample for hundreds or thousands of genes. Naturally, many researchers don't limit themselves to just one of these techniques, as each has its advantages and disadvantages. The presence of mRNA for a cytokine in tissue is meaningful, but its absence is not, as cytokine transcripts are short-lived, and their absence in the tested tissue could reflect rapid processing or degradation rather than the lack of synthesis.^ref
• cytokine microarrays :
• IntelliGene Human Cytokine CHIP (glass slide-based; source : Takara Bio) bears nearly 550 cytokine-related cDNA fragments.
• cytokine macroarrays (printed on large nylon membranes and thus require no special equipment : when combined with colorimetric detection, you can view them by eye if you want to. You don't need a microscope or a scanner, though many researchers do use aids such as these and phosphor imagers for greater accuracy.
• GEArray macroarrays (source : Superarray) produces several ones for cytokine research, each bearing a different selection of as many as 440 genes.
• Th1-Th2-Th3 Gene Array, for instance, queries RNA relevant to these subtypes of CD4⁺ T cells.
• Atlas Human Cytokine/ Receptor Array. (source :BD Biosciences-Clontech of Palo Alto, Calif.)
• Panorama cytokine macroarrays (source : Sigma-Genosys) is no longer produced, though it will custom-design and manufacture them to order
• Message Hunter (source : Geno Technology)
• scientists interested in measuring the RNA of a few cytokines in many samples simultaneously may want to consider multiplex PCR (MPCR), a technique whose sample throughput is limited only by thermocycler capacity.
• Biosource International, a Camarillo, Calif.-based company supplies a range of cytokine detection products. Several suppliers offer MPCR kits that contain reagents optimized to work together to simultaneously amplify multiple cytokine sequences in a single tube (e.g. MAXIM Biotech). The PCR primers have similar T_m's and no obvious 3'-end overlap to enhance amplification of multiple targets, claims Biosource's messageScreen literature. If the cDNA isn't required for other experiments, reverse transcription and PCR can be done as a one-step reaction in a single tube. Most manufacturers engineer their primers to recognize cDNA sequences that span an intron splice boundary, so that contaminating genomic DNA (if present) will not be amplified. Amplicons are designed to be of sufficiently different sizes so as to be distinguishable by standard agarose gel electrophoresis. But results can also be imaged via other means such as fluorescence or radiolabeling. MPCR is considered semiquantitative : detecting a twofold difference may be difficult; 10-fold is much more likely. That's because the technique relies on a reaction that essentially goes to completion (endpoint PCR). Real-time PCR, in contrast, catches the reaction in its logarithmic phase, when the amount of product generated is proportional to the amount of template in the original sample. The technique uses specialized tags that fluoresce as they are incorporated into the amplicons; fluorescence is assayed as the reaction proceeds, using dedicated thermocycler-detectors such as the ABI PRISM Sequence Detection systems from Applied Biosystems, Foster City, Calif. Real-time PCR is probably the most widely used technique for measuring message these days. Reagents can readily be found, or easily synthesized from published sequences. It used to be that we begged other investigators to give us probes for certain cytokines. Today, they're widely commercially available for most cytokines and chemokines. Because real-time PCR uses distinct fluorescent tags rather than size to distinguish one amplicon from another, the technique can't really amplify > 2-3 genes at a time
• ribonuclease protection assays (RPA), on the other hand, can examine a dozen or so genes simultaneously. In RPA, labeled RNA probes are hybridized with sample RNA. Digestion of the sample with ribonuclease destroys unhybridized RNA while leaving, or "protecting," hybridized fragments that can be resolved electrophoretically. With RiboQuant multiprobe template sets from BD Biosciences-Pharmingen you can look at a lot of cytokines at the same time, and it's very sensitive. San Diego-based Pharmingen offers 8 RiboQuant multiprobe template sets, each of which will make up to 10 different anti-sense probes.
• at least 2 manufacturers offer systems to detect cytokine RNA in ELISA-like assays. In Minneapolis-based R&D Systems' Quantikine mRNA kits, RNA is mixed with 2 oligonucleotides, one biotin-labeled probe and one digoxygenin (DIG)-labeled probe. The solution is transferred to a streptavidin-coated plate, where the RNA is visualized using an anti-DIG-alkaline phosphatase conjugate to catalyze an enzymatic reaction. Color is generated in proportion to the amount of specific RNA in the starting material, and it can be read in any standard ELISA reader. Probe kits are designed to query multiple samples for the presence of a single cytokine message species. Fremont, Calif.-based Genospectra's QuantiGene Reagent System Screen Kit can also quantify a single RNA species directly from cell lysates or tissue homogenates. Cytokine-specific oligonucleotide probes bound to the wells of a 96-well plate capture the RNA. A second set of oligonucleotide probes then hybridizes to another sequence on the bound RNA. The probes are in turn recognized by "branched DNA" conjugated to alkaline phosphatase, which catalyzes a chemiluminescent reaction that is read using a luminometer. Genospectra recently introduced a multiplex version of this technology, called QuantiGene Plex, in which the primary probes are bound to fluorescent Luminex beads rather than a microtiter plate. The system, which also replaces the standard kit's chemiluminescence detection with fluorescence detection, can probe up to 10 mRNAs at once. The beads are run through a Luminex instrument (from Luminex of Austin, Texas), which simultaneously reads the beads' internal fluorescence (indicating which transcripts were captured) and that of the tags (reporting how much was captured)
• finally, some researchers measure cytokine gene expression using in situ hybridization (ISH), a histologic technique in which tissue sections are incubated with labeled oligonucleotides to probe for the presence of complementary sequences. ISH's primary advantage is that, unlike the other techniques discussed here, it can identify the cells that actually produce a particular transcript. But like northern blotting, ISH is low-throughput, testing a single slide for a small number of genes.
Web resources :
• NHGRI Microarray Project
• Gene expression links by EBI
• Tissue Microarray Project (TMA) by NHGRI
• Tissue Array Research Program samples from several types of cancer.
• Microarray guide by PatBrown Laboratory
• DNA microarray (Genome Chip)
• Microarray links 1 & 2
• Microarrays glossary at Cambridge Healthtech Institute (CHI)
• Microarray-related activities at the European Bioinformatics Institute (EBI)
• Stanford Microarray experiments Database (SMD)
• Gene expression omnibus (GEO) at NCBI
• Microarray Gene Expression Database (MGED)
• Usenet bionet.molbio.genearrays
• companies offering DNA array technologies : Affymetrix, Agencourt Bioscience, Agilent, Alpha Innotech, Applied Biosystems, Beckman Coulter, BioDiscovery, Bio-Rad Laboratories, Brooks Automation (formerly Intelligent Automation Systems), Cartesian Technologies, Clondiag Chip Technologies, CombiMatrix, Corning, GE Healthcare (Amersham), Genaissance, Genomic Solutions, Illumina, Invitrogen, Marligen Biosciences, Mergen, MiraiBio, Molecular Devices, Nanogen, Nimblegen, Orchid Biosciences, Panomics, Parallele Bioscience, Perkin Elmer,

Perlegen, Phalanx Biotechnology, Plexigen, Polymorphic DNA, Promega, Qiagen, Robodesign, Rosetta Biosoftware, Rosetta Inpharmatics (a subsidiary of Merck), SeqWright,Sequenom, Sigma-Genosys, Spectral Genomics, SpectruMedix, Stratagene, TeleChem/ArrayIt.com, TmBioscience, V&P Scientific, Vysis, Xenopore
• Companies offering DNA microarrays, platforms and softwares : Affymetrix; Agilent Technologies; Alpha Innotech; Ambion; Applied Biosystems; Applied Precision; Axon Instruments; BD Biosciences-Clontech; BioDiscovery; Biolog; Bio-Rad Laboratories; Cartesian Technologies; Clinical MicroArrays; Clondiag Chip Technologies; CombiMatrix; Compugen; Corning; CyVera; GE Healthcare (formerly Amersham Biosciences); Genomic Solutions; GeneMachines;GeneScan; Genetix; Genicon Sciences; Illumina; Intelligent Automation Systems; Invitrogen; Mergen; MiraiBio; MWG Biotech; NimbleGen Systems; Panomics; PanVera; Perkin Elmer; Phalanx Biotech; Plexigen; Qiagen; Robodesign; Rosetta Biosoftware; Rosetta Inpharmatics; Silicon Genetics; Sigma-Genosys; Spectral Genomics; Stratagene; SuperArray; SurModics; TECAN; TeleChem/ArrayIt.com; UVP; V&P Scientific; Vysis; Xeotron Corporation; Xenopore
• serial analysis of gene expression (SAGE) : this identifies and quantitates transcripts on the basis of sequencing and does not rely on a priori gene predictions. Short, usually 15-bp sequence tags are isolated from a defined restriction enzyme site near the 3'-end of the cDNAs. These tags contain sufficient sequence information to identify the transcript from which each tag was derived. The 15-bp tags are concatenated, PCR amplified, cloned and sequenced. The abundance of a transcript is estimated by counting the occurrence of each SAGE tag.
• RNA interference (RNAi) has revolutionized functional genomic studies in many model organisms. In particular, the introduction of short double-stranded RNAs (dsRNAs) as mediators of RNAi in mammalian cells has moved loss-of-function studies in these experimental systems to a new plane^ref. Now the 2 most commonly used RNAi triggers in mammalian cells are chemically or in vitro synthesized short interfering RNAs (siRNAs)^ref and in vivo expressed short hairpin RNAs (shRNAs)^ref. We, and others, have pioneered an alternative technology, which is considerably more cost-efficient and less laborious than siRNA and shRNA methodologies. Our method is based on the generation of siRNAs by digestion of long dsRNAs with recombinant Escherichia coli RNase III or Dicer^{ref1, ref2, ref3, ref4} (Buchholz, F., Drechsel, D., Ruer, M. & Kittler, R. Production of siRNA in vitro by enzymatic digestion of double-stranded RNA. In Gene Silencing by RNA: Technology and Application (ed. Sohail, M.) 87?99 (CRC Press, Boca Raton, Florida, USA, 2004)). The resulting endoribonuclease-prepared siRNAs (esiRNAs) have proven to be efficient and specific mediators of RNAi in mammalian cells^{ref1, ref2, ref3}. Here we describe a robust and simple protocol for the production of esiRNA : a cDNA fragment tagged with T7 promoter sequence by PCR is transcribed in vitro to produce dsRNA. By limited digestion, the long dsRNA is then converted to siRNAs of <30 base pairs (bp) and spin-purified in a single column. Because of its ease, speed and cost-efficiency, this protocol can be easily scaled for the generation of libraries for (sub)genomic screens. Flowchart of esiRNA production :


Materials :
• reagents :
• agarose, electrophoresis grade (Invitrogen)
• BIOTAQ Red DNA polymerase (Bioline)
• complementary DNA (cDNA) clones or cDNA preparations
• deoxynucleoside triphosphate (dNTP) mix (10 mM; Bioline)
• dsRNA digestion buffer (20 mM Tris-HCl, 0.5 mM EDTA, 5 mM MgCl₂, 1 mM Diothiothreitol (DTT), 140 mM NaCl, 2.7 mM KCl, 5% (vol/vol) glycerol (pH 7.9))
• elution buffer (20 mM Tris-HCl, 1 mM EDTA, 520 mM NaCl, (pH 8.0))
• 70% (vol/vol) ethanol, cold
• equilibration buffer (20 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl (pH 8.0))
• esiRNA loading dye (3 mg/ml Orange G, 5% (wt/vol) Ficoll; both from Sigma-Aldrich)
• GST-RNase III (prepared as previously described)
• isopropanol
• marker DNA (for example, 25-bp DNA ladder, HyperLadder V; Bioline)
• MEGAscript in vitro transcription kit (Ambion)
• MgCl₂ (50 mM; Bioline)
• 10 ammonium reaction buffer (160 mM (NH4)2SO4, 670 mM Tris-HCl (pH 8.8), 0.1% (vol/vol) Tween 20; Bioline)
• primer oligonucleotides containing the T7 promoter sequence, as described in Step 2
• Q Sepharose FastFlow (Amersham Biosciences)
• Wash buffer, (20 mM Tris-HCl, 1 mM EDTA, 400 mM NaCl (pH 8.0))
• equipment
• 96-well filter plates UNIFIL 98-800U for large-scale synthesis (Whatman)
• 96-well PCR plates (Nerbe)
• empty spin columns (for example, Micro Bio-Spin Chromatography Columns; BioRad Laboratories)
• deep-well plates (1.1 ml) for large-scale synthesis (Nerbe)
• Multifuge 4KR, for large-scale synthesis (Kendro)
• silicon seals for deep-well plates (Nerbe)
• thermal cycler programmed with the desired amplification protocols (MJ Research)
Procedure :
• generation of a template for in vitro transcription by PCR :
• 1. The template for in vitro transcription can be generated from a cDNA clone or directly from cDNA using vector- or target-specific primers appended with T7 promoter sequences. cDNA clones have the advantage that universal vector-specific primers can be used to amplify the insert. Conversely, cDNA sequence-specific primers allow a high degree of flexibility to choose the target region for esiRNA production. For the selection of the esiRNA target region, we recommend using the web server DEQOR12, which allows the identification of cDNA regions containing a high percentage of efficient silencers and a low number of cross-silencers^ref. This web server uses accession numbers from GenBank or raw cDNA sequences. Selection of an optimal esiRNA target region with DEQOR :


The web server DEQOR allows the identification of efficient silencers and cross-silencers using state-of the-art siRNA design criteria. The in silico analysis of a given mRNA sequence is graphically shown: efficient silencer windows (green), inefficient silencer windows (black), perfect cross-silencer windows (red) and imperfect cross-silencer windows (yellow). Suitable sequence regions can be dragged (depicted by the blue bar) and copied from a field below containing the marked sequence for subsequent primer design.
• 2. After selecting a 400-600-bp esiRNA target region, design primers appended 5' with the T7 promoter sequence 5'-CGTAATACGACTCACTATAGGGAGAG-3'. When using cDNA clones with the same vector backbone, a universal primer pair can be used. For primer design we routinely use the Primer3 program with the provided default parameters^ref. The web server for Primer3 is available online^ref. When choosing cDNA clones or designing cDNA-specific primers bear in mind that the amplicon length is critical for the downstream digestion of the dsRNA. We obtained best results with amplicon lengths between 400 and 600 bp. For shorter templates, the streamlining of RNase III digestion conditions seems to be more difficult, and for templates longer than 800 bp, we occasionally observe problematic annealing between the two single-stranded RNAs
• 3. Set up the amplification reaction :
• 10X NH₄ reaction buffer 5 ml
• MgCl₂ (50 mM) 2 ml
• dNTP mix (10 mM) 4 ml
• T7 forward primer (10  mM) 1 ml
• T7 reverse primer (10 mM) 1 ml
• BIOTAQ Red polymerase (1 u/ml) 2 ml
• water 34 ml
• 4. Amplify the template as follows:

cycle number	denaturation	annealing	polymerization
1	2 min at 94°C	30 s at 60°C	30 s at 72°C
2-39	30 s at 94°C	30 s at 60°C	30 s at 72°C
40	30 s at 94°C	30 s at 60°C	5 min at 72°C

Annealing and extension times may need to be adapted for individual amplicons. PCR products can be stored at -20 °C for at least one year.
• 5. Analyze 3 ml of PCR product on a 1.5% (wt/vol) agarose gel with an appropriate marker. An intense band should be observed indicating high yield of amplification (typically >100 ng/ml). The amount of PCR product is limiting for the yield of in vitro transcription. Therefore at least 250 ng of DNA should be used as template for a 10-ml in vitro transcription reaction. We obtain our highest yields using >400 ng DNA.
• generation of long dsRNA by in vitro transcription :
• 6. Using components from the MEGAscript kit and the PCR product obtained in Step 5, set up an in vitro transcription reaction as follows:
• 10X T7 reaction buffer 1 ml
• uridine triphosphate (UTP; 75 mM) 1 ml
• adenosine triphosphate (ATP; 75 mM) 1 ml
• guanine triphosphate (GTP; 75 mM) 1 ml
• cytosine triphosphate (CTP; 75 mM) 1 ml
• PCR product 4 ml
• T7 enzyme mix 1 ml
• 7. Perform the in vitro transcription and annealing reaction in a thermal cycler as follows:
• in vitro transcription : 4-12 h at 37°C
• denaturation :
• 3 min at 90°C
• ramp to 70°C with 0.1°C/s
• 3 min at 70°C
• ramp to 50° with 0.1°C/s
• 3 min at 50°
• annealing : ramp to 25°C with 0.1°C/s
Do not freeze the long dsRNA as this will promote the formation of aggregates, which hamper subsequent digestion. This is especially true for dsRNAs of >800 bp.
8. Check 0.5 ml of the product on a 1.5% (wt/vol) agarose gel against an appropriate marker. A very intense band should be observed indicating high yield of in vitro transcription (4-10 mg/ml). This gel check can be omitted when using the BIOTAQ Red polymerase from Bioline, especially for the production of esiRNAs in 96-well formats. A marker dye included in the polymerase indicates an efficient in vitro transcription reaction in a color shift from red to yellow. This presumably occurs as a result of a change in the pH of the reaction as the NTPs are consumed. If there is a low yield of dsRNA ...

A cDNA fragment (452 bp) tagged with T7 promoter sequence was in vitro transcribed to produce a long dsRNA, which was digested with different amounts of GST-RNase III. The long dsRNA, digestion products, purified esiRNA and a chemically synthesized siRNA (21mer) were separated on a 4% agarose gel. M, 25-bp ladder, the 25- and 50-bp bands are indicated; lane 1, 0.5-ml long dsRNA; lanes 2 and 3, 3 ml of digestion product generated with the 5 mg (recommended) and 2 mg (too little) GST-RNase III, respectively; lane 4, 15 mg (too much) GST-RNase III; lane 5, 500 ng esiRNA purified from the digestion product shown in lane 2; and lane 6, 500 ng of a 21-nt chemically synthesized siRNA.
.. that means the amount of DNA template was too low. Use at least 250 ng DNA for in vitro transcription.
• digestion of long dsRNA :
• 9. Set up the digestion reaction as follows: mix 10 ml of in vitro transcription product from Step 7 with 90 ml of dsRNA digestion buffer, containing 5 mg GST-RNase III, and mix vigorously. The amount of GST-RNAse III is critical for optimal digestion. The recommended amount of 5 mg GST-RNase III is optimal for the digestion of 30-100 g dsRNA. Under the digestion conditions described in Step 10, 90% of all samples should have digestion product smears of <30 bp. Because the specific activity of GST-RNase III may differ between preparations, we recommend performing an initial titration of each enzyme stock to ensure optimal digestion results.
• 10. Perform the digestion reaction as follows: incubate with agitation for 4 h at 20-25 °C, then shift the temperature to 37 °C for 2 h more. Digestion products can be stored at -20 °C for at least 1 month.
• 11. Mix 3 ml of digestion product with 3 ml of esiRNA loading dye and analyze the sample on a 4% (wt/vol) agarose gel against an appropriate marker. The 4% agarose gels are somewhat difficult to prepare with a microwave. A good solution is to dissolve the agarose in a water bath set to 95 °C, which takes about 90 min. The digestion product smear should be shorter than 30 bp, whereas most of the smear should range between 18 and 25 bp
• if the digestion products are too long, the amount of GST-RNase III was too low or the amount of dsRNA was too high. If the digestion product smear is <50 bp, add 2 g of GST-RNase III and incubate for another hour at 37 °C. If the digestion product smear is >50 bp, add 4 mg of GST-RNase III and incubate for another hour at 37 °C.
• if the digestion products are too short, the amount of dsRNA was too low, or the amount of GST-RNase III was too high. Repeat the digestion with more dsRNA or decrease the amount of GST-RNase III.
• esiRNA purification :
• 12. Prepare spin columns for purification as follows: add 200 ml of Q-Sepharose (slurry) to an empty spin column or to a Whatman 96-well filter plate (for large-scale synthesis). Put the column/filter plate in/on an empty 2-ml microcentrifuge tube or deep-well plate. Add 500 ml of equilibration buffer to the column. Centrifuge at 1,000g for 1 min and discard the flowthrough. Add another 500 ml of equilibration buffer, centrifuge at 1,000g for 1 min and discard the flowthrough.
• 13. Load all of the digested dsRNA onto the column and incubate for 5 min at 15-25 °C.
• 14. Centrifuge at 1,000g for 1 min and discard the flowthrough.
• 15. Add 500 ml of wash buffer, centrifuge at 1,000g for 1 min and discard the flowthrough.
• 16. Add 300 ml of elution buffer (270 l for deep-well plates), centrifuge at 1,000g for 1 min and collect the flowthrough in a new microcentrifuge tube or deep-well plate.
• 17. Repeat Step 16.
• 18. Add 500 ml of isopropanol (400 l for deep-well plates), seal and vortex. Store at -20 °C for at least 1 h. The isopropanol-esiRNA mixture can be stored at -20 °C for at least 3 d.
• 19. Centrifuge at 16,000g for 15 min at 4 °C (or at 4,400g for 60 min at 4 °C for deep-well plates). Gently discard the supernatant and wash the pellet with 500 ml of cold 70% (vol/vol) ethanol. Care should be taken not to lose the pellet when decanting isopropanol after centrifugation. The same applies to Steps 20 and 21
• 20. Centrifuge at 16,000g for 5 min at 4 °C (or at 4,400g for 5 min at 4 °C for deep-well plates). Gently discard the supernatant and wash the pellet with 500 ml of cold 70% (vol/vol) ethanol.
• 21. Centrifuge at 16,000g for 5 min at 4 °C (or at 4,400g for 5 min at 4 °C for deep-well plates). Gently discard the supernatant and dry the esiRNA pellet in a Speed Vac.
• 22. Dissolve the pellet in 100 ml of water.
• 23. Mix 3 ml of purified esiRNA with 3 ml esiRNA loading dye and check on a 4% (wt/vol) agarose gel with an appropriate marker.
• 24. Measure OD₂₆₀ to quantify the esiRNA concentration. For quantification of esiRNA, we routinely use the extinction factor of dsDNA. The average esiRNA yield should be 40 mg.
• 25. Store the esiRNA at -20 °C. Under these conditions esiRNA can be stored for > 6 months with no detectable reduction in silencing efficiency.
• 26. The esiRNA can be used directly for the transfection of mammalian cells using appropriate transfection methods, such as lipofection and electroporation. The amount of esiRNA for each transfection depends on the cell type and the transfection method. For HeLa cells we routinely transfect with Oligofectamine (Invitrogen) 200-400 ng esiRNA/ml medium, that is, 20-40 ng for a single transfection in 96-well plates.
RNAi-based gene function analyses in mammalian cells require transient or stable knockdowns depending on protein half-life or the onset of downstream effects. shRNA expression vectors are the method of choice for persistent knockdown studies and positive selection screens^ref. For many experiments, however, transient gene silencing is desirable. The most commonly used mediator for transient RNAi is chemically synthesized siRNAs. The advantages of siRNAs over shRNA expression vectors are that they are more efficiently introduced into cells, are less toxic and offer far greater control over interfering dsRNAs at the cellular level. One major drawback of chemically produced siRNAs, however, is their high cost. The protocol described here for the production of esiRNAs offers a very fast and cost-efficient alternative. Initial comparative studies between siRNA and esiRNAs showed similar silencing efficiencies, with a knockdown efficiency of >70% at the mRNA level for >90% of all tested esiRNAs. EsiRNA technology is especially useful for the rapid generation of large libraries. Using the described protocol we have previously generated a library of 15,000 human esiRNAs^ref10 and another library of 25,000 mouse esiRNAs^ref
Web resources : companies offering systems and reagents for analysis of gene expression : Abgene, Active Motif, Agilent Technologies, AlleLogic, Biosciences Corp., Althea Technologies, Ambion, Apogent, Applied Biosystems, Artus Biotech, Beckman Coulter, Biochain, Biogene, BioGenex, Biognostik, BioHelix Corp. (NEB), Bioneer, Bio-Rad Laboratories, Biosearch Technologies, Biosource, Biotrove, Bio S&T, Brinkmann Instruments,
Chemicon International, Cepheid, Clontech (Takara Bio), Corbett Research, DxS Limited, Enzo Life Sciences, Epicentre, Epoch Biosciences, Eurogentec,
Exiqon, Fermentas Inc., Finnzymes, Fluoresentric, Genetic Research Instruments, Genetix, Gene Express, Gene Therapy Systems, Inc., GenScript, GenoMechanixm, GeneWorks, Genzyme Corporation, Idaho Technology Inc., Integrated DNA Technologies, Invitrogen, DNAform,Lark Technologies,
Lucigen, Luminex, Maxim Biotech Inc., MJ Research (Bio-Rad), Midwest Scientific, MobiTec Molecular Biotechnologies, Molecular Probes (Invitrogen),
MWG Biotech, New England BioLabs, Novagen (EMD Biosciences), Origene, Panomics, PGC Scientifics, PerkinElmer, Premier Biosoft International, Promega, Qiagen, Quantum Dot (Invitrogen), Roche Applied Science, Seegene, Serologicals Corporation, SeqWright, Sigma-Aldrich,
Sigma-Genosys, Solexa, Stratagene, SuperArray Bioscience Corp., Synthegen, TagSorter, Takara Mirus Bio, Thermo Electron Corporation, Techne, USB Corp., VTT
• molecular methods :
• SNP detection systems :
• dye-labeled oligonucleotide ligation (DOL) : method for detection of SNP using ligation reaction. Template is mixed with a universal downstream primer and two alternate upstream primers. The SNP allows the correct hybridisation of one upstream primer, and prevents ligation of the alternate primer. In the presence of ligase the down- and upstream oligo ligate to a new molecule having the correct fluorescent labels. Specific signal detection is based on FRET. Chen X., P.-Y. Kwok (1997) Template-directed dye-terminator incorporation (TDI) assay: A homogeneous DNA diagnostic method based on fluorescence resonance energy transfer. Nucleic Acids Res. 25:2347-353.
• microtiter array diagonal gel electrophoresis (MADGE) : an easily scored difference in the electrophoretic migration of a PCR product can be obtained if an SNP alters the recognition sequence for a restriction endonuclease. This principle has been used in the high through put, low-resolving microtiter array diagonal gel electrophoresis technique (MADGE). Here, restriction-digested PCR products are loaded on stackable horizontal gels with wells arranged in a microtiter format. The electric field is applied at an angle relative to the columns and rows of wells in the gel, allowing products from large numbers of reactions to be resolved between the downstream wells^ref.
• multiplexed allele-specific diagnostic assay (MASDA) : allele-specific oligo-nucleotide (ASO) hybridizations are frequently per-formed with either the DNA samples or the oligo-nucleotides arrayed on a surface, sometimes referred to as formats 1 and 2, respectively. Format 1 is suitable for screening many patient samples for the presence of a few disease-causing alleles, as in the multiplexed allele-specific diagnostic assay (MASDA), in which a pool of mutation-specific oligonucleotides are hybridized to spots of pooled PCR products from individual patients, and positive reactions are eluted and sequenced to determine which probe bound to a particular patient sample^ref.
• molecular beacons (MB) : these oligonucleotide hybridization probes have two complementary DNA sequences flanking the target-specific sequence, and a donor-acceptor dye pair is present at opposite ends of each probe. When not hybridized to a target sequence, the probes adopt a hairpin-loop conformation, bringing the fluorophore and quencher pair close together, thereby extinguishing the donor fluorescence. On the other hand, when hybridized to the correct target sequence, the two dyes are separated and fluo-rescence increases by up to 900-fold. A valuable side effect of the preorganized hairpin probe design is that mismatch hybridization is further destabilized, providing increased allele selectivity in SNP analyses^{ref1, ref2}.
• oligonucleotide ligation assay (OLA) : in the OLA, the sequence surrounding the SNP is first amplified by PCR, whereas in LCR, genomic DNA can be used as a template. By use of allele-specific ligation probes differentially labeled with lanthanide chelates, and a multipronged solid support for parallel sample handling, sets of ligation reactions can be conveniently analyzed via time-resolved fluorometry^ref. Dual-color detection of allele-specific ligation products is also possible in a regular spectrophotometer^ref
• TaqMan : the TaqMan assay takes advantage of the 58-nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interact via FRET. Cleavage of the TaqMan probe dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time during PCR^ref.
• hybridization based marker :
• cleavase fragment length polymorphism (CFLP) : technique for mutation detection in DNA stetches with unknown sequences. The method is an alternative to SSCP or DGGE. The principle relies on denaturation (melting) of DNA. The ssDNA will assume folded hairpin-like structures which are unique to the nucleotide sequence. The endonuclease cleavase I specifically cleaves the junction between ss and ds regions. Fragments are 5' labelled, electrophoretically separated and visualized. Polymorphic fragments indicate a mutation; their length is indicative for the position of the mutated site. Brow M.A.D. et al (1996) Focus (Life technology) 18(1):2-5
• genomic mismatch scanning (GMS) :
• RNase A mismatch mleavage method (RAMCM) : for detecting single base substitutions in rna:rna or rna:dna hybrids with ribonuclease A^{ref1, ref2}.
• restriction fragment length polymorphism (RFLP) : a DNA fragment used to probe Southern blots of restricted genomic DNA from different strains of the same species. This results in the visualisation of variation in the size and/or number of detected restriction fragments generated from the different strains. The detected length variation is based on DNA sequence variation caused by insertions, deletions or changes in restriction sites^ref. Soller M. and Beckmann J.S. (1983) Genetic polymorphism in varietal identification and genetic improvements. Theor. Appl. Genet. 67:25-33
• allele specific sequencing using restriction enzyme and biotinylation (ASSURE B) : direct sequencing of PCR products to reveal mutations is only possible if the PCR product represents the target allele. If the PCR mixture is a heterogeneous mix of several alleles this method allows allele specific sequencing. Biotinylated PCR products are digested with a frequent cutter. The unaffected allele is captured and PCR-able because it has both primers sites and is captured on the magnetic beads^ref.
• differential display reverse transcriptase-PCR (DDRT-PCR) / differential display polymerase chain reaction (DD-PCR)  : this technique enables the amplification of differentially expressed mRNA deriving from identical genotypes exposed to different treatments^ref. It uses PCR to amplify and display cDNAs derived from the mRNAs of a given cell or tissue type. First-strand-synthesis is primed with an anchored primer complementary to 13 nucleotides (nt) of the poly(A) tail of mRNA and the adjacent 2 nucleotides of the transcribed sequence. Anchored primers therefore anneal to the junction between the poly(A) tail and the 3'-untranslated region of mRNA templates. A second primer, an arbitrary sequence of 10 nt, is then added to the reaction mixture, and double-stranded cDNAs are produced by PCR carried out at low stringency. By comparing the banding patterns of the resulting cDNA products, it is sometimes possible to identify the products of differentially expressed genes. Procedure^{ref1, ref2} :
• optimization of amplification conditions :
• 1| set up a series of trial PCR to estabilish the optimum concentrations of "control" and "test" RNAs required to produce a pattern of 100-300 amplified cDNA bands after gel electrophoresis and autoradiography. Make 5-fold serial dilutions of the total RNA preparations in H₂O to produce concentrations between 1 mg/ml and 100 mg/ml.
• 2| choose one or more anchored 3' oligonucleotides and set up a series of annealing reactions that combine 8 ml of template RNA (1-100 mg/ml) with 2 ml (6 pmol) of anchored 3' primer
• 3| add the following to the annealing reactions
• 5X reverse transcruptase buffer : 4 ml
• 100 mM dithiothreitol : 2 ml
• 200 mM deoxynucleoside triphosphate (dNTP) solution : 2 ml
• placental RNAse inhibitor (25 units/ml) : 0.25 ml
• reverse transcriptase (200 units/ml) : 0.25 ml
• water : to 20 ml
Incubate at 37°C for 1 h, then at 97°C for 10 min to inactivate the reverse transcriptase. Control for contaminating DNA by setting up one or more reactions without reverse transcriptase and carry through step 7. If necessary, treat the RNA with RNase free DNase I
• 4| set up 2 series of 8 0.5 ml amplification reactions. Each tube should contain the following :
• 10X amplification buffer : 2 ml
• anchored 3' oligonucleotide primer : 2 ml
• dNTPs (20 nM solution containing all 4 dNTPs) : 1 ml
• [a-³³P]dATP (deoxyadenosine triphosphate) or [a-³⁵S]dATP (3,000 Ci/min) : 1 ml
• water : 9 ml
• thermostable DNA polymerase (5 units/l) : 1 unit
To each tube, add 2 ml of a different arbitrary 5' primer. Mix by tapping the sides of the tubes.
• 5| into one series of 8 tubes, dispense 3-ml aliquots of the reverse transcriptase reaction containing the test RNA. Into the other series of 8  tubes dispense 3-ml aliquots of the reverse transcriptase reaction containing the control RNA. Mix the contents and place in the thermal cycler.
• 6| amplify the nucleic acids using the following program :

cycle number	denaturation	annealing	polymerization
1-30	15 s at 94°C	30 s at 42°C	15 s at 72°C
31	15 s at 94°C	30 s at 42°C	2 min at 72°C

Times and temperatures may need to be adapted to suit the particular reaction conditions
• 7| separate the radiolabeled products by electrophoresis through an electrolyte gradient polyacrylamide gel of the type used for DNA sequencing. Continue electrophoresis at constant electrical power until the tracking dye has migrated about two thirds of the length of the gel. Dry the gel and expose it to autoradiographic film. Examine the pattern of DNA bands; a good display contains between 100 and 250 well-resolved bands. Select the conditions that work well with the largest number of primer pairs.
• amplification of cDNA for differential dysplays
• 8| repeat the annealing, reverse transcription and amplification reactions using all combinations of primer pairs and the optimum amount of RNA templates and separate products as in step 7
• 9| using the autoradiograph as a guide, cut target bands from the dried gel. Soak each gel sliver overnight in a separate 0.5-ml tube containing 50 ml of sterile H₂O.
• 10| puncture the base of each tube with a small-gauge needle and place each tube inside a 1.5-ml microcentrifuge tube. Centrifuge for 20 s to transfer the eluate into the larger tube
• 11| amplify the eluted fragment in a reaction containing the following :
• 10X amplification buffer : 2 ml
• DNA eluted from polyacrylamide gel : 3 ml
• arbitrary 5' oligonucleotide primer : 2 ml
• anchoring 3' oligonucleotide primer : 2 ml
• 20 mM solution of 4 dNTPs (pH 7.0) : 1 ml
• water : 9.5 ml
• Taq thermostable DNA polymerase (5 units/ml) : 2 units
• 12| Place the tubes in a thermal cycler and amplify the nucleic acids using the program from Step 6. Polymerization should be carried out for 1 min for every 1,000 base pairs of length of the target DNA
• 13| estimate the concentration of the reamplified DNA fragment by analyzing 5-10% of the product on a 1% agarose gel
• 14| amplified DNA products can now be ligated into a dT-tailed vector for transformation into Escherichia coli and further analysis
• an in vitro linkage technique based on screening approximately HAploid amounts of DNA by the PolYmerase chain reaction (HAPPY) : how HAPPY mapping of bacterial genomes works: Intact genomic DNA is broken by irradiation (I) to give a pool of random fragments, which are size selected by PFGE (II). A mapping panel of 96 aliquots is taken (III) from this pool. Each aliquot contains ~1 haploid genome's worth of DNA, so each marker is present in only a subset of the aliquots. (IV) The panel is pre-amplified a few hundred-fold by primer extension preamplification (PEP). (V) Subfractions of the pre-amplified panel are screened for specific markers using nested PCR. Markers A and B, (e.g.), are found to cosegregate frequently. This implies that they tend to lie on the same fragment of DNA, and so must be tightly linked. A map may be constructed (VI) by examining cosegregation frequencies between all markers. he number of polymorphic bands resembling the recurrent parent are use as the selection criterium^ref.
• marker addition through substraction (MATS) : a procedure based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs). This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome. Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis^ref.
• primed in situ labelling (PRINS)
• representational display analysis (RDA)
• restriction enzyme mediated integration (REMI) : method to stimulate transformation. The enzyme enters the cell during electroporation and facilitates intergration of linearized foreign DNA with compatible einds into the host chromosomes^ref
• restriction enzyme mediated integration - restr. fragm. length polym. (REMI-RFLP) : length differences caused by REMI are used as a polymorphic marker locus^ref.
• rapid isolation of c-DNA by hybridization (RICH) : method depends on solution hybridization, enzymatic modification and amplification/ selection of sequences present in both cDNA populations and the genomic clones^ref.
• strand displacement amplification (SDA) : DNA is amplified by a DNA-polymerase lacking exo-nuclease activity. Therefore, the newly synthesized strand displaces the former strand. The DNA-polymerase can start again from a single-stand nick generated by a restiction enzyme. The other strand is protected from the restriction enzyme by modified nucleotides incorporated during DNA synthesis^ref
• miscellaneous :
• bacterial artificial chromosome (BAC) : cloning vector for large DNA fragments^ref.
• yeast artificial chromosome (YAC) :
• plant artificial chromosome (PAC) :
• P1-derived artificial chromosome (PAC) : one type of vector used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia coli cells. Based on bacteriophage (a virus) P1 genome.
• bulked segregant analysis (BSA) : a rapid mapping strategy suitable for monogenic qualitative traits. When DNA of ten plants are bulked into one pool, all alleles must be present. 2 bulked pools of segregants differing for one trait, will differ only at the locus harbouring that trait^ref.
• genomic selection (GS) : the number of polymorphic bands resembling the recurrent parent are use as the selection criterium^ref
• log odds ratio (LOD) : a statistical measure indicating the significance of linkage. The log₁₀ of the odds ratio. The odds ratio (OR) is the probability (given H₀) devided by the probability (given H_a = unlinked)^ref
• marker assisted selection (MAS) : a breeding strategy applying indirect selection
• marker assisted recurrent selection (MARS) : a breeding strategy with the repeated application of indirect selection
• miniature inverted-repeat transposable element (MITE) : a class of transposable elements, which share features of both class I and class II elements, and, therefor, remain unclassified. The transposons in this group share structural, but not sequence similarity^ref.
• methylation sensitive amplification polymorphism (MSAP) : a class of transposable elements, which share features of both class I and class II elements, and, therefor, remain unclassified. The transposons in this group share structural, but not sequence similarity^{ref1, ref2}
• polymorphic information content (PIC) : a measure for the probability of polymorphism at a (STMS-) locus, calculated from the number of alleles and their respective allele frequencies in a population^ref.
• quantitative trait locus (QTL) : single locus from a series of polygenes which are involved in a quantitative trait^ref

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