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 quantific