Bandstab: A PCR-Based Alternative to Cloning PCR Products

In many DNA amplification reactions, extra fragments arising from nonspecific mispriming, a mixture of target templates or allelic variation may be generated in addition to the desired product. Some nonspecific bands can be reduced by more stringent annealing conditions, but in cases of allelic variation or other mixed targets, the amplification products may be of the same length but differ by only a single base in the DNA sequence. In these situations, it is often desirable to purify the expected product(s) from contaminating fragments or alleles in order to undertake further analysis such as direct DNA sequencing. The conventional cloning-based approach for preparing a pure template for DNA sequencing involves ligation of the polymerase chain reaction (PCR) products (usually gel-purified) into an appropriate vector, followed by transformation and then propagation in a suitable host (4). Several steps are required to generate the recombinants, after which it is necessary to sequence several independent clones to ensure that any observed base changes reflect genuine allelic variation and not mistakes introduced during the PCR, especially if Taq DNA polymerase is used in the amplification (3). Problems with Taq DNA polymerase-induced errors can be minimized by using high-fidelity amplification systems such as the GeneAmp® XL PCR Kit (PerkinElmer, Norwalk, CT, USA) and the Expand High Fidelity PCR System (Boehringer Mannheim, Indianapolis, IN, USA). It is possible to isolate pure species of PCR products by electrophoretically separating the mixture on the basis of length or shape (single-strand conformation polymorphism [SSCP]) and then eluting the bands of interest from the gel slice. A simpler and more efficient approach is to stab the band(s) of interest with a pipet tip(s) and use this to inoculate a second round of DNA amplification. This procedure, called “bandstab”, can readily generate sufficient material for direct DNA sequencing, for use as a hybridization probe or for other detailed analysis. This bandstab approach requires much less effort and is quicker and easier than even band slicing or excision techniques, where the desired band(s) is cut out of a gel with a scalpel blade, and the gel slices are transferred to another tube before elution or other manipulations. The limitation on how many fragments can be bandstabbed from a gel is often determined by the number of individual PCRs that can be set up conveniently. This bandstab approach is easier, faster and more convenient than cloning, and the ability to analyze a product directly from a gel can help to overcome any problems encountered with errors that may have arisen during the amplification reaction. This report provides two examples where the bandstab method was used to select and re-amplify individual bands from a mixture of amplification products after electrophoretic separation based on SSCP analysis (5) and length. DNA fragments may be detected and bandstabbed after staining in ethidium bromide (EtdBr; 0.5 μg/mL) and visualization on a transilluminator or after silver staining. Since the bandstab procedure can be carried out very quickly, there is minimal exposure of the PCR products to damage from the ultraviolet light. Cross-contamination with other PCR products was not a problem if fresh EtdBr was used each time and an excessive number of thermal cycles was not carried out in the re-amplification. In situations where EtdBr staining was not sufficiently sensitive (short DNA fragments or single-stranded material such as that found after SSCP analysis), a simple silver staining method was used where the polyacrylamide gel was soaked in approximately 20 vol of 10% ethanol for 3 min, 1% nitric acid for 3 min and 0.1% silver nitrate for 8 min. The gel was kept in developer (6% sodium carbonate, 0.12% formaldehyde) until a satisfactory signalto-background ratio was achieved (usually 4–5 min), at which time the reaction was stopped by placing the gel in 10% acetic acid for 10 min. (The gel was rinsed twice with distilled water between each solution). Although it is preferable to carry out the bandstab reamplification soon after the gel has been stained/developed, we have reamplified fragments from silver-stained gels that had been developed 48 h earlier. In cases where the silver-stained gel could not be bandstabbed immediately, the gel was rinsed in water and then kept in a 1% glycerol solution in subdued light at room temperature. Bandstab Amplification. After gel fractionation and staining (either EtdBr or silver), the gel was rinsed briefly with distilled water before a sterile micropipet tip (yellow P200 tip; Quality Scientific Plastics, USA) was used to stab the band of interest. A single stab contained sufficient material for re-amplification from either polyacrylamide or agarose gels. The pipet tip was left in