Sequencing PCR Products

The products of Polymerase Chain Reaction (PCR) can have structures which make them difficult to sequence. One of the most common problems associated with sequencing of PCR products is the presence of stops or BAFLs, where the sequence pauses or stops at artifactual ends in the template (actually the ends of truncated PCR product). This kit incorporates label by way of a radiolabeled dideoxy terminator so that only the fragments which were properly terminated are visible in the sequence. No labeled bands are formed at ‘ends’ in the template, eliminating many of these artifacts and enabling sequences to extend to essentially the last base of a PCR product. Artifacts caused by appearance of double-stranded PCR product on denaturing gels are similarly eliminated since they are not labeled. Following is information which should assist in producing high quality, reliable sequence information even with PCR product templates which have been very difficult to sequence with standard methods.

It is essential that PCR products are of high quality and quantity in order to obtain high quality sequence information. Problems with high background, low signal intensity and ambiguities can often be traced to the PCR step. Not every PCR will yield a product which can be sequenced. Analysis of the PCR product on agarose gels and optimization of the PCR may be necessary to obtain quality sequences.

Enzymatic pre-treatment of PCR products

The key step in this method for sequencing PCR products consists of treating the PCR product with a combination of Exonuclease I and Shrimp Alkaline Phosphatase∞ to eliminate any primer or dNTPs which were not incorporated into the PCR product. These enzymes are available from USB in a reagent pack (70995) or pre-mixed (ExoSAP-IT™, 78200) with detailed protocols for their usage. It is recommended that this enzymatic clean-up of the PCR product be used with this sequencing method.

Elimination of compressions

Some DNA sequences, especially those with dyad symmetries containing dG and dC residues, are not fully denatured during electrophoresis. When this occurs, the regular pattern of migration of DNA fragments is interrupted; bands are spaced closer than normal (compressed together) or sometimes farther apart than normal and sequence information is lost. The substitution of a nucleotide analog (dITP) for dGTP which forms weaker secondary structure has been successful in eliminating most of these gel artifacts (18, 19). Two examples are shown in figure 2 in the sequences run with dGTP.

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