DNA template as a source of artifact in the detection of p53 gene mutations using archived tissue.

data not shown). Figure 1 demonstrates the two alleles of exon 15 of hMLH1 obtained with and without the modifications described above. The modified technique resulted in unequivocal resolution in the complete absence of artifactual bands. Representative samples of hMLH1 exon 15 were subcloned (TA Cloning® Kit; Invitrogen, San Diego, CA, USA) and sequenced following manufacturer’s instructions on the automated Model 373 Fluorescent Sequencer (PE Applied Biosystems, Foster City, CA, USA). Sequencing confirmed a single base-pair substitution resulting in the two alleles detected. In all cases, the modified asymmetric-PCR SSCP significantly improved resolution of different alleles compared to standard symmetric-PCR SSCP. Carryover of primers from symmetric PCR as well as using unequal portions of both primers in asymmetric PCR presumably resulted in amplification of both strands of DNA, thereby confounding the resolution of different alleles. The above described modifications of PCR product purification and use of one primer only in the asymmetric reaction eliminated unwanted bands, produced clear bands with easily distinguishable homozygotes and heterozygotes and yielded no artifacts. As with PCR cycle conditions, the choice of sense vs. antisense primer in the asymmetric step is determined empirically. The asymmetric-PCR SSCP modifications described here consistently and reproducibly result in improved amplification of DNA segments with much improved resolution of products in the absence of artifacts. This technique allows unambiguous assignment of genotypes and can clarify or confirm findings of other techniques of DNA polymorphism analysis.