Recombinant DNA sequences generated by PCR amplification.

Abstract

The polymerase chain reaction (PCR) has greatly enhanced the field of molecular biology by making numerous regions of the genome (coding and noncoding), in both extant and extinct taxa, accessible for detailed analysis. PCR is especially well suited for applications in systematic biology because conserved regions that flank variable portions of the genome can be used as primer sites for the amplification and sequencing of variable regions from a wide variety of species (see Palumbi 1996). In addition, PCR has been used successfully to examine polymorphisms within populations. Although many advantages are offered by PCR, the technique is not error-free. In fact, the replication-like process that drives PCR can lead to erroneously amplified products. For example, recombinant amplification products (Saiki et al. 1988; Scharf 1990) can be produced in vitro when amplifying elements of multi-gene families. Thus, the amplified product may not represent a sequence that actually exists in a single continuous stretch of DNA within any organism. Although this phenomenon was reported shortly after the development of PCR, it appears to be poorly known by many evolutionary biologists who use PCR in studies of gene evolution. Here we show that in vitro recombination also can occur when amplifying alleles of single-gene loci in heterozygous individuals, and that recombination frequency can vary as a function of using different polymerases in the extension step of PCR. In vitro recombination occurs when DNA polymerases incompletely extend a segment of DNA and the partially amplified sequence acts as a primer during subsequent amplification cycles (fig. 1). If the partially amplified segment hybridizes to an alternative form of a template strand, then subsequent PCR products will be recombinants of the two original template sequences. Partial amplification is most likely to occur when polymerases pause or prematurely disassociate from the template strand during the extension reaction, although the recombination may also be enhanced by short extension cycles or long amplifications. Although this phenomenon was initially identified in amplification of multi-gene families (Scharf, Long, and Erlich 1988; Scharf et al. 1988), we identified products of in vitro recombination in pocket gophers (Geomys) that were heterozygous for the alcohol dehydrogenase1 (Adh1) locus (Bradley et al. 1993). This experiment was designed to determine the DNA sequences of three Adh1 alleles (designated K, M, and N) that had been identified from allozyme studies. Initially, we determined the sequences of