Restriction analysis of polymerase chain reaction (PCR) products is one of the earliest techniques used for analyzing amplification products (1). This approach is applicable for distinguishing alleles in which the polymorphic residue results in the creation or removal of a restriction enzyme site. Unfortunately, many polymorphisms are not associated with restriction enzyme site change and thus are not amenable to this analysis. However, by using site-directed mutagenesis using primers with mismatches near the 3' ends, it is possible to create an artificial restriction fragment length polymorphism (A-RFLP) for almost all naturally occurring DNA polymorphisms 2-5. Figure 1 illustrates the principles of this approach. Fig. 1. Principles of artificial RFLP. Reprinted with permission from ref. 6.
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