Fluorescein PAGE analysis of microsatellite-primed PCR: a fast and efficient approach for genomic fingerprinting.

partial digests are difficult to control and are not feasible when the number of internal sites is high. A second solution would be to amplify the desired region with PCR using a primer set that flanks the terminal EcoRI and HindIII sites. In this case, a unique restriction site would be incorporated into the downstream primer in such a way that HindIII digestion would not be required for the cloning of the PCR product. Typically, this approach results in the addition of a portion of the primer sequence to the end (the HindIII-end in this case) of the desired DNA fragment. This would be problematic if, for example, one was trying to reconstitute an open reading frame (after subcloning of the PCR product into a vector) that ran through the terminal HindIII site. Here, we present a class of restriction enzymes, referred to as the BbvI class, that permits specific cleavage of DNA at an otherwise non-unique restriction site. A partial list of such enzymes is shown in Table 1. All of the BbvI class enzymes cut DNA outside of their recognition sequences. As illustrated in Figure 1B, the proximity of the BbvI recognition sequence to the terminal HindIII restriction site allows digestion with BbvI equivalent to that with HindIII. Note that the recognition sequence of BbvI and other enzymes of its class are non-palindromic. Thus, depending on the orientation of the recognition sequence within the primer, they are useful when incorporated into either the upstream or downstream primer for PCR. The non-palindromic nature of these recognition sequences also avoids problems associated with the inclusion of possible secondary structures into primers. The recognition sequences of most of the BbvI class enzymes are five or six nucleotides. With many such enzymes from which to choose, it should not be difficult to find one that is unique to the region of interest. The only obvious limitation of the BbvI enzyme class is that it does not include any enzymes that give a fournucleotide 3′ overhang or a two-nucleotide 5′ overhang. Thus, these enzymes are not viable alternatives for cleavage of the recognition sites of PstI (5′-CTGCA▼G), ClaI (5′-AT▼CGAT) and related enzymes. Address correspondence to Dr. Robert S. Cohen, University of Kansas, Dept. of Molecular Biosciences, Division of Biology, Haworth Hall, Lawrence, KS 66045-1630, USA. Internet: [email protected]