Rapid PCR-based characterization of sequences flanking microsatellites in large-insert libraries

Microsatellite polymorphisms are widely being employed as a major tool in genome analysis. Analysis of length polymorphisms in the PCR depends on the elucidation of the sequence of their flanking regions. Different methods have been developed for the efficient characterization of the flanking sequences. Small insert genomic libraries directly suitable for sequencing of the entire insert and highly enriched for the desired microsatellite sequence may be obtained by a number of strategies (Ostrander et al.; Karagyozov et al., 1993; Kandpal et al., 1994). The limitation of small insert libraries is, however, that the inserts are too small for physical mapping by in situ hybridization. Microsatellites from large insert genomic libraries suitable for the construction of both a genetic and a physical map are routinely analyzed by subcloning into small insert libraries. This elaborate approach may be circumvented by direct outward sequencing with a set of six primers complementary to the repeat motif having one of the six theoretically possible 3' flanking bases (Yuille et al., 1991). Disadvantages of the latter procedure are that for both flanks twelve sequence reactions are required, and that ambiguous results can be obtained if the repeat is imperfect, compound or palindromic. We have developed an alternative method that is more generally applicable to analysis of flanking sequences of microsatellites and have compared the results with those of outward sequencing according to Yuille et al. (1991).