Rapid isolation of DNA sequences flanking microsatellite repeats

Genetic linkage analysis is routinely used to focus on a defined region of DNA containing a gene of interest. This strategy requires the isolation of highly informative markers either from YACs, cosmids, P1 clones, or other vectors such as lambda . The contemporary approach is to isolate sequence flanking polymorphic microsatellite repeats d(C.A) sub(n) for the polymerase chain reaction (PCR). The process of identifying suitable clones containing repeats, is a lengthy one, and requires serial rounds of screening with radiolabelled d(C.A) sub(n) heteropolymer and subcloning of positive fragments derived from libraries, followed by sequencing. A method is described which enables the rapid and efficient subcloning of fragments from small amounts of P1 clone DNA, and which contain CA-repeats of a size suitable for sequencing. The technique can also potentially be applied to YACs or cosmids, and is adapted from a linker-based method used to enrich for cDNA clones.