Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites

Microsatellite loci are highly informative genetic markers useful for popul ation genetic studies,linkage mapping and parentage determination. Methods to identify novel microsatellite loci commonly use subtractive hybridizatio n to enrich small-insert genomic libraries for repeat sequences. A critical step in enrichment is attachment of an oligonucleotide linker,to genomic D NA fragments so that repeat-containing sequences can be recovered by PCR fo r cloning. Current linkers and ligation methods rely on single restriction enzymes to size-fraction genomic DNA and generate complementary ends. These restriction enzyme/linker combinations ave often species-specific, give po or recovery of repeat-enriched DNA and yield library inserts that are not a broad sample of the genome. We have developed a blunt-end linker; named SN X for its restriction sites, that allows the use of combinations of restric tion enzymes to digest the majority of genomic DNA into the 200-1000-bp ran ge. SNX is attached to genomic DNA with a simultaneous ligation/restriction reaction that is highly efficient and improves recovery of sequences after subtractive hybridization. SNX can be used for microsatellite enrichment i n any species, since ligation is independent of the restriction enzymes use d to size-fraction genomic DNA. These methods improve current repeat-enrich ment strategies, resulting in representative small-insert libraries with a very high proportion of positive clones.