Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome

Abstract

Trinucleotide (AGC)n microsatellites are found as 3' tails of the artiodactyl short insterspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)n microsatellites with 2-4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)n microsatellites. Enrichment for (AGC)n microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.