HYBRIDIZATION CAPTURE OF MICROSATELLITES DIRECTLY FROM GENOMIC DNA

A rapid approach for isolation of microsatellites and other tandem rep eated sequences in described. The method is based on hybridization cap ture of repetitive elements from digested genomic DNA using biotinylat ed oligonucleotide probes in solution and subsequent attachment to mag netic beads coated with streptavidin. Captured fragments are amplified by adapter polymerase chain reaction (PCR) and the PCR products enric hed for microsatellites cloned directly into a T-vector for sequencing . The results presented here show that this approach is highly effecti ve, allowing di-and trinucleotide repeats to be isolated and sequenced directly from fish and mammalian genomic DNA within four to five days . Assuming a density and relative abundance of repeats with AC/GT moti fs corresponding to that found in the human genome, the protocol prese nted gives at least a 35-fold enrichment of AC/GT microsatellites usin g an (AC)(10) oligo probe. In addition, four out of five sequences cap tured by a (CAG)(9) oligo probe contained one or several CAG repeat ar rays. The efficiency of this direct approach suggests that it can be u sed for extracting other types of tandem and interspersed repeated seq uences (including transposons, rRNA and tRNA genes and proviruses) fro m vertebrate genomes.