Hybridization capture of microsatellites directly from genomic DNA.
A rapid approach for isolation of microsatellites and other tandem repeated sequences in described. The method is based on hybridization capture of repetitive elements from digested genomic DNA using biotinylated oligonucleotide probes in solution and subsequent attachment to magnetic beads coated with streptavidin. Captured fragments are amplified by adapter polymerase chain reaction (PCR) and the PCR products enriched for microsatellites cloned directly into a T-vector for sequencing. The results presented here show that this approach is highly effective, 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 motifs corresponding to that found in the human genome, the protocol presented gives at least a 35-fold enrichment of AC/GT microsatellites using an (AC)10 oligo probe. In addition, four out of five sequences captured by a (CAG)9 oligo probe contained one or several CAG repeat arrays. The efficiency of this direct approach suggests that it can be used for extracting other types of tandem and interspersed repeated sequences (including transposons, rRNA and tRNA genes and proviruses) from vertebrate genomes.
Refseth
U H
UH
University of Oslo, Department of Biology, Norway. u.h.refseth@bio.uio.no
Fangan
B M
BM
Jakobsen
K S
KS
D000818:Animals; D019921:Biotinylation; D004247:DNA / Q000032:analysis*; D005589:Foxes; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D018598:Minisatellite Repeats; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012474:Salmon
D015345:Oligonucleotide Probes; D004247:DNA