Hybridization capture of microsatellites directly from genomic DNA

Abstract

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.

Construction Fractional Genomic Libraries and Screening Microsatellites DNA of <I>Esox reieherti</I> Dybowski

“…As a result, 192 (97.96%) clones were found including microsatellite sequences, Quan (Quan et al, 2006) sequenced 173 clones and 178 (97.19%) clones included microsatellite sequence; Lu et al (2005) sequenced 138 clones and 149 (92.62%) clones included microsatellite sequences, prior to obtaining the ratio in the above results. As shown in this paper, 1300 positive clones were acquired from 1600 microsatellite clones, the ratio was 81.25%, far greater than congeneric results Quan et al, 2006;Lu et al, 2005;Refseth et al, 1997).…”

Short Communication Microsatellite markers for Amazon pellona Pellona castelnaeana (Clupeiformes: Pristigasteridae)

“…To assess the genetic parameters of Pellona in the Amazon Basin, we developed a partial genomic library of P. castelnaeana enriched for microsatellites using selective hybridization with biotinylated probe types (CT) 8 and (GT) 8 , conjugated to streptavidin-coated magnetic beads (Refseth et al, 1997). After hybridization, the microsatellite-enriched sequences were amplified by polymerase chain reaction (PCR), ligated in to the pGEM-T Easy Vector, and transformed into Escherichia coli XL1-BLUE electroporated competent bacteria.…”

Methods for Development of Microsatellite Markers: An Overview

“…These protocols generally involve the fragmentation of DNA either by sonication (Karagyozov et al, 1993;Kandpal et al, 1994;Geng et al, 2010) or restriction enzymes (Brown et al, 1995;Chen et al, 1995;Edwards et al, 1996;Prochazka., 1996;Refseth et al, 1997;Fischer and Bachmann, 1998;Hamilton et al, 1999;Glenn and Schable, 2005;Nunome et al, 2006) or nebulisation (Kumpatla et al, 2004;Connell et al, 1998) and its subsequent ligation to a known sequence (linker or adaptors) or directly to a vector. DNA is then denatured and subjected to enrichment by hybridization with a) biotinylated oligos followed by capture of biotinylated hybrids (oligo bound DNA fragments) in vectrex-avidin matrix (Kandpal et al, 1994) or b) oligonucleotides bound to nylon membrane (Karagyozov et al, 1993;Chen et al, 1995;Edwards et al, 1996) or c) 5′ biotinylated repeat oligos and subsequent capture of biotinylated hybrids by streptavidin coated magnetic beads (Brown et al, 1995;Refseth et al, 1997;Fischer and Bachmann, 1998;Connell et al, 1998;Hamilton et al, 1999;Kumpatla et al, 2004;Dixit et al, 2005;Glenn and Schable, 2005;Nunome et al, 2006;Geng et al, 2010) or d) 'biotinylated SSR probe-streptavidin coated magnetic bead complex' ('Triplex affinity capture' protocol; White and Powell, 1997). The enriched DNA fragments were then amplified, either cloned and sequenced or sequenced directly and searched for the presence of SSR motifs.…”