Application of a double-enrichment procedure for microsatellite isolation and the use of tailed primers for high throughput genotyping

Fábio Mendonça Diniz,Norman Maclean,Paul Bentzen,Paulo Sarmanho Da Costa Lima,Arati Iyengar

doi:10.1590/s1415-47572007000300014

Abstract

The number of microsatellite loci and their allelic diversity contribute to increase accuracy and informativity of genetic estimates, however, the isolation of microsatellite loci is not only laborious but also quite expensive. We used (GATA)n and (GACA)n tetranucleotide probes and single- and double-enrichment hybridization to construct and screen a genomic library with an increased proportion of DNA fragments containing repeat motifs. Repeats were found using both types of hybridization but the double-enrichment procedure recovered sequences of which 100% contained (GATA)n and (GACA)n motifs. Microsatellite loci primers were then designed with an M13R-tail or CAG-tag to produce scorable PCR products with minimal stutter. The approach used in this study suggests that double-enrichment is a worthwhile strategy when isolating repeat motifs from eukaryotic genomes. Moreover, the use of tailed microsatellite primers provides increased resolution for compound microsatellite loci, with a significant decrease in costs.

Highlights

Microsatellites, known as simple sequence repeats (SSRs), are present throughout the eukaryotic genome, often at high concentrations
In an effort to increase the proportion of genomic DNA fragments containing repeat motifs a simple and inexpensive methodology is described and optimized for the construction of an enriched genomic library
Microsatellite loci primers were designed on the unique flanking regions of each locus using PRIMER 3 (Rozen & Skaletsky, 2000) and an oligonucleotide tail corresponding to the 5’-GGAAACAGCTATGACCAT-3’ M13-R universal primer (Oetting et al, 1995; BoutinGanache et al, 2001) or the 5’-CAGTCGGGCGTCATC A-3’ CAG tag (Hauswaldt & Glenn, 2003) was added to the 5’ end of one primer of each pair to facilitate fluorescent labeling of the polymerase chain reaction (PCR) products during amplification

Summary

Introduction

Microsatellites, known as simple sequence repeats (SSRs), are present throughout the eukaryotic genome, often at high concentrations. In an effort to increase the proportion of genomic DNA fragments containing repeat motifs a simple and inexpensive methodology is described and optimized for the construction of an enriched genomic library.

Results

Conclusion