A modified enrichment method to construct microsatellite library from plateau pika genome (Ochotona curzoniae)

Abstract

A microsatellite-enriched library of plateau pika (Ochotona curzoniae) was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster (Cricetulus griseus) and small abalone [Haliotis diversicolor (Reeve)] with high rates of positive clones, demonstrating its feasibility and stability.

Figure 1

Hybridization-enriched DNA band. The microsatellite-containing DNA fragments were captured by magnetic beads, and single-stranded DNA was amplified in a single-primer PCR. PCR products (Lane 1) were check up on 1% agarose gel with a DL2000 (Lane 2) marker as standard.

Figure 2

Experimental flow for microsatellite-enrichment library construction.