A Modified Enrichment Method to Construct Microsatellite Library from Plateau Pika Genome (Ochotona curzoniae)

• Published in: Genomics, proteomics & bioinformatics proteomics & bioinformatics, 2010-03, Vol.8 (1), p.72-76
• Main Authors: Geng, Jianing, Li, Kexin, Zhang, Yanming, Hu, Songnian
• Format: Article
• Language: English
• Subjects: enriched library; Escherichia coli; Haliotis; Haliotis diversicolor; magnetic bead; Method; microsatellite; Ochotona; ultrasonication
• ISSN: 1672-0229; 2210-3244
• DOI: 10.1016/S1672-0229(10)60007-1

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.