A Modified Enrichment Method to Construct Microsatellite Library from Plateau Pika Genome(Ochotona curzoniae)
10.1016/S1672-0229(10)60007-1
- Author:
Geng JIANING
1
;
Li KEXIN
;
Zhang YANMING
;
Hu SONGNIAN
Author Information
1. CAS Key Laboratory of Genome Sciences and Information,Beijing Institute of Genomics,Chinese Academy of Sciences,Beijing 100029,China
- Keywords:
microsatellite;
enriched library;
magnetic bead;
ultrasonication
- From:
Genomics, Proteomics & Bioinformatics
2010;8(1):72-76
- CountryChina
- Language:Chinese
-
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.
