A modified enrichment method to construct microsatellite library from plateau pika genome (Ochotona curzoniae).
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.
Geng
Jianing
J
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China.
Li
Kexin
K
Zhang
Yanming
Y
Hu
Songnian
S
D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D015723:Gene Library; D016678:Genome; D015698:Genomic Library; D007793:Lagomorpha / Q000235:genetics*; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction
D004247:DNA
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