An Efficient Procedure for Isolating Microsatellite DNAs from Sea Cucumber (Apostichopus japonicus)

摘要

The construction of enrichment library proves to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of sea cucumber was digested with HaeⅢ and size-selected DNA fragments (250-700bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of GA and CA probes, which were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 700 clones. Two hundred and thirty-two clones reacted positively after the library screening procedure. Of the 50 clones sequenced, all contained at least one microsatellite and one duplicate clone was found. Approximately 86% of the sequenced fragments permitted to design primers for sequence tagged microsatellite site (STMS).