A Novel Anchored PCR-Based Strategy For Microsatellite Isolation

Microsatellite DNA is a widely used genetic marker in gene mapping, population geneticanalysis, evolution and phylogenetics, cancer mechanism, QTL identifiaction, molecularassisted breeding and forensic science. The application requires enoumers microsatellitesisolated from genome of target species. Despite of extensive employement, current strategies ofmicrosatellite isolation still possess a pack of drawbacks, including high cost and technicalrequirement in library construction, extremely short flanking sequences left due to randomdistribution of cutting sites of restriction enzymes, as well as economic and labour consumingdue to high repeatativity of a same microsatellite. We here created a novel anchored-PCR basedstrategy for microsatellite isolation. The application of this strategy in isolating microsatellitesfrom Amur tiger genome proved a high efficiency and overcoming of drawbacks as possessedby other strategies.In this strategy, a single oligo primer Str-ACN designed in the repeating region with ananchoring nucleotide was used to amplify lower stream sequence of multiple microsatellites.Terminal Deoxynucleotidy Transferase was used to add a poly C tail to the PCR products. Asecond primer GG-N complement to the poly C tail was used with Str-ACN together to amplifyPCR products for a second round. PCR products were then subjected to cloning andsequencing. Secondly, a reverse primer Nest 1 was designed on the down stream sequence ofmicrosatellties that is cloned as above to amplify its upper stream sequence, and PCR productswere subjected to the similar procedure as first round cloning. Finally up and down streamsequences were aligned to produce full sequence of a microsatellite. 147 differentmicrosatellite loci were obtained from the 149 sequenced positive clonies in the first roundcloning. Twenty-four microsatellites with full repeating region and flanking sequences wereobtained from the second round cloning of 26 randomely chosen loci.