%0 Journal Article
%T A simple and efficient method for isolating polymorphic microsatellites from cDNA
%A Gen Yue
%A Ze Zhu
%A Chun Wang
%A Jun Xia
%J BMC Genomics
%D 2009
%I BioMed Central
%R 10.1186/1471-2164-10-125
%X The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65¡ãC for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ¡À 0.54, while the expected heterozygosity was 0.56 ¡À 0.03. All the isolated microsatellites inherited in a Mendelian pattern.Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.Microsatellites are short segments of DNA in which a specific motif of 1¨C6 bases is repeated [1,2]. Due to their high polymorphism, codominant inheritance, ease of scoring and dense distribution throughout eukaryotic genomes, microsatellites are now generally considered to be the most powerful genetic markers for genetic mapping and evolutionary studies [3]. One perceived difficulty with microsatellites is the long lead time in identifying and characterizing microsatellites in new taxonomic groups. This problem is alleviated by developing novel protocols for enriching repeat DNA from genomic DNA [4]. However, most microsatellit
%U http://www.biomedcentral.com/1471-2164/10/125