Isolation of microsatellite with enrichment by magnetic beads in grass carp, Ctenopharyngodon idella

Enrichment by magnetic beads is a simple and efficient method for rapid isolation of microsatellite DNA. In this experiment, we isolated microsatellite DNA from grass carp （ Ctenopharyngodon idella ） genome with this method. Grass carp genomic DNA was extracted and digested with restriction enzyme Sau3AI. Fragments of 400 - 900 bp were isolated and purified and then ligated withshort linkers （20 bp）. They were used to create a “whole genome PCR library”. This genomic DNA was hybridized with a biotin-labeled microsatellite probe （CA）15. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SRS fragments, the eluted single-stlanded DNA contains the selected microsatellite DNA. The selected DNAs are then amplified using primers designed complementary to the linkers, cloned into the pGEM - T vector and seq. uenced. In this experiment, we performed the second screening with a radiolabeled （CA）15 probe and obtained 132 positive clones. From these positive clones, we isolated 130 microsatellite sequences. This allowed us to design 83 pairs of primers with the software Primer Premier 5.0.