Microsatellite enrichment by magnetic beads in Silurus meriaionalis

Microsatellite enrichment by magnetic beads is a fast and efficient method for isolating this molecular genetic marker.Some microsatellite markers of Silurus meriaionalis were obtained by using the method.First,the DNA was cut by the enzyme of Sau3AI,and targeted segments were collected with the size of 400-900 base pairs by centrifugation of sucrose density gradient.Then the segments were purified in a low melting agarose gel and ligated with a short linkers(20 bp),from which,the genomic PCR library was created.These DNA fragments were hybridized with a biotin-labeled SRS(simple repeat sequence) 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-stranded DNA contained the selected microsatellite DNA.The selected DNAs were then amplified using primers designed complementary to the linkers,cloned into the pMD18-T vector and transformed into competent E.coli DH5α.In this experiment,we performed the second screening with a radio labeled(CA)_(15) probe and obtained 178 positive clones.From these clones,173 microsatellite sequences(about 97.19%) were isolated, among which 90.60% microsatellites repeated more than 10 times and 75.98% microsatellites were perfect repeats.This allowed us to design 120 pairs of primers with the software Primer Premier 5.0.In addition,40 pairs of primers were composed and screened.38 pairs were used successfully to amplify special fragments,among which 31 pairs were polymorphism within species.These show microsatellite enrichment by magnetic beads is a suitable method to acquire microsatellite and it should become a main way to develop this molecular maker.At the same time,the microsatellite obtained can offer useful genetic markers for studying Silurus meriaionalis in the future.