磁珠富集法分离草鱼微卫星分子标记

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

磁珠富集法是一种快速、高效的分离微卫星分子标记的方法。本研究通过该方法分离草鱼的微卫星分子标记。将草鱼(Ctenopharyngodon idella)基因组DNA经Sau3AI酶切,回收纯化400～900bp片段,连上接头,构建“基因组PCR文库”。用生物素标记的简单重复序列(CA)15作探针与其杂交,杂交复合物结合到包被有链霉亲和素的磁珠上,经一系列的洗涤过程,去除磁珠表面不含有微卫星的片段。将吸附在磁珠上的片段洗脱,PCR扩增放大,再进行克隆和测序,根据微卫星两端的保守序列设计引物,即可得到微卫星分子标记。本研究又通过同位素标记的探针(CA)15进行二次杂交筛选,获得阳性克隆132个,所得到的阳性克隆经测序,86.36%含有微卫星序列,共获得130个微卫星DNA序列。用引物设计软件PrimerPremier5.0设计引物83对。

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.