磁珠富集法筛选白鲢的微卫星分子标记  

Microsatellite Enrichment by Magnetic Beads in Silver Carp(Hypophthalmichtys molitrix)

作　　者：鲁翠云[1] 孙效文[1] 曹洁[2] 梁利群[1] 

机构地区：[1]中国水产科学院黑龙江水产研究所,哈尔滨150070 [2]大连水产学院,大连116023

出　　处：《农业生物技术学报》2005年第6期772-776,共5页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.863-101-05-02-01)资助

摘　　要：通过磁珠富集法筛选白鲢(Hypophthalmichtysmolitrix)的微卫星分子标记。从血液中提取白鲢大片段基因组DNA,限制性内切酶Sau3AI部分消化,使大部分片段介于400～900bp,低熔点琼脂糖凝胶回收。在筛选的基因组片段上连接1个20bp的双链接头,构建白鲢全基因组PCR文库,用生物素标记的微卫星探针(CA)15进行筛选,磁珠富集含有微卫星序列的DNA片段,获得含有微卫星的单链序列;将这些序列通过PCR扩增,连接pGEM-T载体,转化入感受态大肠杆菌(Escherichiacoli)DH5α,得到一个微卫星序列文库;又经同位素标记的微卫星探针(CA)15进行第2次筛选,获得149个阳性克隆,经测序分析,获得微卫星序列138个,这可用引物设计软件PrimerPremier5.0设计引物81对。It was a study to isolate microsatellite DNA from silver carp （Hypophthalmichtys molitrix） genome with enrichment by magnetic beads. Genomic DNA was extracted from blood by normal methods and digested with restriction enzyme Sau3AI. The fragments of 400-900 bp were isolated and purified in a low melting agarose gel and then ligated short linkers （20 bp）. A whole genome PCR library was created. This genomic DNA was hybridized with a biotin-labeled SSR （simple sequence repeat） probe （CA）15. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNA contained the selected microsatellite DNA was obtained. The selected DNAs were then amplified using primers designed complementary to the linkers, cloned into the pGEM-T vector and transformed into competent Escherichia coli DH5α. In this experiment, performing the second screen with a radiolabeled （CA）15 probe, One hundred and forty-nine positive clones were obtained. From these positive clones, one hundred and thirty-eight microsatellite sequences were isolated. This allowed to designed 81 pairs of primers with the software Primer Premier 5.0.

关 键 词：磁珠富集 白鲢 微卫星 

分 类 号：S512.1[农业科学—作物学] Q75[生物学—分子生物学]