黑斑原(鱼兆)微卫星DNA富集文库构建与鉴定  

CONSTRUCTION AND IDENTIFICATION OF DNA LIBRARIES ENRICHED FOR MICROSATELLITE REPEAT SEQUENCES OF GLYPTOSTERNUM MACULATUM

作　　者：郭宝英[1,2] 谢从新[2] 祁鹏志[2] 吴常文[1] 邓一兵[1] 

机构地区：[1]浙江海洋学院浙江省海洋养殖装备与工程技术重点实验室,浙江舟山316004 [2]华中农业大学水产学院,武汉430070

出　　处：《水生生物学报》2011年第6期908-912,共5页Acta Hydrobiologica Sinica

基　　金：国家海洋公益性行业科研专项(201005013);国家自然科学基金项目(30471324);海洋渔业科学与技术浙江省重中之重学科开放课题(20100105);农业部海洋与河口渔业资源及生态重点开放实验室开放课题(开-10-01)资助

摘　　要：采用磁珠富集法,利用生物素标记的(CA)12寡核苷酸探针从黑斑原(鱼兆)基因组DNA MboI酶切的400—1000 bp片段中筛选CA/GT微卫星位点,洗脱的杂交片段克隆到pMD18-T载体上构建富集微卫星基因组文库后,通过PCR筛选检测出720个阳性克隆,占所有克隆的89.2%,从阳性克隆中随机选取139个进行测序,序列分析发现,124个克隆含有7个以上的重复序列,其中完全的为80个(64.5%),不完全的为40个(32.3%),复合的为15个(3.2%),重复次数范围为7—165次,平均为52次。在124条序列中共59条可以设计引物。Microsatellite marker(SSR) has been widely used in population genetics and genetic map construction.In order to determine the genetic diversity of G.maculatum,this study was undertaken to develop and characterize the microsatellite sequence firstly for further to develop the microsatellite markers.Genomic DNA was extracted from muscle tissue using a traditional proteinase K digestion and phenol-chloroform extraction procedure with RNA removed by RNase.Approximately 2 μg of total genomic DNA was digested with MboI,then ligated to the adapters(Linker A and Linker B).The treated DNA sample was then pooled and fragments were separated on a 1.5% agarose gel prior to size selection.The resulting fragments(400–1000 bp) were extracted from the gel matrix using a column and amplified 20 cycles with Linker B primers.The amplified DNA was hybridized with 5 μL of 5’-biotinylated(CA)12 repeat oligos in a total volume of 100 μL of 6× SSC and 0.1% SDS.The mixture was incubated at 95℃ for 5min,followed by anneal at 65℃ for 60min and cooled to room temperature.During this hybridization,the 100 μL(per treatment) of Streptavidin coated beads was resuspended in 300 μL 1× hybridization buffer(6× SSC + 0.1% SDS) and washed three times.The hybridization mixture was added to the washed beads and incubated for 30 min at room temperature.The beads were washed twice with 1× hybridization buffer,twice with high wash solution(0.2× SSC + 0.1% SDS) and twice with high wash solution at 10℃ below the annealing temperature used.The enriched fragments were recovered from the beads by resuspending the beads in 50 μL of 1× TE(pH=8.0) and heating to 95℃ for 5min.The enriched solution was removed from the beads immediately using a magnetic stand.DNA containing repeats was amplified with Linker B primers.Cy-cle conditions were: an initial denaturalization at 94℃ 2min,followed by 20 cycles of 94℃ for 30s,55℃ for 30s,72 ℃ for 60s,with a final extension at 72℃ for 10min.Cleaned products were ligated into a pMD18-T vector(

关 键 词：黑斑原 微卫星 磁珠富集 DNA文库 构建 

分 类 号：Q785[生物学—分子生物学]