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| 细角螺微卫星DNA 富集文库构建及特征分析 |
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李清荟1,2, 陈晓姣1,2, 黎中宝1,2, 曹媛钰1,2, 陈丽娜1,2, 戴 刚1,2
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1.集美大学 水产学院;2.福建省海洋渔业资源与生态环境重点实验室
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| 摘要: |
| 采用生物素磁珠富集法,用生物素标记的(GT)15 和(CT)15两种探针与细角螺基因组MseI酶切的300～1200bp片段杂交,杂交复合物与链霉亲和素磁珠结合,捕获含有重复序列的微卫星片段。最后将磁珠捕获到的重复序列与PMD19-T载体连接后克隆到DH5α中构建微卫星基因组文库。通过PCR检测出354个阳性克隆,从中随机选取248个片段大于500bp的阳性克隆进行测序,结果显示,在220个成功测序的阳性克隆中共获得278个微卫星序列,其中完美型171个,占61.5%;非完美型81个,占29.1%;复合型26个,占9.4%。除探针使用的GT和CT的重复序列外,还筛选到三碱基GTT、TGG、GAA、CAA;四碱基TCTA、ACAG、TAGA、GTGA、GTCT、GATA及五碱基TTTTG的重复序列。在278条序列中共有82条可以设计引物。 |
| 关键词: 细角螺(Hemifusus ternatanus) 磁珠富集 微卫星 构建 DNA 文库 |
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| 基金项目:集美大学创新团队基金项目(2010A004) |
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| Construction and Characterization of DNA Libraries enriched for Microsatellites repeat sequences in Hemifusus ternatanus |
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| Abstract: |
| Microsatellites enriched genome library for Hemifusus ternatanus was constructed by bio-(GT)15 and bio-(CT)15 using FIASCO. Genomic DNA was digested with restriction enzyme MseI. Fragments ranging from 300 to 1200 bp were selected to ligate with short linkers. Fragments containing microsatellite repeats were captured with Streptavidin-coated Magnetic Sphere Particles. The target fragments were eluted, PCR amplified, purified and then ligated to pMD-19T vector and transformed into Escheri- chia coli. A total of 248 clones with DNA fragments above 500 bp were selected for sequencing. As the result, 278 microsatellite sequences were isolated. Among the microsatellites, 171 repeating motifs were perfect, occupying 61.5 %; 81 repeating motifs were imperfect, occupying 29.1%; 26 repeating motifs were compound, occupying 9.4%. Besides the GT and CT repeats, several other types of repeats were also detected, such as GTT, TGG, GAA, CAA, TCTA, ACAG, TAGA, GTGA, GTCT, GATA and TTTTG. There are 82 sequences among the 278 microsatellites can be designed with primers and these primers could be used to test polymorphism in further research. |
| Key words: Hemifusus ternatanus enrichment by magnetic beads microsatellite DNA construction DNA library |