| 限制性显示-聚合酶链反应扩增苏云金杆菌基因片段的克隆与分析 |
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| 引用本文： | 李侠,马文丽,庞义,张宝,姜立,郑文岭.限制性显示-聚合酶链反应扩增苏云金杆菌基因片段的克隆与分析[J].第一军医大学学报,2003,23(4):323-325. |
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| 作者姓名： | 李侠 马文丽 庞义 张宝 姜立 郑文岭 |
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| 作者单位： | [1]第一军医大学分子生物学研究所，广东广州510515 [2]中山大学生命科学学院，广东广州510632 [3]广州军区广州总医院分子肿瘤学研究所，广东广州510010 |
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| 摘 要： | 目的 运用限制性显示-PCR技术(RD-PCR)扩增苏云金杆菌(Bacillus thuringiensis，Bt)基因片段，并进行克隆、测序分析。方法 设计特异性引物扩增长片段Bt基因，对扩增产物进行RD-PCR扩增，扩增后的产物克隆至pMD18-T载体并进行快速鉴定。提取阳性克隆质粒进行测序分析。结果 运用RD-PCR技术，将长片断的基因(2000—3000bp)分为多个短的片段，片段长度较为均一(200～600bp)。测序结果表明，所扩增的片段均属于特异扩增的Bt基因。结论 运用RD-PCR技术可以将长片段基因进行片段化，使其适用于基因芯片的探针。
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| 关 键 词： | 苏云金杆菌 限制性显示-聚合酶链反应 基因克隆 基因芯片 |
Cloning and sequence analysis of Bacillus thuringiensis gene fragments isolated by restriction digest PCR. |
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| Xia Li,Wen-li Ma,Yi Pang,Bao Zhang,Li Jiang,Wen-Ling Zheng.Cloning and sequence analysis of Bacillus thuringiensis gene fragments isolated by restriction digest PCR.[J].Journal of First Military Medical University,2003,23(4):323-325. |
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| Authors: | Xia Li Wen-li Ma Yi Pang Bao Zhang Li Jiang Wen-Ling Zheng |
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| Institution: | Institute of Molecular Biochemistry, First Military Medical University, Guangzhou 510515, China. Wenli@fimmu.com |
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| Abstract: | OBJECTIVE: To clone and analyze Bacillus thuringiensis gene fragments isolated by restriction digest PCR (RD-PCR). METHOD: Specific primers were designed to amplify the genes of Bacillus thuringiensis israelensis (Bti), and the PCR products were classified and re-amplified by RD-PCR to obtain the fragments for subsequent purification and cloning into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. RESULTS: Sequence analysis showed that all the fragments amplified were Bti genes. CONCLUSION: RD-PCR is reliable in breaking down large gene fragments into confined and shorter gene fragments for preparing microarray probes. |
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