线虫PCR:直接针对秀丽线虫进行PCR扩增基因组DNA的新方法  

A New Method to Amplify the Genomic DNA by Using PCR on the Caenorhabditis elegans Directly

作　　者：李媛[1,2] 任长虹[2] 石锦平[2] 李伟光[2] 刘虎岐[1] 张成岗[1,2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]军事医学科学院放射与辐射医学研究所蛋白质组学国家重点实验室,北京100850

出　　处：《生物技术通讯》2011年第1期66-70,共5页Letters in Biotechnology

基　　金：国家重点基础研究发展计划(2006CB504100);国家自然科学基金(30900862;30800196;30771230);国家科技重大专项(2009ZX09503-002;2009ZX09301-002;2009ZX09103-616)

摘　　要：目的:直接针对秀丽线虫进行PCR反应,以便快速扩增基因组DNA,从而提高钓取目的基因和鉴定基因组是否发生突变的效率。方法:根据生物信息学分析,针对不同基因设计单重或多重PCR引物;在不含Taq DNA聚合酶的PCR反应体系中加入蛋白酶K消化秀丽线虫染色体中的组蛋白,然后加入Taq酶,直接针对野生型或突变型秀丽线虫个体进行PCR反应,无须提前制备基因组DNA。结果:用该方法能直接从秀丽线虫体内的基因组DNA中扩增出目的基因片段,与常规预先制备基因组DNA为模板的扩增产物条带位置一致,且特异性更高。同时,针对不同基因的多重PCR亦能扩增出理想的目的基因片段。结论:直接针对秀丽线虫进行PCR能够实现基因组DNA的快速扩增和鉴定,在保证反应特异性的同时,还能显著提高PCR反应的灵敏度,为秀丽线虫基因组的快速鉴定提供了一种便捷方法。Objective：In order to improve the efficiency of target gene amplification and mutant identification,the Caenorhabditis elegans was used directly in the PCR.Methods：According to bioinformatics analysis,primers for single or multiplex PCR were designed.Firstly,the proteinase K and the C.elegans（either wild type or mutants） were added into the PCR system without Taq DNA polymerase.Secondly,proteinase K was inactivated by heating.Finally,the Taq polymerase was added and the PCR procedure started to amplify the target genes.Results：Our data showed that C.elegans can be used directly in the PCR amplification to acquire and identify target genes with expected sizes and higher specificity.Moreover,by using multiplex C.elegans PCR,more than one gene could be amplified at the same time.Conclusion：This new method is time-consuming and practical and much more specific and sensitive compared with the traditional operation which require preparation of the C.elegans genomic DNA at first.Our protocol thus provides a new and effective method to facilitate genomic DNA analysis of C.elegans

关 键 词：秀丽线虫 线虫PCR 多重PCR 基因扩增 突变株系鉴定 

分 类 号：Q78[生物学—分子生物学]