【摘要】 结合笔者等多年积累的经验详细介绍环境微生物学研究中变性梯度凝胶电泳(DGGE)技术应用全步骤:从环境样品中直接提取总DNA,利用5′端含GC夹子的引物PCR 16S rDNA的部分片段,将PCR产物在含有浓度线形递增的变性剂的聚丙烯酰胺凝胶中进行电泳分析,使不同来源的DNA片段在电泳胶中分离,从胶中切出分离的DNA片段测定碱基序列,进行分类分析。PCR-DGGE技术能够检测不可培养的微生物基因,而DGGE技术与克隆等其他技术结合更能发挥其分离作用。更多还原

【Abstract】 The aim is to introduce detailed procedures for the application denaturing gradient gel electrophoresis(DGGE) in the study of environmental microorganisms based on our current experience in molecular ecology.The procedure for DGGE includes: Extracting DNA directly from environmental samples;PCR in the region of 16S rDNA using a primer with GC clamp in 5′;analyzing the PCR products in a polyacrylamide gel with a chemical denaturing gradient which could separate the different DNA fragments and then extracting bands from the gel and performing sequencing and phylogenetic analysis.PCR-DGGE could detect the gene of unculturable microoganisms,and the combination of DGGE with clone library and other molecular techniques is more appropriate to exert its predominance in separating DNA.更多还原