凝胶条带内DNA回收测序评估DGGE可靠性  

ASSESSING THE RELIABILITY OF DGGE BY RETRIEVING AND SEQUENCING DNA FRAGMENTS IN GEL BANDS

作　　者：倪加加[1,2] 余育和[1] 

机构地区：[1]中国科学院水生生物研究所中国科学院水生生物多样性与保护重点实验室,武汉430072 [2]中国科学院大学,北京100049

出　　处：《水生生物学报》2014年第3期454-458,共5页Acta Hydrobiologica Sinica

基　　金：国家重点基础研究发展计划(2009CB118705);国家自然科学基金(30970358,31071896)资助

摘　　要：为了评估DGGE的可靠性,对DGGE条带中回收的DNA片段进行了测序比较分析,并引入了DGGE可靠性指数的概念评价其可靠性。结果显示同一条DGGE条带回收的DNA来自同一属的概率为64.7%,相同位置的DGGE条带可以被认为是同一OTU;不同的DGGE条带回收到类似的DNA序列(16S rDNA V3区差异小于4 bp)的概率为10.5%;DGGE可靠性指数为74.8%。以上结果表明尽管DGGE技术与理论预期存在一定的差距,但是DGGE技术基本能够反映微生物群落的多样性。To assess the reliability of DGGE,the V3 region fragments of prokaryotic 16S rDNA sequences were amplified by PCR and separated by DGGE with 40%—70% denaturing gradient in present study,and the differences of DNA sequences retrieved from DGGE bands were compared,and the concept of DGGE reliable index（RI） that was calculated by the equation RI =（Nss – Nsd ＋ Ndd – Nds）/Ns,was introduced.The results showed that the probability of a single band retrieving similar sequences（the sequence diversity of the V3 region of 16S rDNA is less than 4 bp） was 64.7%,and that the bands located at the same level of denaturing gradient could be considered as the same OTU.The probability that different bands retrieve similar sequences was 10.5%.The DGGE reliable index was 74.8%.Therefore,the results of DGGE still reflect the molecular diversity of environmental microbiota,although there was a disparity comparing with theoretical expectations.

关 键 词：DGGE可靠性指数 DNA指纹 变性梯度凝胶电泳 微生物群落结构 

分 类 号：Q-331[生物学]