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

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.