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

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.