Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 44, Issue 3, Pages 253-262Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0167-7012(01)00220-2
Keywords
16S ribosomal DNA; denaturing gradient gel electrophoresis; bias; degenerate primers; inosine
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Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGCE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers. (C) 2001 Elsevier Science B.V. All rights reserved.
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