Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.
Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). 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.
Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Iwate 026-0001, Kamaishi City, Japan. kazuya.watanabe@kamaishi.mbio.co.jp
K
K
Watanabe
Y
Y
Kodama
S
S
Harayama
D001419:Bacteria / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods* / Q000592:standards; D017931:DNA Primers / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010578:Petroleum; D016133:Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014874:Water Pollutants, Chemical / Q000032:analysis
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D010578:Petroleum; D014874:Water Pollutants, Chemical