Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting

• Published in: Journal of microbiological methods 2001-04, Vol.44 (3), p.253-262
• Main Authors: Watanabe, Kazuya, Kodama, Yumiko, Harayama, Shigeaki
• Format: Article
• Language: English
• Subjects: 16S ribosomal DNA; Bacteria - chemistry; Bacteria - classification; Bacteria - genetics; Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Bias; Biological and medical sciences; Degenerate primers; Denaturing gradient gel electrophoresis; DNA Fingerprinting - methods; DNA Fingerprinting - standards; DNA Primers - chemistry; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Inosine; Microbiology; Molecular Sequence Data; Petroleum; Polymerase Chain Reaction - methods; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Water Pollutants, Chemical - analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(01)00220-2

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.