DNA amplification fingerprinting of bacteria

Brant J. Bassam, Gustavo Caetano-Anollés, Peter M. Gresshoff

Research output: Contribution to journalArticlepeer-review

Abstract

We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·μl-1 and high MgCl2 levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.

Original languageEnglish (US)
Pages (from-to)70-76
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume38
Issue number1
DOIs
StatePublished - Oct 1 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

Fingerprint

Dive into the research topics of 'DNA amplification fingerprinting of bacteria'. Together they form a unique fingerprint.

Cite this