Optimization of arbitrarily primed PCR for the identification of bacterial isolates

Eileen Maura Jutras, Raina M. Miller, Ian L. Pepper

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Arbitrarily primed polymerase chain reaction (AP-PCR) has been used extensively for genetic mapping, and the identification of bacterial isolates. To ensure that the results will be reproducible and due to true genetic variations, the AP-PCR reaction conditions must be optimized. In this study, three cultured bacterial isolates were screened with 100 arbitrary primers. Of these, five were chosen for the optimization study. The parameters optimized included: the operating conditions of the thermal cycler, the agarose gel concentration, the annealing temperature, and the concentrations of Taq polymerase enzyme, magnesium chloride, primer, and template. The final optimized PCR reaction conditions were 1 × buffer (3.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl and 0.1 mg ml-1 gelatin), 200 μM dNTP, 0.4 μM primer, 2.5 U AmpliTaq® (Perkin-Elmer Cetus) polymerase enzyme, and 5 μl of template (at least 106 lysed bacterial cells). The Perkin-Elmer Gene-Amp™ 9600 PCR System was used with the following cycling conditions; a 94°C 15 s denaturation step, a 45°C 15 s annealing step, and 72°C 30 s extension step for a total of 35 cycles. Reproducible, unique fingerprints were generated for the three isolates using each of the five arbitrary primers.

Original languageEnglish (US)
Pages (from-to)55-63
Number of pages9
JournalJournal of Microbiological Methods
Volume24
Issue number1
DOIs
StatePublished - Nov 1995

Keywords

  • AP-PCR
  • Bacterium
  • Identification
  • Optimization

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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