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Published online 12 January 2004

Nucleic Acids Research, 2004, Vol. 32, No. 1 e7
© 2004 Oxford University Press

Method

PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism

Francesco Pomati and Brett A. Neilan*

Cyanobacteria and Astrobiology Research Laboratory, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, NSW, Australia

*To whom correspondence should be addressed. Tel: +61 2 9385 3235; Fax: +61 2 9385 1591; Email: b.neilan{at}unsw.edu.au
+AY445143–AY445188

A PCR-based positive hybridization (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of Anabaena circinalis, a cyanobacterium able to produce saxitoxin (STX). The PPH technique is based on the same principles of suppression subtractive hybridization (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridized at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A.circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed the recovery of DNA libraries that were mainly specific for each of the two STX-producing strains used. Several candidate sequences were found by PPH to be in common between both the STX-producing testers. The PPH technique performed using unsubtracted genomic libraries proved to be a powerful tool to identify DNA sequences possibly transferred laterally between two cyanobacterial strains that may be candidate(s) in STX biosynthesis. The approach presented in this study represents a novel and valid tool to study the genetic basis for secondary metabolite production in microorganisms.


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