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

  • ️Thu Jan 01 2004

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

Francesco Pomati et al. Nucleic Acids Res. 2004.

Abstract

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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Figures

Figure 1
Figure 1

Schematic diagram of the procedure applied in this study. Toxic A.circinalis strains were used as testers, while non-toxic strains were used as drivers. Dashed lines indicate unsubtracted tester DNA.

Figure 2
Figure 2

(A) Standard SSH procedure, as described previously by Akopyants et al. (12). (B) PPH method, based on adaptor-mediated PCR. Solid lines represent tester DNA fragments, boxes represent terminal adaptors 1 and 2R. Adaptor 2R was excised from tester DNA 1 (strain 131C) by digestion with EagI, and adaptor 1 was removed from tester DNA 2 (strain 344B) with NotI. *Although there is an identical primer binding sequence on both ends of the type c molecules, the short overall homology at the two ends negates the suppression PCR effect (12).

Figure 3
Figure 3

Electrophoretic comparison of fragments 179 (lanes 1–6) and 109 (lanes 7–12) amplified by PCR from toxic and non-toxic strains of A.circinalis. Gel lanes 1–12 as follows: 131C (1 and 7), 344B (2 and 8), 134C (3 and 9), 279B (4 and 10), 306A (5 and 11) and 271C (6 and 12). PCR products were loaded as a total of 4 µl per sample and run with 1 kb Plus DNA Ladder (Invitrogen) as standard (lanes M).

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