Contamination, Error, and Nonspecific Molecular Tools

Abstract

ABSTRACT Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are widely used in studies of genetic variation. Although it is recognized that contamination should be avoided in DNA samples, little is known about the potential hazards of low level bacterial contamination of samples from which DNA is extracted for RAPD or AFLP analyses. We found that contamination of Aphanomyces cochlioides cultures with a prokaryote at visibly undetectable levels markedly altered the results of RAPD and AFLP analyses. The contamination resulted in seven contaminant-specific RAPD products and in the suppression of eight products characteristic of uncontaminated A. cochlioides cultures. Prokaryote contamination resulted in 39 contaminant-specific AFLP products, but did not cause suppression of AFLP products. Comparing A. cochlioides samples with outgroup A. euteiches did not clearly indicate the presence of contaminant DNA, because uneven product suppression in RAPD analysis increased the apparent similarity between contaminated samples and A. euteiches and because a high proportion of the contaminant-specific amplified products comigrated with products from A. euteiches in both RAPD and AFLP analyses. Work with organisms that are prone to contamination should employ techniques such as restriction fragment length polymorphism or DNA sequence comparisons rather than relying solely on RAPD or AFLP analyses.