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Contamination, error, and nonspecific molecular tools
Phytopathology
|October 24, 2008
Summary
Low-level bacterial contamination can significantly alter genetic analysis results. Even invisible prokaryote contamination in DNA samples can lead to inaccurate findings in Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) studies.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are common molecular markers for genetic variation.
- The impact of low-level bacterial contamination on DNA extraction for RAPD and AFLP analyses is not well understood.
Purpose of the Study:
- To investigate the effects of subtle prokaryote contamination on RAPD and AFLP analyses.
- To assess the reliability of RAPD and AFLP in detecting contamination when comparing closely related species.
Main Methods:
- DNA extraction from Aphanomyces cochlioides cultures.
- Analysis of genetic variation using RAPD and AFLP techniques.
- Comparison of contaminated and uncontaminated DNA samples, including outgroup comparison with Aphanomyces euteiches.
Main Results:
- Bacterial contamination introduced specific RAPD products and suppressed native A. cochlioides products.
- Prokaryote contamination generated numerous specific AFLP products but did not suppress existing ones.
- Contamination masked true genetic differences, leading to apparent similarity between contaminated samples and the outgroup A. euteiches in both RAPD and AFLP.
Conclusions:
- Visibly undetectable bacterial contamination can severely compromise the accuracy of RAPD and AFLP analyses.
- RAPD and AFLP may not be reliable for detecting contamination in organisms prone to microbial presence.
- Alternative methods like restriction fragment length polymorphism or DNA sequencing are recommended for contaminated samples.
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