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United States Department of Agriculture

Agricultural Research Service

Title: Contamination, Error, and Nonspecific Molecular Tools

Authors
item Dyer, Alan - UNIVERSITY OF MINNESOTA
item Leonard, Kurt

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 18, 2000
Publication Date: N/A

Interpretive Summary: DNA samples are often compared by methods that amplify specific DNA fragments so that they can be seen in laboratory preparations that separate the fragments according to their sizes. This technique is used to distinguish genetic differences between different species of organisms or between different members of a single population in biological as well as forensic investigations. Scientists who use these methods take precaution to keep the DNA samples reasonably free from mixtures with foreign DNA. Tests with intentional mixtures of foreign DNA showed that at small proportions, the contaminating DNA did not interfere with analyses. However, we found that a very small quantity of bacteria in a sample from which DNA is obtained can significantly change the results of analyses with DNA samples from plants or animals. This is because, relative to their size, bacteria have much larger amounts of DNA than plant or animal cells. A small amount of bacteria that may escape detection in a plant or animal sample can interfere with the methods used to amplify DNA fragments from that sample. Our results serve as a caution to other scientists using these methods. They should not rely on a single method of DNA comparison when they suspect that samples may be contaminated with bacteria. Bacterial contamination can give the appearance of genetic differences that do not actually exist between different individuals of the plant or animal species being compared.

Technical Abstract: RAPD analysis with DNA extracted from of 121 cultures Aphanomyces cochlioides isolated from soil of sugar beet fields in Minnesota and North Dakota revealed three distinct phenotypes. Phenotypes A and C had Dice similarity of 0.99, while phenotypes A and B had Dice similarity of 0.80. AFLP analysis with representative cultures of each phenotype revealed that cultures of RAPD phenotype B had 39 AFLP products not found in A or C, which suggested possible contamination even though none of the original cultures showed evidence of bacterial growth. When these cultures were grown on media amended with rifampicin at 50 mg/L rather than 30 mg/L and with 50 mg/L penicillin G, they were identical to phenotype A in RAPD analysis, and the 39 AFLP products specific to phenotype B disappeared. Thus, phenotype B was concluded to be contaminated with DNA of a bacterium associated with the fungus in soil. The bacterial DNA suppressed expression of some RAPD products found in phenotype A as well as contributing RAPD products unique to the contaminated phenotype B. In addition, several phenotype B specific RAPD and AFLP products associated with the contaminant co-migrated with products from A. euteiches. These products of a prokaryote could not be homologous with products of similar molecular size that appear in A. euteiches but not A. cochlioides, so they can be attributed to nonhomologous RAPD or AFLP products migrating identically. The co-migrational error accounted for Dice similarity values of 0.23 for RAPD and 0.15 for AFLP comparisons between A. euteiches and contaminated A. cochlioides cultures.

Last Modified: 4/17/2014
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