Use of the DiversiLab repetitive sequence-based PCR system for genotyping and identification of Archaea.
Repetitive elements are short stretches of DNA that are randomly distributed throughout the chromosomes of prokaryotes. The use of PCR primers to amplify intervening sequences of DNA between specific repetitive elements in Bacteria has become a standard method for rapidly genotyping bacterial strains and providing good resolution between multiple strains within a single species. Rapid, standardized methods for high resolution genotyping of Archaea are not widely available. We evaluated the DiversiLab system from Bacterial Barcodes that utilizes a kit-based repetitive sequence-based (rep-PCR) method that has been optimized for genotyping DNA was extracted from the source organisms using either a standard chemical DNA extraction kit or Whatman FTA paper. Rep-PCR was performed using an archaeal primer set and, the products were run on an Agilent, Lab-on-a-Chip DNA analyzer. Results were analyzed and compared using DiversiLab web-based software from Bacterial Barcodes. Seventy-nine strains representing 27 genera of Crenarchaeota and Euryarchaeota were analyzed. All the organisms could be successfully genotyped and the results were reproducible. We could not detect differences in rep-PCR profiles between DNA extracted using the chemical extraction kit and FTA paper. Thus far, 14 genera and 32 species of methanogens have been analyzed, and all yielded unique genotypes. For halophiles, 11 genera and 28 different species were analyzed, and all yielded unique genotypes. A comparison of 7 different strains of Halobacterium salinarium demonstrated that 6 of the 7 strains had a unique genotype. A comparison of 4 strains of Methanosarcina mazei indicated that each strain produced a unique genotype. There was little systematic inference that could be made from dendrograms comparing different strains, species, and genera of Archaea based on UPGMA cluster analysis. Based on these results, rep-PCR was a useful tool for the genotyping and strain identification of Archaea.
Cleland
David
D
Bacteriology Program, American Type Culture Collection, Manassas, VA, United States. dcleland@atcc.org
Krader
Paul
P
Emerson
David
D
D016000:Cluster Analysis; D019615:Crenarchaeota / Q000145:classification* / Q000235:genetics*; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics*; D019605:Euryarchaeota / Q000145:classification* / Q000235:genetics*; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D019641:DNA, Archaeal