Evaluating Two Methods for Fingerprinting Genomes of Actinobacillus Actinomycetemcomitans
polymerase chain reaction
restriction fragment length polymorphism
The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.