EVALUATING 2 METHODS FOR FINGERPRINTING GENOMES OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS

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.