Rapid isolation of chromosomal DNA from oral streptococci and polymerase chain reaction-oriented restriction fragment-length polymorphism analysis for genetic heterogeneity.
We have extensively modified the published method for the lysis of gram-positive bacteria to isolate chromosomal DNA from only 1 ml of oral streptococcal overnight culture. Cells were incubated with lysozyme and R Nase A in the presence of polyethylene glycol. After centrifugation, cells were lysed with sodium dodecyl sulfate and proteinase K. Following ethanol precipitation, sodium dodecyl sulfate solution was added to the residue, and the pellet was completely dispersed by incubating at 65 degrees C. The chromosome was purified by extraction over phenol and chloroform. Two regions corresponding to the ribosomal RNA (rrn) operon and the glucosyltransferase gene were amplified using the chromosome from Streptococcus mutans and Streptococcus sobrinus by polymerase chain reaction (PCR). Genetic heterogeneity was assessed by restriction fragment-length polymorphism (PCR-RFLP). The PCR-RFLP analysis readily allowed us to subtype each strain, suggesting that the strategy presented here will provide a useful tool to verify epidemiological studies at the molecular level.
Shiroza
T
T
Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Shinozaki
N
N
Watanabe
T
T
Ikemi
T
T
Fukushima
K
K
Abiko
Y
Y
D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005798:Genes, Bacterial / Q000235:genetics; D018740:Genetic Heterogeneity; D005964:Glucosyltransferases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009055:Mouth / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D016986:Streptococcus sobrinus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D014774:Virulence / Q000235:genetics; D012407:rRNA Operon / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D005964:Glucosyltransferases