Development of a fast DNA-DNA hybridization method based on melting profiles in microplates.
DNA-DNA hybridization is still the "gold standard" for the genotypic delineation of bacterial species. However, it is not widely used because traditional DNA-DNA hybridization techniques are rather time-consuming and not easy to perform in routine laboratories. In the present study, DNA of reference strains was digested with Sau3A, ligated with linker oligonucleotides S1/2 and in vitro amplified. The amplified DNA fragments were immobilized on MaxiSorb 96-well plates. DNA isolated from target strains was also digested with Sau3A, ligated with linker oligonuleotides P1/2 and in vitro amplified in the presence of digoxygenin modified dUTP. The labeled amplificate was hybridized to the immobilized reference DNA under isothermal conditions. Thermal denaturation curves of the DNA-DNA hybrids were obtained by using washing solutions of increasing stringency. Remaining hybrids were colorimetrically detected with anti-digoxygenin-horseradish peroxidase anti-bodies. The new method was validated with strains of the genus Pedioccocus for which DNA-DNA similarities have also been determined by the filter hybridization method. In addition, DNA-DNA hybridizations were performed with genotypically defined Enterobacter species.
Mehlen
André
A
Department of Microbiology, Technical University Munich, Freising, Germany. mehlen@mikro.biologie.tu-muenchen.de
Goeldner
Marcia
M
Ried
Sabine
S
Stindl
Sibylle
S
Ludwig
Wolfgang
W
Schleifer
Karl-Heinz
KH
D001419:Bacteria / Q000145:classification* / Q000302:isolation & purification*; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004754:Enterobacter / Q000145:classification / Q000302:isolation & purification; D006358:Hot Temperature; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010376:Pediococcus / Q000145:classification / Q000302:isolation & purification; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases