Optimization of the rate of DNA hybridization and rapid detection of methicillin resistant Staphylococcus aureus DNA using fluorescence polarization.
The hybridization rate of two complementary single-stranded DNA 24-mers was determined at various NaCl concentrations using a fluorescence polarization method. The rate was taken as the rate constant of the second order reaction, obtained by mathematical fitting of the time course curves of hybridization. The rate increased with the NaCl concentration, plateauing in the concentration range of about 1-2 M. Over the temperature range of 46 degrees C to 56 degrees C, it was found that in 0.8 M NaCl, hybridization was complete in under 10 min and that the difference in the polarization values between specific hybridization and non-specific binding was greater at lower temperatures. Under the optimized conditions of 0.8 M NaCl at 46 degrees C, the time for DNA hybridization to reach 90% completion decreased to less than 5 min. The assay time for one sample was 10 min and the detection limit was of the order of 10(-10) M (40 fmol per assay). Under the optimized conditions, the DNA of methicillin resistant Staphylococcus aureus (MRSA), which had been multiplied by PCR, could be detected within 10 min.
Tsuruoka
M
M
Research Center for Advanced Science and Technology, University of Tokyo, Japan.
Yano
K
K
Ikebukuro
K
K
Nakayama
H
H
Masuda
Y
Y
Karube
I
I
D001483:Base Sequence; D001709:Biotechnology; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D005454:Fluorescence Polarization / Q000379:methods*; D016106:Methicillin Resistance / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012965:Sodium Chloride; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics*; D013696:Temperature
D015342:DNA Probes; D004269:DNA, Bacterial; D012965:Sodium Chloride