DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA.
A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the target DNA and then enzymatically digested. As these probe hybridization and digestion cycle reactions are repeated at a fixed temperature, digested probes (shortened probes) accumulate in the reaction mixture in a manner similar to a DNA polymerase chain reaction. Investigation of the digestion characteristics of the DNA probe showed that a slight digestion of a free single-stranded probe produces a large background signal, which results in low detection sensitivity. The digestion of single-stranded DNA probes is caused by double-stranded formations in the molecules. This digestion decreases and the double-stranded-specific digestion increases with increasing reaction temperature. When the reaction occurs at 45 degrees C, the association rate of the enzyme on the double-stranded DNA is 700 times faster than that on single-stranded DNA. This enables selective digestion of double-stranded DNA. The detection limit is 9 x 10(-19) mol for a M13-phage DNA.
Okano
K
K
Central Research Laboratory, Hitachi, Limited, Tokyo, Japan.
Kambara
H
H
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000032:analysis* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000032:analysis; D005090:Exodeoxyribonucleases; D006801:Humans; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III