Detection of single-base mutations by fluorogenic ribonuclease protection assay.
The ribonuclease protection assay is a generally applicable technique for the detection of known mutations. We have developed a simple and rapid method for mutation detection based on the ribonuclease protection assay using fluorescently labeled oligodeoxyribonucleotide probes. The fluorogenic ribonuclease protection (FRAP) assay uses two differently labeled oligodeoxyribonucleotides, a donor probe and an acceptor probe, to obtain a fluorescence resonance energy transfer (FRET) signal. We have utilized the FRAP assay for the detection of a single-base mutation in the YMDD motif of the hepatic B virus DNA polymerase gene. The occurrence of mismatch-selective RNA cleavage was successfully discriminated by measuring the FRET signal between the donor and acceptor probes. Moreover, mutation sensing was successfully visualized by a UV transillumination. This simple and rapid mutation sensing method should facilitate a high-throughput mutation analysis.
Ichinose
Hirofumi
H
Japan Science and Technology Agency, Fukuoka 814-0001, Japan.
Kitaoka
Momoko
M
Okamura
Nobuko
N
Maruyama
Tatsuo
T
Kamiya
Noriho
N
Goto
Masahiro
M
D020137:Base Pair Mismatch; D001483:Base Sequence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D012260:Ribonucleases