title: Mutation detection in DNA oligonucleotides based on a Guanine quenching method coupled with enzymatic digestion of single-stranded DNA.
abstract: Fluorescence quenching by guanine allows DNA hybridization to be monitored and any point mutations in oligonucleotides to be detected. However, fluorescence quenching is often affected by untargeted guanine located in a protruding end (single-strand DNA) of the probe-target DNA duplex resulting in an unsatisfactory sensitivity. In the present study, we used enzymatic digestion of the protruding end of a probe-target DNA duplex to avoid interference by untargeted guanine on fluorescence quenching for detection of a nucleobase mutation. Enzymatic digestion of the protruding end of the DNA duplex fully prevented interference by untargeted guanine, and produced a marked difference in the quenching ratios (36% for wild-type, and 0% for mutant).
authors.affiliation: Department of Applied Chemistry Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, 812-8581, Fukuoka, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Maruyama
authors.forename: Toshimitsu
authors.initials: T
authors.lastname: Shinohara
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ichinose
authors.forename: Momoko
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Nobuko
authors.initials: N
authors.lastname: Okamura
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000235:genetics; D006515:Hepatitis B virus / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D012235:Rhodamines / Q000737:chemistry
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D004277:DNA, Single-Stranded; D005452:Fluoresceins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D012235:Rhodamines; C045132:4-carboxyfluorescein; D006160:Guanosine Triphosphate; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C046264:gene b exonuclease; D016650:Fluorescein-5-isothiocyanate