Masking oligonucleotides improve sensitivity of mutation detection based on guanine quenching.
Guanine quenching of a fluorescence-labeled DNA probe is a powerful tool for detecting a mutation in a targeted site of a DNA strand. However, a different guanine adjacent to a targeted site can interfere with detection of a point mutation, resulting in unsatisfactory sensitivity. In the current study, we developed a simple method to improve sensitivity of the guanine quenching method using a masking DNA oligonucleotide. The simple addition of a masking DNA oligonucleotide was found to mask the interference of a different guanine in a target oligonucleotide on fluorescence and to enhance difference in the quenching ratio between wild-type and mutant oligonucleotides. Based on this strategy, we succeeded in discriminating various mutations from the wild-type YMDD motif of the hepatitis B virus DNA polymerase gene using guanine quenching with a masking oligonucleotide.
Maruyama
Tatsuo
T
Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, Fukuoka 812-8581, Japan.
Shinohara
Toshimitsu
T
Hosogi
Takuya
T
Ichinose
Hirofumi
H
Kamiya
Noriho
N
Goto
Masahiro
M
D004252:DNA Mutational Analysis / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006147:Guanine / Q000737:chemistry*; D006515:Hepatitis B virus / Q000201:enzymology; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry*; D017354:Point Mutation; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012235:Rhodamines; D006147:Guanine; D004259:DNA-Directed DNA Polymerase