Detection of point mutations in the HBV polymerase gene using a fluorescence intercalator in reverse micelles.
We report a novel and simple method for mutation detection in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) in nanostructured molecular assemblies, called reverse micelles. The intercalation of SYBR Green I into the duplex DNA exhibits fluorescent emission in a CTAB/isooctane reverse micellar system as well as in an aqueous solution. We found marked differences in the fluorescence intensity between perfectly matched and mismatched 52-mer synthetic oligonucleotides, which were designed to contain the YMDD motif of the hepatitis B virus (HBV) polymerase gene, in a reverse micellar solution. Using this method, we successfully detected a mutation in PCR-amplified oligonucleotides of the HBV polymerase gene in sera of four patients with chronic hepatitis B. This detection method does not require DNA immobilization, chemical modification of DNA, or any special apparatus; it only needs a normal fluorescence spectrophotometer, an inexpensive dye, and just 10 pmol of sample DNA.
Maruyama
Tatsuo
T
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Takata
Tomoaki
T
Ichinose
Hirofumi
H
Kamiya
Noriho
N
Kuma
Hiroyuki
H
Hamasaki
Naotaka
N
Morita
Hiroyuki
H
Goto
Masahiro
M
D020137:Base Pair Mismatch; D001483:Base Sequence; D015342:DNA Probes; D005456:Fluorescent Dyes / Q000737:chemistry*; D015684:Gene Products, pol / Q000235:genetics*; D007364:Intercalating Agents / Q000737:chemistry*; D008823:Micelles; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D005456:Fluorescent Dyes; D015684:Gene Products, pol; D007364:Intercalating Agents; D008823:Micelles; C066027:P protein, Hepatitis B virus