Simple detection of point mutations in DNA oligonucleotides using SYBR Green I.
A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (it tercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 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
Park
Lian-Chun
LC
Kamaiya
Noriho
N
Goto
Masahiro
M
D020137:Base Pair Mismatch / Q000235:genetics; D052160:Benzothiazoles; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D003959:Diamines; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D009930:Organic Chemicals; D017354:Point Mutation / Q000235:genetics*; D011804:Quinolines; D016415:Sequence Alignment / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D009841:Oligonucleotides; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I