DNA length evaluation using cyanine dye and fluorescence correlation spectroscopy.
To develop a high-performance method for measuring the length of double-stranded DNA (dsDNA) fragments, the capability of fluorescence correlation spectroscopy (FCS) was examined. To omit troublesome and time-consuming labeling operations such as PCR with fluorescently labeled mononucleotides or primers, intercalation of dimeric cyanine dye YOYO-1 iodide (YOYO) to dsDNA was utilized as a simple labeling method. Various lengths of dsDNA fragments were prepared and mixed with YOYO prior to FCS, and the dependence of the diffusion time of a dsDNA-YOYO complex on the length of dsDNA fragment and the dsDNA/YOYO ratio was investigated. It was successfully demonstrated that the dsDNA length can be measured using YOYO and FCS, and the calibration curve was developed taking into account the rewinding and expansion of the dsDNA fragment caused by YOYO intercalation.
Shimizu
Masafumi
M
School of Bionics, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan.
Sasaki
Satoshi
S
Tsuruoka
Makoto
M
D001583:Benzoxazoles / Q000494:pharmacology; D002138:Calibration; D002232:Carbocyanines / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000494:pharmacology; D004058:Diffusion; D019281:Dimerization; D005456:Fluorescent Dyes / Q000494:pharmacology; D007454:Iodides / Q000494:pharmacology; D008956:Models, Chemical; D017785:Photons; D011806:Quinolinium Compounds / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D001583:Benzoxazoles; D002232:Carbocyanines; D017931:DNA Primers; D005456:Fluorescent Dyes; D007454:Iodides; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA