title: Photodestruction of fluorophores and optimum conditions for trace DNA detection by automated DNA sequencer.
abstract: Although automated DNA sequencers are becoming popular, their sensitivity in detecting DNA bands is still around 10(-17) mole/band. The sensitivity of a system depends on the laser power, labeling fluorophore, and the fluorescence-collecting yield. The emission and photodestruction cross-sections of the fluorophores are critical in optimizing the irradiated laser power and the migration speeds of DNA fragments to achieve high sensitivity. We investigated photodestruction cross-sections of various fluorophores to optimize the irradiation laser power. In addition, we used a cylindrical lens system to improve the fluorescence-collecting yield of a DNA sequencer using side entry laser irradiation. Fluoresceine isothiocyanate (FITC) commonly used in fluorescence studies, is very photo-destructive, the cross-section of the destruction being about 3.8 x 10(-20) cm2 in buffer solution while that of Texas Red is 1.5 x 10(-21) cm2. When the time for DNA fragments to transit through the irradiated region is 11 s, the optimum laser powers are 0.9 mW, with an Ar laser (488 nm) for FITC-DNA, and 18 mW, with an He-Ne laser (594 nm) for Texas Red DNA. We have developed a DNA sequencer, with a cylindrical lens system which improves the fluorescence-collecting efficiency by a factor of 4, and an He-Ne laser (5 mW). Although the sequencer uses a slab gel, an ultra-high sensitivity of 5 x 10(-20) mole/band (S/N-4) was achieved under optimized conditions.
authors.affiliation: Central Research Laboratory, Hitachi Ltd., Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kambara
authors.forename: K
authors.initials: K
authors.lastname: Nagai
authors.forename: K
authors.initials: K
authors.lastname: Kawamoto
mesh_terms: D001322:Autoanalysis; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005453:Fluorescence; D007834:Lasers; D010777:Photochemistry; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA; D016650:Fluorescein-5-isothiocyanate