Photodestruction of fluorophores and optimum conditions for trace DNA detection by automated DNA sequencer.
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.
Kambara
H
H
Central Research Laboratory, Hitachi Ltd., Tokyo, Japan.
Nagai
K
K
Kawamoto
K
K
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
D004247:DNA; D016650:Fluorescein-5-isothiocyanate