ISSN:
0173-0835
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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 × 10-20 cm2 in buffer solution while that of Texas Red is 1.5 × 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 m W, 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 × 10-20 mole/band (S/N˜4) was achieved under optimized conditions.
Additional Material:
3 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.11501301111
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