High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner.
A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)
Quesada
M A
MA
University of California, Berkeley.
Rye
H S
HS
Gingrich
J C
JC
Glazer
A N
AN
Mathies
R A
RA
D001483:Base Sequence; D052160:Benzothiazoles; D002874:Chromosome Mapping / Q000379:methods; D003201:Computers; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004996:Ethidium; D005453:Fluorescence; D007834:Lasers; D008969:Molecular Sequence Data; D011804:Quinolines; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013844:Thiazoles
D052160:Benzothiazoles; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange; D004247:DNA; D004996:Ethidium