Ultrasensitive fluorescence detection of DNA. Progress report, February 1, 1991--January 31, 1992

Technical Report · Tue Dec 01 04:00:00 EST 1992

DOI:https://doi.org/10.2172/10190856· OSTI ID:10190856

Mathies, R A; Glazer, A N

We have shown that a number of polycationic highly fluorescent dyes form complexes with double-stranded DNA (dsDNA) which are stable to electrophoresis and have characterized in detail such dsDNA complexes with TOTO (1,1{prime}-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole). TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with dsDNA, up to a maximum dye to DNA bp ratio of 1:4, with >1000-fold fluorescence enhancement. We have developed an assay using YOYO for the quantitation of single-stranded and dsDNA in solution applicable over a range of DNA concentrations from 0.5 to 100 ng per ml. The fluorescent dsDNA-dye complexes allow detection of dsDNA on agarose and acrylamide gels with picogram sensitivity. We have applied these complexes in multiplex mapping experiments for accurate sizing and quantitation of restriction fragments. We have shown that in gel shift experiments the stable dsDNA-dye complexes can be used to detect heteroduplex-Muts complexes with a sensitivity comparable to radioisotopic detection.

• Research Organization: California Univ., Berkeley, CA (United States). Dept. of Chemistry
• Sponsoring Organization: USDOE, Washington, DC (United States)
• DOE Contract Number: FG03-91ER61125
• OSTI ID: 10190856
• Report Number(s): DOE/ER/61125--2; ON: DE93004145
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
BIOCHEMISTRY
DERIVATIZATION
DNA
ELECTROPHORESIS
FLUORESCENCE SPECTROSCOPY
GENETICS
OLIGONUCLEOTIDES
PROGRESS REPORT
QUINOLINES