A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system

Various assays are available for quantification of DNA in solution, but non e has been described that is both sensitive and specific for double-strande d (ds) DNA and features practical properties such as low dye and equipment costs, speed, and highly parallel microplate formats. Here we show that qua ntitative and sensitive measurement of ds DNA in solution is achieved using a 96-well microplate SYBR Green I assay and a standard uv transilluminatio n-based gel-imaging system for detection. Specific detection of ds DNA was obtained over a broad concentration range of 0.5-500 ng using a single low dye concentration of up to 1/6250. Measured SYBR Green I fluorescence was n ot significantly affected by pH variation (4-10), assay volume (50-250 mu l ), and time (4-15 min), and measurements were appreciably compatile with co mmonly encountered concentrations of contaminating salts, organics, deterge nts, and other substances. ds DNA yielded up to 13-fold higher fluorescence compared to single-stranded DNA or RNA, but this ratio was dependent somew hat on GC content and fragment size. Of note, linear ds DNA fluoresced sign ificantly stronger than supercoiled plasmid DNA. Our method should be broad ly applicable for sensitive, rapid, and inexpensive ds DNA quantification i n the average molecular biology laboratory. (C) 1999 Academic Press.