A Quantitative Method of Determining Initial Amounts of DNA by Polymerase Chain Reaction Cycle Titration Using Digital Imaging and a Novel DNA Stain

Abstract

A new nonradioactive method is described for quantitative determination of small amounts of DNA by PCR, examplified with mitochondrial DNA. The method represents a combination of serial dilution PCR and kinetic PCR and avoids the use of radioactivity by applying the fluorescent dye SYBR Green I, allowing visualization of PCR amplified bands on agarose gels in a broad exponential range of PCR cycles. After recording agarose gel images with a video camera in a computer, the band intensities are processed with the NIH image program and analyzed by a new graphical method. This nonradioactive method allows calculation of small original amounts of specific DNA in samples at high accuracy.