Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA

Isogenic NK603 DNA was degraded by sonication or heat and quantified using A260 and two fluorescent dye methods. Quantitative PCR (qPCR) experiments were conducted by amplifying an SSIIb-3 endogenous control and an NK603 transgene in untreated, sonicated, and heat treated samples. qPCR reactions on sonicated DNA samples, based on A260 quantification, provided 0.125%, 1.14% and 2.15% NK603; while heat treated samples, provided results of 0.128%, 1.42%, and 2.73% NK603. qPCR reactions on sonicated DNA samples, based on the fluorescent dye method, provided results of 0.18%, 0.861% and 1.74% NK603; while heat treated DNA samples, provided results of 0.18%, 1.02%, and 2.16% NK603. The data suggested that fluorescent dye-based quantifications yielded more accurate determinations of the percent genetically engineered (GM) content at higher concentrations, most likely because fluorescent dye quantifications resulted in additional copies of template added into the qPCR. The data in this study suggested that neither fluorescent dye nor spectrophotometric methods of quantification on highly degraded DNA translated into concordant measurements of qPCR amplifiable DNA and accurate Ct values.