Comparison of Fluorometric and Spectrophotometric DNA Quantification for Real-time Quantitative PCR of Degraded DNA
Luke A. Shokere, Marcia J. Holden, G. Ronald Jenkins
Isogenic NK603 DNA was degraded by sonication or heat and quantified using A260 and a fluorescent dye method. 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 GM-content at higher concentrations most likely because fluorescent dye quantifications provided additional copies of template into the qPCR.
A260, degraded DNA quantitative real-time PCR, DNA quantification, Hoescht dye, NK603 maize, Picogreen fluorescence.
, Holden, M.
and Jenkins, G.
Comparison of Fluorometric and Spectrophotometric DNA Quantification for Real-time Quantitative PCR of Degraded DNA, Food Control
(Accessed November 29, 2023)