Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA
Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA
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DOI：
10.1016/j.foodcont.2008.07.009
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发表时间：
2009-04-01
期刊：
影响因子：
6
通讯作者：
Jenkins, G. Ronald
中科院分区：
文献类型：
--
作者：
Shokere, Luke A.;Holden, Marcia J.;Jenkins, G. Ronald
通过超声或加热降解同基因NK 603 DNA，并使用ANO和两种荧光染料方法进行定量。通过在未处理、超声处理和热处理的样品中扩增SSIIb-3内源对照和NK 603转基因来进行定量PCR（qPCR）实验。基于A（260）定量，超声处理的DNA样品的qPCR反应提供了0.125%、1.14%和2.15%的NK 603;而热处理的样品提供了0.128%、1.42%和2.73%的NK 603的结果。基于荧光染料法，超声处理的DNA样品的qPCR反应提供了0.18%、0.861%和1.74% NK 603的结果;而热处理的DNA样品提供了0.18%、1.02%和2.16% NK 603的结果。数据表明，基于荧光染料的定量在较高浓度下可更准确地测定基因工程（GM）含量百分比，这很可能是因为荧光染料定量导致qPCR中添加了额外的模板拷贝。本研究中的数据表明，荧光染料和分光光度法对高度降解DNA的定量均不能转化为qPCR可扩增DNA的一致测量和准确的Q值。由爱思唯尔有限公司发布
Isogenic NK603 DNA was degraded by sonication or heat and quantified using ANO 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 A(260) 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 Q values. Published by Elsevier Ltd.