DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair.
DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair.
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DNA超螺旋抑制了实时PCR：一种定量线粒体DNA损伤和修复的新方法。
DOI：
10.1093/nar/gkm010
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发表时间：
2007
影响因子：
14.9
通讯作者：
Chen JZ
中科院分区：
文献类型：
--
作者：
Chen J;Kadlubar FF;Chen JZ
实时荧光定量PCR作为核酸起始量定量的金标准，在医学研究中越来越多地用于mtDNA拷贝数的定量分析。利用体外和癌细胞中修饰的超螺旋质粒DNA和mtDNA，我们证明了超螺旋DNA的构象变化对实时PCR定量有深远的影响。我们发现实时PCR信号是DNA松弛形式（开放圆形和/或线性）而不是超螺旋形式的正函数，并且DNA链断裂介导的构象转变是敏感检测松弛DNA的主要基础。这一新发现随后被用于结构介导的mtDNA损伤和修复的敏感检测，以及当所有超卷曲DNA通过先前的热变性步骤转化为松弛形式时，用于准确定量总mtDNA拷贝数。新方法揭示了氧化应激对前列腺癌细胞mtDNA的动态响应，不仅涉及过氧化氢诱导的早期结构损伤和修复，还涉及持续的拷贝数减少。最后，超卷绕效应应该在使用实时PCR进行DNA定量时提高警惕。
As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.
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影响因子：
4.7
作者：
Chen, JJZ;Gokden, N;Kadlubar, FF
通讯作者：
Kadlubar, FF
影响因子：
8.8
作者：
Gahan, ME;Miller, F;Wesselingh, SL
通讯作者：
Wesselingh, SL
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
影响因子：
4.8
作者：
Ayala-Torres, S;Chen, YM;Van Houten, B
通讯作者：
Van Houten, B
影响因子：
14.9
作者：
Harrison JF;Hollensworth SB;Spitz DR;Copeland WC;Wilson GL;LeDoux SP
通讯作者：
LeDoux SP