An improved method of competitive PCR for quantitation of gene copy number.
An improved method of competitive PCR for quantitation of gene copy number.
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一种改进的竞争性 PCR 定量基因拷贝数的方法。
DOI：
10.1093/nar/21.20.4848
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发表时间：
1993
影响因子：
14.9
通讯作者：
Smith,HS
中科院分区：
文献类型：
--
作者：
Deng,G;Yu,M;Smith,HS
先前的研究使用差异 PCR 来定量人类肿瘤组织中靶基因的扩增，并利用第二个非扩增基因的共扩增作为内标 (1)。然而，这种方法没有考虑到当在同一样品中扩增多于一组引物时PCR扩增可能出现问题的问题。最近，在通过竞争性 PCR 定量基因拷贝数时，使用与靶基因相同的引物进行扩增的片段已被用作内标 (2-4)。当这些内标片段（此处缩写为“Std”）在PCR前与DNA混合时，PCR后的目标序列与标准序列的比率将指示目标序列的相对量。确定给定 DNA 样本的基因拷贝数需要与含有已知目标基因拷贝数的第二个 DNA 来源进行比较。为了使该技术准确，必须将来自已知拷贝数的 DNA 来源的等量基因组 DNA 和测试 DNA 添加到这两个单独的 PCR 混合物中。然而，当测试DNA来自肿瘤标本时，通过光密度判断的DNA量并不一定代表添加到PCR混合物中的目标序列的量，因为肿瘤源性DNA制剂经常被RNA污染，并且经常表现出不同程度的DNA降解。因此，通过竞争性 PCR 确定的拷贝数可能不准确。在本报告中，我们通过添加第二个 PCR 反应来规避与肿瘤来源 DNA 相关的问题，该反应扩增肿瘤 DNA 中未扩增的第二个基因，该基因与目标片段大小相似，因此它应具有与目标序列相同程度的降解。因此，该反应对照的 PCR 产物应反映 PCR 中使用的靶标的量。为了更准确地测量对照DNA的量，将另一个内标片段（缩写为“Std”）添加到第二个PCR反应中，该内标片段被构建为使用与对照基因相同的引物进行扩增。分别用PCR扩增靶序列和反应对照序列后，靶序列与Stdt的比值除以反应对照与Std的比值的商，即为细胞中靶基因的相对拷贝数：
Previous studies using differential PCR to quantitate amplification of a target gene in human tumor tissue utilized co-amplification of a second nonamplified gene as an internal standard (1). However, this approach does not take into account the problem that PCR amplification can be problematic when more than one set of primers are amplified in the same sample. More recently, fragments which are constructed so that they can be amplified with the same primers as the target gene have been used as an internal standard when quantitating gene copy number by competitive PCR (2-4). When these internal standard fragments (here abbreviated'Std,') are mixed with DNA before PCR, the ratio oftarget to Std, after PCR will indicate the relative amount of target sequence. Determination of gene copy number for a given DNA sample requires comparison with a second source of DNA containing a known copy number for the target gene. For this technique to be accurate, equivalent amounts ofgenomic DNA from the source of DNA with known copy number and test DNA must be added to these two separate PCR mixture. However, when the test DNA is derived from tumor specimens, the DNA amountjudged by optical density does not necessarily represent the amount oftarget sequence added to the PCR mixture because tumor-derived DNA preparations frequently are contaminated with RNA, and often show variable degrees of DNA degradation. Thus, the copy numbers determined by competitive PCR could be inaccurate. In this report, we circumvent the problems associated with tumor-derived DNA by adding a second PCR reaction which amplifies a second gene which is not amplified in tumor DNA and is similar in size to the target fragment so that it should have the same degree of degradation as the target sequence. Thus, the PCR product of this reaction control should reflect the amount of the target used in PCR. For more accurate measurement of the amount of control DNA, another internal standard fragment (abbreviated'Std,'), constructed so that it amplifies with the same primers as the control gene, was added to the second PCR reaction. After the target and reaction control sequences were amplified by PCR separately, the quotient of the ratio of target to Stdt divided by the ratio of reaction control to Std, will represent the relative copy number ofthe target gene in the cells: