A simple method for elimination of false positive results in RT-PCR.
A simple method for elimination of false positive results in RT-PCR.
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消除 RT-PCR 中假阳性结果的简单方法。
DOI：
10.5483/bmbrep.2002.35.2.248
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
E. Schömig
中科院分区：
文献类型：
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作者：
F. Martel;D. Gründemann;E. Schömig
在进行逆转录酶-聚合酶链式反应（RT-PCR）时，mRNA的扩增和污染基因组DNA之间的区分是常见的问题。即使用DNA酶处理样品后，阴性对照（未添加逆转录酶的样品）也可能得到阳性结果。这表明存在DNA扩增，其不是在逆转录酶步骤中产生的。Taq DNA聚合酶有可能充当逆转录酶，在PCR步骤中从RNA生成cDNA。为了检验这一假设，我们在cDNA合成后将样品与无DNAse的RNAse孵育。比较从这些样本（与或不与无DNA酶的RNA酶一起孵育）获得的结果证实，当对无内含子基因进行RT-PCR时，TaqDNA聚合酶I的逆转录酶活性可能是假阳性结果的来源。此外，我们在这里描述了一种简单而快速的方法来克服由Taq聚合酶的这种活性引起的假阳性结果。
Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.