Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR
Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR
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低浓度模板下的逆转录酶 (RT) 抑制 PCR 及其对定量 RT-PCR 的影响
DOI：
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发表时间：
1998
影响因子：
4.4
通讯作者：
H. Bolton
中科院分区：
文献类型：
--
作者：
D. Chandler;Christina A. Wagnon;H. Bolton
摘要在发展非定量解偶联RT-PCR技术原位检测固氮酶和氨单加氧酶基因表达时，观察到大量的PCR逆转录酶（RT）抑制的情况。随着模板浓度的增加超过105至106个拷贝，RT对PCR的抑制作用被去除。在RT-PCR反应的逆转录阶段包括T4基因32蛋白使RT-PCR产物产率增加多达483%;如果在逆转录之后但在PCR阶段之前引入基因32蛋白，则没有观察到产物产率的改善。加入1 μg外源小牛胸腺DNA或酵母tRNA对解除PCR对基因组DNA和mRNA模板的RT抑制作用很小。这些结果表明，PCR的RT抑制是通过与特异性引物-模板组合（DNA和RNA）的直接相互作用介导的，并指出了用于估计RT抑制程度和抵消一些抑制作用的特异性测定修改。此外，RT抑制低于105至106拷贝阈值的工作假设对定量RT-PCR研究具有重要意义。特别是，竞争性的定量RT-PCR系统将始终低估实际的RNA浓度。因此，低于105至106个拷贝的RNA模板的计数将相对于内标物，而不是原位RNA丰度的绝对量度。
ABSTRACT Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ. The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 105 to 106 copies. Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed. Addition of 1 μg of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates. These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect. Furthermore, the working hypothesis of RT inhibition below a 105 to 106 copy threshold has important implications for quantitative RT-PCR studies. In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration. Hence, enumerations of RNA templates below 105 to 106 copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.
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噬菌体 T4 DNA 螺旋不稳定蛋白的一级结构。
DOI：
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发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Williams,KR;LoPresti,MB;Setoguchi,M
通讯作者：
Setoguchi,M
影响因子：
56.9
作者：
CRAIK, CS;LARGMAN, C;RUTTER, WJ
通讯作者：
RUTTER, WJ
噬菌体 T4 基因 32 和 45 的产物提高了 DNA 体外复制的准确性。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Topal,MD;Sinha,NK
通讯作者：
Sinha,NK
影响因子：
14.9
作者：
ZACHAR, V;THOMAS, RA;GOUSTIN, AS
通讯作者：
GOUSTIN, AS