REVERSE-TRANSCRIPTASE INHIBITS TAQ POLYMERASE-ACTIVITY
REVERSE-TRANSCRIPTASE INHIBITS TAQ POLYMERASE-ACTIVITY
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DOI：
10.1093/nar/20.7.1487
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发表时间：
1992-04-11
影响因子：
14.9
通讯作者：
MACKENZIE, JS
中科院分区：
文献类型：
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作者：
SELLNER, LN;COELEN, RJ;MACKENZIE, JS
通过聚合酶链式反应 (PCR) 检测病毒 RNA 需要事先对病毒 RNA 进行逆转录。为了最大限度地减少处理大量样品所需的手动操作次数，我们尝试设计一种系统，将逆转录和扩增所需的所有试剂添加到一个管中，并执行单个不间断的热循环程序。在尝试用 Taq 聚合酶（Taq；Biotech International）和禽成肌细胞增多症病毒（AMV）逆转录酶（RT）建立这样的单管系统时，我们注意到病毒 RNA 检测的灵敏度大幅下降。对这一现象的研究揭示了 RT 对 Taq 聚合酶的直接干扰。支持这一结论的证据包括以下观察结果：（1）增加 Taq 与 RT 的比率可提高灵敏度； (2)添加非同源RNA提高灵敏度； (3) 在添加 Taq 之前已热灭活的 RT 不会发挥此作用； (4) 效果不受顺序限制； (5) Mg2+ 离子不会被 RT 隔离。此外，影响不仅仅限于AMV RT，莫洛尼鼠白血病病毒RT也影响Taq活性。
Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.