High sensitive one-step RT-PCR using MMLV reverse transcriptase, DNA polymerase with reverse transcriptase activity,and DNA/RNA helicase.
High sensitive one-step RT-PCR using MMLV reverse transcriptase, DNA polymerase with reverse transcriptase activity,and DNA/RNA helicase.
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使用 MMLV 逆转录酶、具有逆转录酶活性的 DNA 聚合酶和 DNA/RNA 解旋酶进行高灵敏度一步 RT-PCR。
DOI：
10.1016/j.bbrc.2017.04.030
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Yasukawa,K.
中科院分区：
文献类型：
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作者：
Okano,H.; Baba,M.;Yamasaki,T.;Hidese,R.;Fujiwara,S.;Yanagihara,I.;Ujiiye,T.;Hayashi,T.;Kojima,K.;Takita,T.;Yasukawa,K.
在一步RT-PCR中，cDNA合成和随后的PCR在单个封闭管中进行。与两步法RT-PCR相比，一步法RT-PCR显著降低了DNA污染的风险。当逆转录病毒逆转录酶（RT）和耐热DNA聚合酶用于一步RT-PCR时，必须优化反应条件，使得它们分别在cDNA合成和PCR中表现出高性能。然而，这种优化可能难以实现，因为逆转录病毒RT和热稳定DNA聚合酶的最佳反应条件是不同的。一些细菌或古细菌以及野生型或基因工程DNA聚合酶具有RT活性[1]、[2]、[3]、[4]。因为它们甚至在90-100 ℃下也是稳定的，所以可以仅使用这样的DNA聚合酶进行一步RT-PCR。然而，它们的RT活性显著低于逆转录病毒RT [5]。因此，一步RT-PCR不如两步RT-PCR敏感，因此应用不广泛。在一些核酸相关的反应中，同时使用两种或多种酶。这些包括等温RNA特异性扩增[6]，[7]，其中使用逆转录病毒RT，T7 RNA聚合酶和大肠杆菌RNA酶H;和RNA酶H依赖性PCR [8]，其中使用热稳定的RNA酶H2和DNA聚合酶。我们建立了一种新的cDNA合成反应，其中基因工程的莫洛尼鼠白血病病毒（MMLV）RT，MM 4的热稳定变体（E286 R/E302 K/L435 R/D524 A）;具有RT活性的家族A DNA聚合酶的遗传工程化变体，来自极端嗜热细菌嗜石油热袍菌（Thermotoga petrophila）K4的K4 pol L329 A;和来自超嗜热古菌Thermococcus kodakarensis的广古菌特异性DNA/RNA解旋酶Tk-EshA [9]。在该反应中，使用田口统计方法优化了16个因素，包括酶和MgCl 2的浓度[9]。
In one-step RT-PCR, cDNA synthesis and subsequent PCR are carried out in a single, closed tube. Compared with two-step RT-PCR, one-step RT-PCR significantly minimizes the risk of DNA contamination. When retroviral reverse transcriptase (RT) and thermostable DNA polymerase are used in one-step RT-PCR, reaction conditions must be optimized so that they exhibit high performance in cDNA synthesis and PCR, respectively. However, such optimization can be difficult to achieve because the optimum reaction conditions of retroviral RT and thermostable DNA polymerase are different. Some bacterial or archaeal, and wild-type or genetically engineered DNA polymerases have RT activity [1],[2],[3],[4]. Because they are stable even at 90–100 C, one-step RT-PCR can be carried out using only such DNA polymerases. However, their RT activity is considerably lower than that of retroviral RTs [5]. Consequently, one-step RT-PCR is not as sensitive and, therefore, less widely used than two-step RT-PCR.In some nucleic acid-related reactions, two or more enzymes are simultaneously employed. These include isothermal RNA-specific amplification [6],[7], wherein retroviral RT, T7 RNA polymerase, and Escherichia coli RNase H are employed; and RNase H-dependent PCR [8] in which thermostable RNase H2 and DNA polymerase are used. We established a novel cDNA synthesis reaction in which the genetically engineered thermostable variant of Moloney murine leukemia virus (MMLV) RT, MM4 (E286R/E302K/L435R/D524A); the genetically engineered variant of family A DNA polymerase with RT activity, K4pol L329A from the hyperthermophilic bacterium Thermotoga petrophila K4; and the euryarchaeota specific DNA/RNA helicase Tk-EshA from the hyperthermophilic archaeon Thermococcus kodakarensis are used [9]. In this reaction, 16 factors, including the concentrations of enzymes and MgCl 2 were optimized using the Taguchi statistical method [9].