Glukhov A I, Grebennikova T V, Kiselev V I, Severin E S
Mol Biol (Mosk). 1995 Jul-Aug;29(4):942-9.
The RT/PCR method was applied to study a possible use of Tth DNA-polymerase for coupled reaction of reverse transcription and polymerase chain reaction (RT/PCR) on the CD-4 receptor mRNA template in the total cellular RNA. The conditions for detecting the CD-4 receptor mRNA were optimized. The pH-optimum for RT reaction was 8.8. The influence of Mn2+, Cu2+, Co2+, and Cd2+ cations in RT and PCR reaction was investigated. The efficiency of the RT reaction was shown to be the highest in the presence of Mn2+ (optimal concentration 1 mM). At Mn2+ concentration > or = 3 mM complete inhibition of RT/PCR was observed. The Tth DNA polymerase in RT/PCR was shown to be more effective than Taq DNA polymerase. The Tth DNA polymerase allows observation of the specific product in the gel containing ethidium bromide using 20 ng of the total RNA. High sensitivity and specificity of RT/PCR performed with the Tth DNA polymerase allow its wide application in the detection, quantitative analysis and cloning of cellular and viral RNAs.
采用逆转录/聚合酶链反应（RT/PCR）方法，研究了嗜热栖热菌DNA聚合酶（Tth DNA聚合酶）在以细胞总RNA中CD-4受体mRNA为模板进行逆转录与聚合酶链反应偶联反应（RT/PCR）中的可能应用。优化了检测CD-4受体mRNA的条件。RT反应的最适pH为8.8。研究了Mn2+、Cu2+、Co2+和Cd2+阳离子在RT和PCR反应中的影响。结果表明，在存在Mn2+（最佳浓度1 mM）时，RT反应效率最高。当Mn2+浓度≥3 mM时，观察到RT/PCR完全抑制。RT/PCR中的Tth DNA聚合酶比Taq DNA聚合酶更有效。使用20 ng细胞总RNA，Tth DNA聚合酶可在含溴化乙锭的凝胶中观察到特异性产物。用Tth DNA聚合酶进行的RT/PCR具有高灵敏度和特异性，使其在细胞和病毒RNA的检测、定量分析及克隆中得到广泛应用。
