Myers T W, Gelfand D H
PCR Division, Cetus Corporation, Emeryville, California 94608.
Biochemistry. 1991 Aug 6;30(31):7661-6. doi: 10.1021/bi00245a001.
A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2. Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full-length products can be obtained. The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme. The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol). The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA. Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line. Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA. The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA.
人们发现，源自嗜热真细菌嗜热栖热菌（Tth pol）的重组DNA聚合酶在存在氯化锰的情况下具有非常高效的逆转录酶（RT）活性。通过使用热稳定DNA聚合酶进行逆转录，通常与RNA中高度二级结构相关的许多问题得以最小化，并且主要可以获得全长产物。cDNA也可以用同一种酶在聚合酶链反应（PCR）中进行扩增。观察到Tth pol在耦合RT/PCR中的效率比来自嗜热水栖菌的类似DNA聚合酶（Taq pol）高出100倍以上。Tth pol进行的反应的灵敏度使得从低至100份合成cRNA开始就能检测到溴化乙锭染色的产物。从费城染色体阳性细胞系的RNA也获得了类似结果。从80 pg总细胞RNA开始就能够检测到IL-1α mRNA。Tth pol进行逆转录和DNA扩增的能力无疑将在细胞和病毒RNA的检测、定量和克隆中证明是有用的。
