Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase.
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.
Myers
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TW
PCR Division, Cetus Corporation, Emeryville, California 94608.
Gelfand
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DH
D001483:Base Sequence; D002460:Cell Line; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D007375:Interleukin-1; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase