[Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction for detecting CD4 receptor mRNA].
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.
D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics*; D002413:Cations, Divalent; D002460:Cell Line; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004795:Enzyme Stability; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*; D014158:Transcription, Genetic
D015704:CD4 Antigens; D002413:Cations, Divalent; D017931:DNA Primers; D012333:RNA, Messenger; D004259:DNA-Directed DNA Polymerase
Ispol'zovanie termostabil'noĭ DNK-polimerazy iz Thermus thermophilus KTP v sovmeshchennoĭ reaktsii obratnoĭ transkriptsii i amplifikatsii dlia detektsii mRNK retseptora CD-4.