Reverse transcription, amplification and sequencing of poliovirus RNA by Taq DNA polymerase.
A model for virion RNA of the poliomyelitis virus, which does not pass the stage of DNA copies during biogenesis, demonstrates that Taq DNA polymerase is capable of synthesizing 960-bp cDNA with the specific primer. When comparing the nucleotide sequence of the starting virion RNA and recombinant DNAs, isolated from several independent clones, copying and amplification of virion RNA appear accurate (one substitution per 960 bp). A comparison of Taq and Tth DNA polymerases in RT/PCR indicates that the sensitivity of Taq polymerase seems to be two orders of magnitude higher than that of Tth polymerase. The RNA detection level under the chosen conditions approached 10(4) RNA copies per test. The present investigation indicates the great versatility of Taq polymerase, which promoted the reverse transcription reaction of RNA, cDNA amplification, screening of recombinant clones as well as sequencing of recombinant DNA. Thus application of Taq polymerase is rather promising not only to detect nucleic acids in biological samples, but also for isolating and cloning individual genes, encoded on DNA and/or on RNA templates.
Grabko
V I
VI
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Russia. GRABKO@ibch.siobc.ras.ru
Chistyakova
L G
LG
Lyapustin
V N
VN
Korobko
V G
VG
Miroshnikov
A I
AI
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D019914:Taq Polymerase; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase