Thermal effects on reverse transcription: improvement of accuracy and processivity in cDNA synthesis.
Reverse transcription, coupled with DNA amplification, has been widely used for molecular analysis of RNAs. Reverse transcriptases are retroviral DNA polymerases that can synthesize DNA from both RNA and DNA. In general, because of the lack of 3'-->5' exonuclease activity in retroviral reverse transcriptases, the reverse transcription step is error prone. Mutations created during the reverse transcription step of cDNA synthesis or RT-PCR are delivered to the final products to be analyzed, interfering with accurate analysis of the RNAs. In addition, because reverse transcription uses RNA as a template, processive DNA synthesis by reverse transcriptase is frequently interrupted by secondary structures of the RNA templates, causing difficulties in full-length cDNA synthesis. Here, we report that an increase in reaction temperature greatly enhances both the accuracy and the processivity of reverse transcription catalyzed by murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases.
Malboeuf
C M
CM
University of Rochester Medical Center, Rochester, NY, USA.
Isaacs
S J
SJ
Tran
N H
NH
Kim
B
B
D018076:DNA, Complementary / Q000096:biosynthesis*; D003854:Deoxyribonucleotides / Q000378:metabolism; D015684:Gene Products, pol / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D009052:Leukemia Virus, Murine / Q000201:enzymology; D009154:Mutation; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D018076:DNA, Complementary; D003854:Deoxyribonucleotides; D015684:Gene Products, pol; D012313:RNA; D012194:RNA-Directed DNA Polymerase