Comparison of three RNA amplification methods as sources of DNA for sequencing.
DNA products generated from a region of the measles virus genome by three RNA reverse transcription and amplification methods were cloned and sequenced, and the results were compared in order to evaluate the methods' relative fidelities. The methods were: (i) reverse transcription followed by a nested polymerase chain reaction (RT-nPCR), (ii) a combined RT-PCR using rTth polymerase and (iii) nucleic acid sequence-based amplification (NASBA). NASBA was followed by RT-PCR with rTth polymerase or RT using AMV reverse transcriptase to generate DNA products for cloning. Products from all three sets of reactions were cloned into a vector, pT7Blue, and 790 bp of cloned DNA were sequenced and analyzed for base changes to determine the error rates for each amplification method. Sequence analysis of cloned RT-nPCR products showed no errors, whereas cloned rTth mediated RT-PCR products possessed an error rate of 0.38% and cloned NASBA products 0.38%. Products generated by NASBA followed by RT-PCR with rTth polymerase possessed an error rate of 1.9%. The results indicated that cloned DNA products generated by RT-nPCRs possessed least errors and that for NASBA, RT of reaction products before cloning and sequencing was preferable to using RT-PCR.
Chadwick
N
N
Royal Free Hospital, Hampstead, London, UK.
Wakefield
A J
AJ
Pounder
R E
RE
Bruce
I J
IJ
D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D008459:Measles virus / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014709:Vero Cells
D018076:DNA, Complementary; D012367:RNA, Viral