COMPARISON OF 3 RNA AMPLIFICATION METHODS AS SOURCES OF DNA FOR SEQUENCING

DNA products generated from a region of the measles virus genome by th ree RNA reverse transcription and amplification methods were cloned an d sequenced, and the results were compared in order to evaluate the me thods' relative fidelities. The methods were: (i) reverse transcriptio n followed by a nested polymerase chain reaction (RT-nPCR), (ii) a com bined RT-PCR using rTth polymerase and (iii) nucleic acid sequence-bas ed amplification (NASBA). NASBA was followed by RT-PCR with rTth polym erase or RT using AMV reverse transcriptase to generate DNA products f or cloning. Products from all three sets of reactions were cloned into a vector, pT7Blue, and 790 bp of cloned DNA were sequenced and analyz ed for base changes to determine the error rates for each amplificatio n method. Sequence analysis of cloned RT-nPCR products showed no error s, whereas cloned rTth mediated RT-PCR products possessed an error rat e of 0.38% and cloned NASBA products 0.38%. Products generated by NASB A followed by RT-PCR with rTth polymerase possessed an error rate of 1 .9%. The results indicated that cloned DNA products generated by RT-nP CRs possessed least errors and that for NASBA, RT of reaction products before cloning and sequencing was preferable to using RT-PCR.