FIDELITY OF NUCLEIC-ACID AMPLIFICATION WITH AVIAN-MYELOBLASTOSIS VIRUS REVERSE-TRANSCRIPTASE AND T7 RNA-POLYMERASE

The Nucleic Acid Sequence-Based Amplification (NASBA(TM)) process invo lves alternate steps of DNA synthesis from an RNA template and RNA syn thesis from a DNA template, using avian myeloblastosis virus (AMV) rev erse transcriptase and T7 RNA polymerase, respectively. The overall fi delity of the amplification process was determined by sequence analysi s of cloned DNA products of NASBA reactions. An error frequency of les s than 0.3% was observed in cloned DNA products from two different seg ments of the HIV-1 gag gene. Partial substitution of GTP with ITP in t he NASBA reaction did not significantly change the fidelity of the pro cess. An error rate of 2 x 10(-4) was calculated for the combined effe cts of both polymerases.