The effects of mutations on HIV-1 quantitation by RT/PCR.
Int Conf AIDS 1996 Jul 7-12; 11:7 (abstract no. Mo.A.161) Christopherson CD, Kwok S; Roche Molecular Systems, Alameda, CA, USA. Fax: (510)814-2810.
Primer design and amplification conditions were previously shown to effect 3' terminal mismatch tolerance (Kellog et. al., NAR 1990). In this study, we investigated the effect of internal primer-template mismatches on reverse transcription (RT)-PCR and of probe-template mismatches on probe capture efficiency. Templates were constructed by PCR that contained mutations that represent various HIV-1 subtypes in either the upstream primer binding region (SK462), the downstream primer binding region (SK431), or the probe binding region (SK102). RNA transcripts were generated by in vitro transcription of the amplified DNA products. The effects of mismatches were determined by a quantitative RT/PCR assay. A maximum of 6 mutations in the upstream primer, 5 in the downstream primer and 5 in the probe region were examined. DNA amplification efficiency was not affected by as many as 4 mutations, but a decrease in efficiency was seen when 5-6 mutations were present. Mismatch tolerance was significantly improved by lowering the annealing temperature and/or by incorporating unconventional bases that stabilize DNA duplexes. In the worst case, the presence of 5 mutations in the RT primer reduced product yield by 10-fold, but a "step-up" RT, whereby the temperature increases from 50 degrees -60 degrees C over 30 min., reduced this to 4-fold. As many as 5 mutations can be tolerated in the probe binding region with no effect on hybridization efficiency. These results indicate that subtype B isolates, which are predominant in the U.S. and Europe, will be efficiently amplified and detected in this system given the small number of mismatches to both SK462 and SK431. Similarly, isolates of subtype C, D, F, G, and H are expected to amplify efficiently. Isolates that contain 5-6 mutations (some subtypes A and E) will be amplified less efficiently. However, amplification efficiency of these isolates can be significantly improved by performing a "step up" reverse transcription, by lowering the anneal temperature during PCR, or by incorporating unconventional bases.
Keywords: AEGIS, Polymerase Chain Reaction, HIV-1, Templates, Genetic, Mutation, DNA Primers, Nucleic Acid Amplification Techniques, HIV-1 Reverse Transcriptase, RNA-Directed DNA Polymerase, Oligonucleotide Probes, Transcription, Genetic, DNA, DNA Fingerprinting, Europe, United States, In Vitro, genetics, ICA11
