Do Taq-generated RT-PCR products from RNA viruses accurately reflect viral genetic heterogeneity?
Article first published online: 20 FEB 2004
Journal of Viral Hepatitis
Volume 11, Issue 2, pages 108–114, March 2004
How to Cite
Mullan, B., Sheehy, P., Shanahan, F. and Fanning, L. (2004), Do Taq-generated RT-PCR products from RNA viruses accurately reflect viral genetic heterogeneity?. Journal of Viral Hepatitis, 11: 108–114. doi: 10.1046/j.1365-2893.2003.00455.x
- Issue published online: 20 FEB 2004
- Article first published online: 20 FEB 2004
- Received February 2003; accepted for publication March 2003
- error rate;
- hepatitis C virus;
- reverse-transcribed polymerase chain reaction
Summary. Since the first report of genetically heterogeneous, or quasispecies, populations of RNA viruses, the genetic heterogeneity of the RNA genomes of major viral pathogens has been extensively studied. These studies aim to provide insights into the evolutionary pressures that act upon viruses, in order to define windows where anti-viral therapies will be most effective, to take prognostic values from viral genetic distributions at a given time, and to aid the development of novel therapeutic compounds that may tilt viral replication towards information loss.
Many methodologies are employed to analyse genetic distributions of a virus in a given sample, but all involve the generation, and subsequent analysis, of the sequence information contained in a reverse-transcription-polymerase chain reaction (RT-PCR) product. Despite the fact that the aim of these RT-PCRs is to obtain sequence information from viral genomes, their application to this task is approached without adequate consideration of this end-goal. The establishment of an RT-PCR for a specific viral target genome generally proceeds in the same fashion as one would apply to establishing a PCR to determine the presence or absence of a specific target sequence in a given sample. However, it is becoming increasingly apparent that RT-PCR products generated by amplification with the ubiquitous thermostable DNA polymerase Taq, coupled with standard cloning and sequencing methodologies, has the potential to yield inaccurate and misleading data as pertains to the information content of populations of RNA viral genomes. This review discusses varying approaches employed to analyse heterogeneous populations of hepatitis C virus RNA genomes.