Effect of oligonucleotide primers in determining viral variability within hosts.
BACKGROUND
Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR) based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet.


RESULTS
To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV) populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient). Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers.


CONCLUSIONS
Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.
Bracho
Maria Alma
MA
Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, Edifici Instituts, Polígon La Coma s/n, Paterna, Spain. alma.bracho@uv.es
García-Robles
Inmaculada
I
Jiménez
Nuria
N
Torres-Puente
Manuela
M
Moya
Andrés
A
González-Candelas
Fernando
F
D017931:DNA Primers / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D009841:Oligonucleotides; D012367:RNA, Viral