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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 te...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2004
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC543450/ https://www.ncbi.nlm.nih.gov/pubmed/15588294 http://dx.doi.org/10.1186/1743-422X-1-13 |
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author | Bracho, Maria Alma García-Robles, Inmaculada Jiménez, Nuria Torres-Puente, Manuela Moya, Andrés González-Candelas, Fernando |
author_facet | Bracho, Maria Alma García-Robles, Inmaculada Jiménez, Nuria Torres-Puente, Manuela Moya, Andrés González-Candelas, Fernando |
author_sort | Bracho, Maria Alma |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-543450 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2004 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-5434502005-01-07 Effect of oligonucleotide primers in determining viral variability within hosts Bracho, Maria Alma García-Robles, Inmaculada Jiménez, Nuria Torres-Puente, Manuela Moya, Andrés González-Candelas, Fernando Virol J Methodology 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. BioMed Central 2004-12-09 /pmc/articles/PMC543450/ /pubmed/15588294 http://dx.doi.org/10.1186/1743-422X-1-13 Text en Copyright © 2004 Bracho et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Bracho, Maria Alma García-Robles, Inmaculada Jiménez, Nuria Torres-Puente, Manuela Moya, Andrés González-Candelas, Fernando Effect of oligonucleotide primers in determining viral variability within hosts |
title | Effect of oligonucleotide primers in determining viral variability within hosts |
title_full | Effect of oligonucleotide primers in determining viral variability within hosts |
title_fullStr | Effect of oligonucleotide primers in determining viral variability within hosts |
title_full_unstemmed | Effect of oligonucleotide primers in determining viral variability within hosts |
title_short | Effect of oligonucleotide primers in determining viral variability within hosts |
title_sort | effect of oligonucleotide primers in determining viral variability within hosts |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC543450/ https://www.ncbi.nlm.nih.gov/pubmed/15588294 http://dx.doi.org/10.1186/1743-422X-1-13 |
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