Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes.
Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to ampl...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2006
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| _version_ | 1826286125700874240 |
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| author | Rousseau, C Birditt, B McKay, A Stoddard, J Lee, T McLaughlin, S Moore, S Shindo, N Learn, G Korber, B Brander, C Goulder, P Kiepiela, P Walker, B Mullins, J |
| author_facet | Rousseau, C Birditt, B McKay, A Stoddard, J Lee, T McLaughlin, S Moore, S Shindo, N Learn, G Korber, B Brander, C Goulder, P Kiepiela, P Walker, B Mullins, J |
| author_sort | Rousseau, C |
| collection | OXFORD |
| description | Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization. |
| first_indexed | 2024-03-07T01:39:08Z |
| format | Journal article |
| id | oxford-uuid:9639a753-26ab-437c-b80f-5aaed155259c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:39:08Z |
| publishDate | 2006 |
| record_format | dspace |
| spelling | oxford-uuid:9639a753-26ab-437c-b80f-5aaed155259c2022-03-26T23:51:44ZLarge-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:9639a753-26ab-437c-b80f-5aaed155259cEnglishSymplectic Elements at Oxford2006Rousseau, CBirditt, BMcKay, AStoddard, JLee, TMcLaughlin, SMoore, SShindo, NLearn, GKorber, BBrander, CGoulder, PKiepiela, PWalker, BMullins, JFull-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization. |
| spellingShingle | Rousseau, C Birditt, B McKay, A Stoddard, J Lee, T McLaughlin, S Moore, S Shindo, N Learn, G Korber, B Brander, C Goulder, P Kiepiela, P Walker, B Mullins, J Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. |
| title | Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. |
| title_full | Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. |
| title_fullStr | Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. |
| title_full_unstemmed | Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. |
| title_short | Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. |
| title_sort | large scale amplification cloning and sequencing of near full length hiv 1 subtype c genomes |
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