Variable stringency hybridization of polymerase chain reaction amplified HIV-1 DNA fragments
| Autor: | R. Snart, K. Laaroubi, Jean-François Zagury, G. Agius, Daniel Zagury, V. Kolesnitchenko |
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| Rok vydání: | 1990 |
| Předmět: |
Immunoblotting
Molecular Sequence Data Dot blot Biology Genes env Polymerase Chain Reaction law.invention chemistry.chemical_compound law Virology HIV Seropositivity Humans Nucleotide Polymerase chain reaction chemistry.chemical_classification Strain (chemistry) Hybridization probe Nucleic Acid Hybridization Molecular biology Genes gag Standard curve chemistry DNA Viral Democratic Republic of the Congo HIV-1 Leukocytes Mononuclear Densitometry Oligonucleotide Probes DNA |
| Zdroj: | Journal of virological methods. 30(2) |
| ISSN: | 0166-0934 |
| Popis: | DNA isolated from the peripheral blood mononuclear cells of HIV-1 seropositive individuals was used for polymerase chain reaction (PCR) amplification of gag and envelope regions. Eight aliquots of the amplified DNA fragments have been subjected to Southern/dot blot analysis, hybridizing with 32P-labelled-BH10 (HTV-1 strain IIIB) at low stringency. After the filters had been autoradiographed, they were cut so that each hybridized band/dot could be subject to variable stringency washing using various ionic concentrations at a fixed temperature. The filter was reconstructed so that the effect of the variable stringency wash might be visualized following a second exposure to Kodak film. The level of activity for each band/dot was measured by counting the 32P or by densitometry analysis of the photographic record. The results allow a plot to be made of the decrease in bound radioactivity against ionic strength. By comparison with a standard curve obtained for HBV-1 strain IIIB amplified fragments subject to similar hybridization and analysis, an estimation of the degree of nucleotide mismatch relative to the BH10 DNA probe can be obtained. The technique provides a rapid means of characterizing PCR amplified fragments. |
| Databáze: | OpenAIRE |
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