Capillary electrophoresis: detection of hybridization between synthetic oligonucleotides and HIV-1 genomic DNA amplified by polymerase-chain reaction
| Autor: | Dario Fiorentino, Roberto Gambari, Nicoletta Bianchi, Carlo Mischiati, Sebastiano Di Biase, Giordana Feriotto, Nicola Apicella |
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| Rok vydání: | 1994 |
| Předmět: |
Electrophoresis
Base Sequence Inverse polymerase chain reaction Molecular Sequence Data Oligonucleotides Multiple displacement amplification Nucleic Acid Hybridization Genome Viral Biology Polymerase cycling assembly Polymerase Chain Reaction Molecular biology Polymerase chain reaction optimization Real-time polymerase chain reaction Virology Primer dimer DNA Viral HIV-1 Capillary electrophoresis Molecular diagnosis Polymerase chain reaction Applications of PCR In silico PCR |
| Zdroj: | Journal of Virological Methods. 47:321-329 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/0166-0934(94)90028-0 |
| Popis: | The polymerase chain reaction (PCR) is one of the most efficient techniques for measuring the viral load of HIV-infected samples. Determination of the specificity of PCR products is usually based on Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes specific for sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) for identification of HIV-1 and HTLV-I PCR products appears interesting in light of its reproducibility, sensitivity and because it is fast and suitable for detection of DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of a HIV-1 oligonucleotide probe to single-stranded DNA obtained by unbalanced PCR is detectable by capillary electrophoresis. This enabled us the application of a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. This procedure is simple, reproducible and is suggested as an integral part of automated diagnostic systems based on the use of laboratory work stations for DNA isolation, preparation of PCR reactions and analysis of PCR products. |
| Databáze: | OpenAIRE |
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