Nurpeisov V, Hurwitz SJ, Schinazi RF, Sharma PL; HIV DRP Symposium Antiviral Drug Resistance.
Program Abstr HIV DRP Symp Antivir Drug Resist. 2002 Dec 8-11; 3: abstract no. 49.
Department of Pediatrics and Laboratory of Biochemical Pharmacology, Emory University, School of Medicine and Veterans Affairs Medical Center, Decatur, Georgia
Soon after the role of drug-selected RT and protease mutations on HIV-1 replication fitness became evident, automated sequencing strategies were used for quantification of viral growth fitness. This was performed by comparing the peak heights of two nucleotides at a single locus in a chromatogram. Previous studies have compared sequenase fluorescent dye-labeled dideoxynucleotide system (dye-terminator system) with dye-labeled primer system. It was concluded that the dye-labeled primer system is more appropriate for the quantitative detection of mixture of HIV-1 drug resistance mutations due to a greater correlation of peak heights with the amount of relative DNA present. The dye terminator system has been routinely used for monitoring the development of resistance mutations in HIV-RT and protease genes in humans undergoing antiretroviral therapy. This system was not previously validated for the quantitative determination between peak heights and relative DNA concentrations. We tested this system extensively using various ratios of wild type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures (Tm) of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentrations demonstrated a statistically significant (r2 = 0.99) linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the dye terminator system. These strategies have broader implications for the quantification of replication fitness of viruses particularly those containing RT mutations at codons 74 and 184.
Publication Types:
Keywords:
- Acquired Immunodeficiency Syndrome
- Base Sequence
- Codon
- DNA Replication
- Dideoxynucleosides
- Fluorescent Dyes
- HIV Infections
- HIV-1
- Humans
- Mutation
- Terminator Regions (Genetics)
- genetics
- methods
- reverse transcriptase, Human immunodeficiency virus 1
- virology
Other ID:
UI: 102260099
From Meeting Abstracts