A new method that combines DNA bar coding and pyrosequencing can identify multiple rare HIV drug resistance mutations in a single test, according to a report in the June 18th Nucleic Acids Research.
"Deep HIV drug resistance genotyping should be readily possible using DNA bar coding and pyrosequencing," Dr. Frederic D. Bushman from University of Pennsylvania School of Medicine in Philadelphia told Reuters Health.
Dr. Bushman and associates combined DNA bar coding and massively parallel pyrosequencing to quantify rare HIV drug resistance mutations.
The researchers designed 11 overlapping amplicons to allow analysis of all known positions of protease (PR) and reverse transcriptase (RT) drug resistance mutations while allowing purification of fragments ranging in size from 200 to 400 base pairs.
In 3 samples of multidrug-resistant HIV populations from patients, the method identified all drug-resistant mutations called using the Viroseq genotyping, as well as 4 lower abundance drug resistance mutations not called by the Viroseq pipeline.
The frequency of resistance mutations identified only by the pyrosequencing method represented 11.6% to 0.65% of the total, the authors report.
Analysis of a control mixture of HIV subtypes showed that resistance mutations present as 5% of the population could be readily detected without false positives.
"Looking forward," the investigators write, "it will be important to test more fully the importance of minor HIV drug-resistant populations on antiretroviral responses and the impact of such information on treatment outcomes."
"Using the DNA bar coding strategy, it should be possible to multiplex large numbers of patient samples in single sequencing runs, thereby driving down costs," the authors explain.
"The method can be much cheaper than microarray techniques, but the specifics revolve around how many samples you pool in a single sequencing run (the more the cheaper)," Dr. Bushman said.
Nucleic Acids Research 2007.
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