Abstract
Patients infected with human irnmunodeficiency virus type 1 (H1V-1) are being treated with a number of different combinations of antiretroviral compounds that target the essential viral enzymes reverse transcriptase and protease. Different sets of HIV-1 mutations that confer drug resistance have been well defined; they allow reasonabe prediction of the drug sensitivity pattern from analysis of the HIV-1 genotype in vivo. Since periodical monitoring of genotypic resistance is expected to improve clinical management in a large number of infected patients, practical and cost-effective methods are highly desirable to set at least medium-scale sequencing in clinical diagnostic settings. We present a complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions. Features making the system amenable to routine clinical use include:
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1.
Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR);
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Direct use of the crude unpurified PCR product as the sequencing template; and
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3.
Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.
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Zazzi, M., Riccio, M.L., Venturi, G. et al. Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors. Mol Biotechnol 10, 1–8 (1998). https://doi.org/10.1007/BF02745858
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DOI: https://doi.org/10.1007/BF02745858