Patients infected with human immunodeficiency virus type 1 (HIV-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 reasonable 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: 1. Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR); 2. Direct use of the crude unpurified PCR product as the sequencing template; and 3. Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.

Zazzi, M., Riccio, M.L., Venturi, G., Catucci, M., Romano, L., DE MILITO, A., et al. (1998). Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors. MOLECULAR BIOTECHNOLOGY, 10, 1-10.

Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors.

ZAZZI, MAURIZIO;RICCIO, MARIA LETIZIA;ROMANO, LAURA;VALENSIN, PIER EGISTO
1998-01-01

Abstract

Patients infected with human immunodeficiency virus type 1 (HIV-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 reasonable 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: 1. Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR); 2. Direct use of the crude unpurified PCR product as the sequencing template; and 3. Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.
Zazzi, M., Riccio, M.L., Venturi, G., Catucci, M., Romano, L., DE MILITO, A., et al. (1998). Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors. MOLECULAR BIOTECHNOLOGY, 10, 1-10.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/32543
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