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

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:

• 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.