Long-read direct infrared sequencing of crude PCR products for prediction of resistance to HIV-1 reverse transcriptase and protease inhibitors.
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.
Dipartimento di Biologia Molecolare, Università di Siena, Italy. zazzi@sivax.unisi.it
M
M
Zazzi
M L
ML
Riccio
G
G
Venturi
M
M
Catucci
L
L
Romano
A
A
De Milito
P E
PE
Valensin
D001483:Base Sequence; D004352:Drug Resistance, Microbial; D004586:Electrophoresis / Q000379:methods; D016333:HIV Protease / Q000187:drug effects / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011480:Protease Inhibitors / Q000494:pharmacology*; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D013698:Templates, Genetic
D011480:Protease Inhibitors; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016333:HIV Protease