Limits...
Protocol for nearly full-length sequencing of HIV-1 RNA from plasma.

Nadai Y, Eyzaguirre LM, Constantine NT, Sill AM, Cleghorn F, Blattner WA, Carr JK - PLoS ONE (2008)

Bottom Line: The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL.While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads.This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology, Institute of Human Virology, University of Maryland, Baltimore, Maryland, USA.

ABSTRACT
Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

Show MeSH

Related in: MedlinePlus

Nearly full-length RT-PCR method.Viral RNA was reverse-transcribed by priming with UNINEF 7′ or with VIF-VPUoutR1 using SuperScriptTM III RNase H− RT. The locations of all the primers used for cDNA synthesis and nested PCR were depicted in this diagram with the map of the complete HIV-1 genome. Two different strategies were employed to amplify the nearly full-length genome: one amplified the 2.6-kb (gag-pol) and 7.0-kb (pol-nef) fragments with the overlap of 797-bp, and the other amplified three overlapping fragments of 2.6-kb (gag-pol), 3.7-kb (pol-vpu) and 3.3-kb (env-nef) with the 797-bp and 321-bp over lap regions, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2170516&req=5

pone-0001420-g001: Nearly full-length RT-PCR method.Viral RNA was reverse-transcribed by priming with UNINEF 7′ or with VIF-VPUoutR1 using SuperScriptTM III RNase H− RT. The locations of all the primers used for cDNA synthesis and nested PCR were depicted in this diagram with the map of the complete HIV-1 genome. Two different strategies were employed to amplify the nearly full-length genome: one amplified the 2.6-kb (gag-pol) and 7.0-kb (pol-nef) fragments with the overlap of 797-bp, and the other amplified three overlapping fragments of 2.6-kb (gag-pol), 3.7-kb (pol-vpu) and 3.3-kb (env-nef) with the 797-bp and 321-bp over lap regions, respectively.

Mentions: The methods for RT- PCR are illustrated in Figure 1. Reverse transcription of the RNA was performed by priming with UNINEF7′ (Table 1) close to the 3′ end of the viral RNA or by VIF-VPUoutR1 (Table 1) in the vpu gene. The extracted RNA (3 µl) was reverse transcribed in a total volume of 20 µl with 500 µM dNTP, 2.5 µM primer, 1× RT buffer, 5 mM MgCl2, 10 mM DTT, 40 U RnaseOUT, and 400 U SuperScriptTM III RNase H− RT (Invitrogen, Carlsbad, CA). The RNA, primer and dNTPs were first incubated at 65°C for 5 minutes, then the remaining reagents were added for cDNA synthesis at 50°C for 2 hours, followed by 85°C for 5 minutes. Then 2 U E. coli RNase H (Invitrogen, Carlsbad, CA) was added, and the reaction tubes were incubated at 37°C for 20 minutes followed by 70°C for 15 minutes.


Protocol for nearly full-length sequencing of HIV-1 RNA from plasma.

Nadai Y, Eyzaguirre LM, Constantine NT, Sill AM, Cleghorn F, Blattner WA, Carr JK - PLoS ONE (2008)

Nearly full-length RT-PCR method.Viral RNA was reverse-transcribed by priming with UNINEF 7′ or with VIF-VPUoutR1 using SuperScriptTM III RNase H− RT. The locations of all the primers used for cDNA synthesis and nested PCR were depicted in this diagram with the map of the complete HIV-1 genome. Two different strategies were employed to amplify the nearly full-length genome: one amplified the 2.6-kb (gag-pol) and 7.0-kb (pol-nef) fragments with the overlap of 797-bp, and the other amplified three overlapping fragments of 2.6-kb (gag-pol), 3.7-kb (pol-vpu) and 3.3-kb (env-nef) with the 797-bp and 321-bp over lap regions, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170516&req=5

pone-0001420-g001: Nearly full-length RT-PCR method.Viral RNA was reverse-transcribed by priming with UNINEF 7′ or with VIF-VPUoutR1 using SuperScriptTM III RNase H− RT. The locations of all the primers used for cDNA synthesis and nested PCR were depicted in this diagram with the map of the complete HIV-1 genome. Two different strategies were employed to amplify the nearly full-length genome: one amplified the 2.6-kb (gag-pol) and 7.0-kb (pol-nef) fragments with the overlap of 797-bp, and the other amplified three overlapping fragments of 2.6-kb (gag-pol), 3.7-kb (pol-vpu) and 3.3-kb (env-nef) with the 797-bp and 321-bp over lap regions, respectively.
Mentions: The methods for RT- PCR are illustrated in Figure 1. Reverse transcription of the RNA was performed by priming with UNINEF7′ (Table 1) close to the 3′ end of the viral RNA or by VIF-VPUoutR1 (Table 1) in the vpu gene. The extracted RNA (3 µl) was reverse transcribed in a total volume of 20 µl with 500 µM dNTP, 2.5 µM primer, 1× RT buffer, 5 mM MgCl2, 10 mM DTT, 40 U RnaseOUT, and 400 U SuperScriptTM III RNase H− RT (Invitrogen, Carlsbad, CA). The RNA, primer and dNTPs were first incubated at 65°C for 5 minutes, then the remaining reagents were added for cDNA synthesis at 50°C for 2 hours, followed by 85°C for 5 minutes. Then 2 U E. coli RNase H (Invitrogen, Carlsbad, CA) was added, and the reaction tubes were incubated at 37°C for 20 minutes followed by 70°C for 15 minutes.

Bottom Line: The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL.While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads.This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology, Institute of Human Virology, University of Maryland, Baltimore, Maryland, USA.

ABSTRACT
Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

Show MeSH
Related in: MedlinePlus