title: Protocol for nearly full-length sequencing of HIV-1 RNA from plasma.
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.
authors.affiliation: Department of Epidemiology, Institute of Human Virology, University of Maryland, Baltimore, Maryland, USA.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Nadai
authors.forename: Lindsay M
authors.initials: LM
authors.lastname: Eyzaguirre
authors.forename: Niel T
authors.initials: NT
authors.lastname: Constantine
authors.forename: Anne M
authors.initials: AM
authors.lastname: Sill
authors.forename: Farley
authors.initials: F
authors.lastname: Cleghorn
authors.forename: William A
authors.initials: WA
authors.lastname: Blattner
authors.forename: Jean K
authors.initials: JK
authors.lastname: Carr
mesh_terms: D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood / Q000235:genetics*; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic
chemical_list: D012367:RNA, Viral