Rapid characterization of HIV-1 sequence diversity using denaturing gradient gel electrophoresis and direct automated DNA sequencing of PCR products.

TitleRapid characterization of HIV-1 sequence diversity using denaturing gradient gel electrophoresis and direct automated DNA sequencing of PCR products.
Publication TypeJournal Article
Year of Publication1993
AuthorsAndersson, B, Ying, JH, Lewis, DE, Gibbs, RA
JournalPCR Methods Appl
Volume2
Issue4
Pagination293-300
Date Published1993 May
ISSN1054-9803
KeywordsBase Sequence, DNA, Viral, Electrophoresis, HIV-1, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Analysis, DNA
Abstract

A direct method for visualization and isolation of sequence variants of human immunodeficiency virus type 1 (HIV-1) utilizing denaturing gradient gel electrophoresis (DGGE) combined with automated direct DNA sequencing was developed. Two fragments from the env gene and one from the nef gene of HIV-1, which together constitute approximately 1.0 kb of sequence, were amplified by PCR and analyzed. HIV-1 variants from each region were resolved and excised from the gel; this was followed by direct sequencing of different viral variants. In 9 infected patients, a limited number of dominant sequence variants could be seen in the three regions, together with a faint background of minor variants. The use of DGGE makes it possible to obtain a direct estimate of overall HIV-1 sequence diversity within patient samples without an intermediate DNA cloning step.

Alternate JournalPCR Methods Appl.
PubMed ID8324502
Grant List21-272110105218 / / PHS HHS / United States
5U01AI 30243-03 / AI / NIAID NIH HHS / United States