Open AccessRapid characterization of HIV-1 sequence diversity using denaturing gradient gel electrophoresis and direct automated DNA sequencing of PCR products.
Author(s) -
Björn Andersson,
Jiale Ying,
D E Lewis,
Richard A. Gibbs
Publication year - 1993
Publication title -
genome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.556
H-Index - 297
eISSN - 1549-5469
pISSN - 1088-9051
DOI - 10.1101/gr.2.4.293
Subject(s) - biology , temperature gradient gel electrophoresis , dna sequencing , genetics , gene , sequence analysis , dna , sequence (biology) , sequencing by ligation , microbiology and biotechnology , gel electrophoresis , cloning (programming) , polymerase chain reaction , peptide sequence , genomic library , 16s ribosomal rna , computer science , programming language
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.
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