Article

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

Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
PCR methods and applications 06/1993; 2(4):293-300. DOI: 10.1101/gr.2.4.293
Source: PubMed

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.

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