Distinct variation pattern in the env of macrophage-tropic simian immunodeficiency virus in vivo demonstrated by denaturing gradient gel electrophoresis

Genetic variations, occurring during the infection in the AIDS animal model using a molecular clone virus, often consist of single nucleotide substitu tions distributed sparsely in the viral genome. A highly sensitive method w as developed to detect such mutations, consisting of denaturing gradient ge l electrophoresis (DGGE) and direct sequencing. The genetic variation in th e env of a macrophage tropic simian immunodeficiency virus (SIV) in a macaq ue infected chronically was examined by this technique and compared with th at of a T cell tropic SIV in an animal infected chronically. A whole env se quence was amplified by 11 sets of PCR for DNA ranging from 300 to 420 bp. The amplified DNA was subjected to mutational screening by DGGE and ro subs equent direct sequencing. Imaging analysis of separated DNA bands by DGGE p rovided information on the proportion of each variant DNA. The validity of this technique was confirmed by sequencing of variant DNAs cloned by limiti ng dilutions. Thus, this technique is suitable for analysis of genetic vari ations in the AIDS animal model using a molecular clone virus. (C) 2000 Els evier Science B.V. All rights reserved.