DETECTION OF VARIABILITY IN NATURAL-POPULATIONS OF VIRUSES BY POLYMERASE CHAIN-REACTION

The retroviruses associated with autoimmune deficiency syndrome (AIDS)—namely, human immunodeficiency virus types 1 and 2 (HIV-1, -2), display considerable genome sequence variability not only between isolates from different individuals but also between isolates from the same individual over time. This diversity is largely because of the extensive misincorporation of nucleotides by the viral reverse transcriptase, which lacks the proofreading 3'→5'-exonuclease activity. The conventional methods for molecular characterization of viral isolates involve cultivation of the agent, extraction of nucleic acid, and analysis by restriction enzyme mapping. Higher analytical resolution generally requires more time-consuming procedures, such as molecular cloning and sequencing of entire genomes. The PCR provides a rapid means for analyzing genomic diversity. There are two major advantages in using the PCR to study viral variants— (1) it obviates the need to propagate the virus in culture (a procedure that is expensive, time-consuming, and biohazardous) and (2) PCR allows detecting naturally occurring isolates and circumvents the viral selection, which often occurs during cultivation. The protocols for amplification, cloning, and sequencing of naturally occurring variants are presented in the chapter. © 1993, Elsevier Inc. All rights reserved.