ANALYSIS OF HETEROGENEOUS VIRAL POPULATIONS BY DIRECT DNA-SEQUENCING

The ability of direct PCR sequencing to detect and quantify sequence p olymorphisms was investigated using samples containing mixed populatio ns of HIV-1. A part of the genome encoding the polymorphic variable re gion 3 of the envelope was directly sequenced to yield a consensus seq uence of the virus population. The results were compared with sequence s obtained by analysis of multiple clones derived from the same clinic al samples. The results of five patients suggested that the direct-seq uencing method can be used as a rapid tool to analyze and quantify het erogeneous viral populations. Reconstitution experiments using cloned material demonstrated that it was possible to detect and quantify mino r sequence variants present in as little as 10% of the total virus pop ulation. The use of the method for molecular diagnosis is discussed.