Genetic Analysis of HIV by In Situ PCR-Directed Laser Capture Microscopy of Infected Cells

Abstract

Behind the exponential expansion of the acquired immunodeficiency syndrome (AIDS) epidemic, there is a continuous and progressive molecular evolution of human immunodeficiency virus (HIV)-1. In this regard, the molecular analysis of viral strains infecting several anatomic compartments in humans has become critical to understanding AIDS-related pathologies and to improving emerging therapeutic protocols. Laser capture microdissection provides outstanding results in the genetic analysis of HIV-1 variants detectable in AIDS patients. The ability of the instrument to microdissect infected cells from a heterogeneous tissue compartment allows the investigator to obtain critical information regarding the genetic nature of a specific viral strain. To perform laser capture microdissection with better accuracy, a priori detection techniques may provide useful information about HIV distribution in the tissue specimen. An in situ polymerase chain reaction (PCR) assay on a serial slide results in a detailed map of the viral infection specific for the case under analysis. The knowledge of HIV distribution in the tissue section is critical for improving the dissection of infected cells by laser capture microscopy. This chapter describes laser capture microdissection and in situ PCR and its role in the analysis of the genetic nature of HIV-1 variants and quasispecies.