ABSTRACT
The first step toward a better understanding of gene products of human and simian immunodeficiency viruses (HIV/SIV) is molecular cloning of intact HIV/SIV genomes. Once biologically active clones are obtained, it is quite easy to do a systematic genetic study. It is important to have plasmid clones to perform a functional analysis of HIV/SIV genomes. Care must be taken in selecting an appropriate plasmid vector. Purity of plasmid DNA is essential for efficient transfection. Purification of the DNA by equilibrium centrifugation in CsCl-ethidium bromide gradients always gives good results. Two types of cells have been used for transfection analysis of HIV/SIV genomes. Adherent and nonadherent cells are efficiently transfected by modified calcium-phosphate coprecipitation and DEAE-dextran methods, respectively. Methods for constructing mutants and for making chimeric recombinants between biologically distinct clones are now well established. This chapter describes a rapid and reliable method to subclone long DNA fragments into plasmid vectors.
