Elimination of HIV-1 plasmid DNA from virus samples obtained from transfection by calcium-phosphate co-precipitation

Molecular genetics is a powerful tool to analyze the replication cycle of human immunodeficiency virus type 1 (HIV-1). Culture fluids obtained from HIV-1 plasmid-transfected cells by calcium-phosphate co-precipitation were treated with ethyleneglycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and DNase I to obtain HIV-1 stocks virtually free of input plasmid DNAs. Even after amplification by polymerase chain reaction (PCR), no plasmid DNA was detected in cells following infection with EGTA/DNase I-treated virus samples. This method is particularly useful for the examination of the early replication phase of HIV-1 by PCR.