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.
Koh
K B
KB
Department of Virology, The University of Tokushima School of Medicine, 770-8503, Tokushima, Japan.
Fujita
M
M
Adachi
A
A
D002118:Calcium / Q000737:chemistry*; D002460:Cell Line; D011232:Chemical Precipitation; D004279:DNA, Viral / Q000737:chemistry* / Q000502:physiology; D003851:Deoxyribonucleases / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D010710:Phosphates / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014162:Transfection; D014779:Virus Replication
D004279:DNA, Viral; D010710:Phosphates; D004533:Egtazic Acid; D003851:Deoxyribonucleases; D002118:Calcium