Variable stringency hybridization of polymerase chain reaction amplified HIV-1 DNA fragments.
DNA isolated from the peripheral blood mononuclear cells of HIV-1 seropositive individuals was used for polymerase chain reaction (PCR) amplification of gag and envelope regions. Eight aliquots of the amplified DNA fragments have been subjected to Southern/dot blot analysis, hybridizing with 32P-labelled-BH10 (HIV-1 strain IIIB) at low stringency. After the filters had been autoradiographed, they were cut so that each hybridized band/dot could be subject to variable stringency washing using various ionic concentrations at a fixed temperature. The filter was reconstructed so that the effect of the variable stringency wash might be visualized following a second exposure to Kodak film. The level of activity for each band/dot was measured by counting the 32P or by densitometry analysis of the photographic record. The results allow a plot to be made of the decrease in bound radioactivity against ionic strength. By comparison with a standard curve obtained for HIV-1 strain IIIB amplified fragments subject to similar hybridization and analysis, an estimation of the degree of nucleotide mismatch relative to the BH10 DNA probe can be obtained. The technique provides a rapid means of characterizing PCR amplified fragments.
Agius
G
G
Laboratoire de Physiologie Cellulaire, Université Pierre et Marie Curie, Paris, France.
Kolesnitchenko
V
V
Snart
R
R
Zagury
J F
JF
Laaroubi
K
K
Zagury
D
D
D004279:DNA, Viral / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D015023:Democratic Republic of the Congo; D015752:Genes, env; D015750:Genes, gag; D006679:HIV Seropositivity; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods
D004279:DNA, Viral; D015345:Oligonucleotide Probes