Construction of gag, pol, and env specific riboprobes for confirmation of HIV-1 specific polymerase chain reaction products.
Conserved regions of the gag, pol, and env genes of HIV-1 pBH10 DNA (gag nucleotides(nt)1520-1665, pol nt 2326-2663, env nt 7192-7333; Ratner et al., 1985) were amplified by the polymerase chain reaction (PCR) using oligonucleotides complementary to the termini of these regions as primers. Primer areas of the amplified DNA were then removed by digestion with restriction endonucleases, and the internal fragments purified and cloned in both orientations into the 'riboprobe' transcription vector pGEM-5Z. Riboprobes made from these plasmids did detect the specific sequences of pBH10 DNA and of HIV-1 DNA amplified by PCR from clinical material. The riboprobes will be useful to confirm the specificity of PCR-amplified fragments of lymphocyte DNA obtained from infants of HIV-infected mothers and from high risk, but seronegative contacts of HIV-1-infected individuals.
Dolan
J
J
Regional Virus Laboratory, East Birmingham Hospital, UK.
Desselberger
U
U
D004279:DNA, Viral / Q000235:genetics; D005260:Female; D015752:Genes, env; D015750:Genes, gag; D015751:Genes, pol; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008214:Lymphocytes / Q000821:virology; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D015347:RNA Probes
D004279:DNA, Viral; D015347:RNA Probes