Polymerase chain reaction in situ: intracellular amplification and detection of HIV-1 proviral DNA and other specific genes.
The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology and cell biology. We report here a unique, highly sensitive and specific technique which can be utilized to detect a single copy of human immunodeficiency virus type I (HIV-1) provirus and other genes, at the single cell level, by in situ amplification of a portion of a gene sequence. In this method, a polymerase chain reaction (PCR) can be carried out in situ, in fixed cells, on specially designed glass slides. After amplification one can detect the amplified signals by the in situ hybridization method, utilizing either biotinylated probes or 32P-labelled probes. The early molecular events in the retroviral life-cycle of HIV-1, in specific target cells, are demonstrated utilizing in situ PCR. The techniques utilized in this procedure and various potential uses of this methodology are described.
Bagasra
O
O
Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107.
Seshamma
T
T
Pomerantz
R J
RJ
D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D015497:HIV-1 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral