Problems encountered in detecting a targeted gene by the polymerase chain reaction

• Published in: Gene Vol. 103; no. 2; p. 227
• Main Authors: Kim, H S, Popovich, B W, Shehee, W R, Shesely, E G, Smithies, O
• Format: Journal Article
• Language: English
• Published: Netherlands 22-07-1991
• Subjects: Animals; Base Sequence; Blotting, Southern; Cell Nucleus; Chromosomes, Human, Pair 11; Electric Stimulation; False Positive Reactions; Genetic Therapy - methods; Globins - genetics; Humans; Mice; Microinjections; Molecular Sequence Data; Mutation - genetics; Polymerase Chain Reaction - methods; Recombination, Genetic; Sickle Cell Trait - genetics; Tumor Cells, Cultured
• ISSN: 0378-1119
• DOI: 10.1016/0378-1119(91)90277-I

We have investigated problems encountered when using the polymerase chain reaction (PCR) to detect recombinants in gene targeting experiments in which homologous recombination occurs between incoming DNA and an endogenous target sequence. The targeting system studied was designed to correct a human sickle-cell beta-globin-encoding gene (HBBS) on human chromosome 11 by replacing the defective gene with incoming DNA carrying normal HBB sequences. Two sets of experiments were executed which led to the isolation of a clone of cells having the sickle-cell gene corrected. We found that a positive control system was essential to allow a real targeting event to be distinguished from various types of false positives that arise during the diagnostic PCR.