PCR and Mammalian Cell Selection Assays for Short-Term Genotoxicity Testing

摘要

In order to develop an extremely sensitive test for chemicals that inducechromosomal rearrangements, a polymerase chain reaction (PCR) assay has been optimized for the detection of one or a few molecules of a translocation-containing human DNA sequence in the presence of a vast excess (7 micrograms) of the normal human genome. This procedure avoids blot hybridization by the use of two rounds of PCR with 20-22 cycles of amplification per round and replacement of one of the two primers from the first round of PCR with a different primer in the second round (semi-nested PCR). We demonstrate that very low numbers of the target DNA molecules can be quantitated by this semi-nested PCR. This method can be used to detect a single DNA molecule from one mutant cell displaying a translocation between the bcl-2 proto-oncogene region and a J sub H immunoglobulin gene sequence (t(14;18)) in a background of normal human DNA from 10(exp 6) cells. Carcinogens, Polymerase chain reaction, Chromosomal translocations.