PCR and Mammalian Cell Selection Assays for Short-Term Genotoxicity Testing
Midterm rept. 24 Feb 1992-24 Aug 1993
TULANE UNIV MEDICAL CENTER NEW ORLEANS LA
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In order to develop an extremely sensitive test for chemicals that induce chromosomal 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 t1418 in a background of normal human DNA from 10exp 6 cells.
- Medicine and Medical Research
- Nuclear Physics and Elementary Particle Physics