Detection and quantitation of low numbers of chromosomes containing bcl-2 oncogene translocations using semi-nested PCR.
A 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 by the 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 JH immunoglobulin gene sequence [t(14;18)] in a background of normal human DNA from 10(6) cells.
Zhang
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Tulane Medical School, New Orleans, LA.
Ehrlich
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M
D001483:Base Sequence; D015139:Blotting, Southern; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D014178:Translocation, Genetic
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D004247:DNA; D004996:Ethidium