Oncogene detection at the single cell level.
We describe strategies for the detection of oncogenes at the single-cell level and for the positive identification of under-represented oncogenic alleles in mixed populations of normal and tumor cells. By combining the Polymerase Chain Reaction (PCR) technique with a liquid hybridization and gel retardation assay, we have been able to detect H-ras sequences in single cells, including in one fertilized mouse ovum. We also describe a modification of the PCR protocol involving the use of mismatched primers. This procedure allows for the creation of novel Restriction Fragment Length Polymorphisms (RFLP) diagnostic of specific point mutations. This experimental approach has allowed us to detect ras oncogenes in a single heterozygous cell in the presence of 10(5) normal cells.
Kumar
R
R
Developmental Oncology Section, NCI-Frederick Cancer Research Facility, Maryland 21701.
Barbacid
M
M
D000483:Alleles; D000818:Animals; D002460:Cell Line; D003062:Codon / Q000235:genetics; D046508:Culture Techniques / Q000379:methods; D017931:DNA Primers; D005260:Female; D011905:Genes, ras; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D009857:Oncogenes; D010063:Ovum / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D051381:Rats
D003062:Codon; D017931:DNA Primers