Use of the polymerase chain reaction technique to create base-specific ras oncogene mutations.
A modification of the polymerase chain reaction technique (PCR) technique, a primer-mediated enzymatic amplification of specific target sequences in genomic DNA, was used to introduce point mutations into copies of human ras oncogene sequences, and to amplify these mutated copies approximately 10(6)-fold. Of the two flanking oligomers used to amplify the DNA, one contained a single base mismatch with the targeted gene segment, either codon 12 or 61 from the Kirsten ras oncogene. Double-stranded fragments harboring any point mutation can be generated using the appropriate oligomer and constitute greater than 99.999% of the PCR-amplified fragments. The amplified DNA fragments are readily slot-blotted to nylon membranes to serve as positive hybridization controls in the search for gene mutations in human tissue specimens. The synthesis of single base mismatched DNA fragments was also used to demonstrate that oncogene mutations can be detected from mixed DNA populations as might be present in primary tumor specimens, even when the mutation of interest is present in only 5% of the amplified sample DNA.
Rochlitz
C F
CF
Cancer Research Institute, University of California, San Francisco 94143.
Scott
G K
GK
Dodson
J M
JM
Benz
C C
CC
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D002460:Cell Line; D003062:Codon; D005260:Female; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D014407:Tumor Cells, Cultured
D003062:Codon; D015345:Oligonucleotide Probes