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Use of the polymerase chain reaction technique to create base-specific ras oncogene mutations
C F Rochlitz1, G K Scott, J M Dodson
1Cancer Research Institute, University of California, San Francisco 94143.
Abstract:
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.
Insights
This study introduces a modified polymerase chain reaction (PCR) technique to efficiently introduce and amplify specific point mutations in human ras oncogene sequences. This method enables sensitive detection of oncogene mutations in mixed DNA samples, crucial for cancer research.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Point mutations in oncogenes, such as the Kirsten ras oncogene, are critical in cancer development.
- Detecting these mutations in clinical samples, especially from mixed cell populations, remains a challenge.
Purpose of the Study:
- To develop a modified polymerase chain reaction (PCR) technique for introducing and amplifying specific point mutations in human ras oncogene sequences.
- To establish a sensitive method for detecting oncogene mutations in mixed DNA populations, relevant to primary tumor specimens.
Main Methods:
- Utilized a modified primer-mediated enzymatic amplification (PCR) to introduce single base mismatches into Kirsten ras oncogene sequences.
- Generated double-stranded DNA fragments with specific point mutations, achieving approximately 10(6)-fold amplification.
- Demonstrated the detection of mutations in mixed DNA samples, even when present at 5% of the total amplified DNA.
Main Results:
- The modified PCR technique successfully introduced and amplified mutated ras oncogene sequences with high fidelity (>99.999%).
- Generated DNA fragments served as effective positive hybridization controls for mutation detection.
- Oncogene mutations were detectable in mixed DNA populations, indicating the method's sensitivity for clinical specimens.
Conclusions:
- The developed PCR-based method is highly efficient for introducing and amplifying specific oncogene point mutations.
- This technique offers a sensitive approach for detecting cancer-associated gene mutations in complex biological samples.
- The method has potential applications in cancer diagnostics and research for identifying oncogenic mutations.