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Published on: September 25, 2018
SuperSelective PCR primers for determining cis/trans configurations of mutations within the same gene
Diana Yaneth Vargas1, Fred Russell Kramer1
1Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America.
Abstract:
In non-small cell lung cancer, the choice of an effective targeted therapy often depends on the presence of a somatic mutation that alters an amino acid in a key protein. However, if an additional somatic mutation occurs in the same mutant gene, causing an additional amino acid substitution to occur in that protein, the targeted therapy can become ineffective. A PCR assay has now been developed that utilizes two different SuperSelective primers, one primer for the amplification of the DNA strand containing the original somatic mutation, and a second primer for the amplification of the DNA strand containing the additional somatic mutation. Exponential amplification only occurs if these two somatic mutations occur in cis on the DNA molecule present in the same sister chromosome, and does not occur at all if the two different somatic mutations occur in trans on different DNA molecules in two different sister chromosomes. Significantly, if only one of these two mutations is present, amplification does not occur, thereby ensuring that the assay specifically detects the coexistence of both target mutations being present on the same DNA strand. This assay (demonstrated utilizing EGFR exon 20 mutations T790M and C797S) is extraordinarily sensitive, enabling the early substitution of a more effective therapy if the two mutations occur in a cis configuration. Moreover, these SuperSelective PCR assays can utilize DNA fragments isolated from noninvasive liquid biopsy samples, and they can be carried out on widely available spectrofluorometric thermal cyclers.
Insights
A new PCR assay detects co-occurring mutations in non-small cell lung cancer (NSCLC). This method identifies specific mutation pairs on the same DNA strand, enabling precise targeted therapy selection for improved patient outcomes.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Targeted therapies for non-small cell lung cancer (NSCLC) effectiveness relies on specific somatic mutations.
- Acquired resistance mutations can render targeted therapies ineffective.
- Detecting co-occurring mutations, especially in cis, is crucial for treatment decisions.
Purpose of the Study:
- To develop a highly sensitive assay for detecting specific co-occurring somatic mutations in NSCLC.
- To differentiate between cis and trans configurations of mutations within the same gene.
- To enable early detection of resistance mutations for timely therapy adjustment.
Main Methods:
- Development of a novel Polymerase Chain Reaction (PCR) assay using two SuperSelective primers.
- Primers designed to amplify DNA strands containing specific, co-occurring somatic mutations (e.g., EGFR T790M and C797S).
- Assay relies on exponential amplification only when mutations are in cis configuration on the same DNA molecule.
Main Results:
- The SuperSelective PCR assay specifically detects the co-existence of two target mutations on the same DNA strand.
- Amplification does not occur if mutations are in trans or if only one mutation is present.
- Demonstrated high sensitivity in detecting EGFR exon 20 mutations (T790M and C797S) in cis.
- Assay successfully utilized DNA from noninvasive liquid biopsy samples.
Conclusions:
- The developed SuperSelective PCR assay is a sensitive and specific tool for detecting co-occurring mutations in NSCLC.
- This assay facilitates the identification of resistance mechanisms, enabling timely switching to more effective therapies.
- The method's applicability to liquid biopsies and standard thermal cyclers enhances its clinical utility.
