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Updated: Jun 23, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
A SNaPshot assay for the rapid and simple detection of four common hotspot codon mutations in the PIK3CA gene
Carolyn D Hurst1, Tahlita C M Zuiverloon, Christian Hafner
1Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK. c.hurst@leeds.ac.uk
Background:
Activating mutations in the PIK3CA gene have been identified in a variety of human malignancies and are commonly detected in hotspot codons located in the helical and kinase domains in exons 9 and 20. Existing methodologies for the detection of PIK3CA mutations are time-consuming and/or expensive. In the present study we describe the first application of a PIK3CA SNaPshot assay to the screening of frequent mutations in these exons.
Findings:
A SNaPshot assay for the simultaneous detection of four frequent PIK3CA hotspot mutations (E542K, E545G, E545K and H1047R) has been developed and evaluated. The assay combines multiplex PCR amplification with a multiplex primer extension assay to allow targeted detection of all four mutations in one reaction. The method was tested using samples that had previously been analysed for mutations by high-resolution melting analysis and sequencing. All mutations detected were concordant and no false positive results were obtained. Sensitivity tests showed that the SNaPshot assay could detect mutant DNA when it represents 5-10% of the total DNA present. The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated.
Conclusion:
The SNaPshot assay described here offers a fast, sensitive, inexpensive and specific approach to the analysis of frequent PIK3CA mutations in both fresh and archival patient samples.
Insights
A new SNaPshot assay enables rapid, sensitive, and cost-effective detection of common PIK3CA gene mutations in cancer. This method accurately identifies mutations in various sample types, improving diagnostic capabilities.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Activating mutations in the PIK3CA gene are prevalent in human cancers, often found in exons 9 and 20.
- Current methods for detecting PIK3CA mutations are often costly and time-consuming.
Purpose of the Study:
- To develop and evaluate a novel SNaPshot assay for the rapid screening of frequent PIK3CA hotspot mutations.
- To provide a more efficient diagnostic tool for PIK3CA-driven malignancies.
Main Methods:
- Development of a SNaPshot assay combining multiplex PCR and primer extension for simultaneous detection of four common PIK3CA mutations (E542K, E545G, E545K, H1047R).
- Validation using samples previously analyzed by high-resolution melting analysis and sequencing.
- Assessment of sensitivity and application to formalin-fixed paraffin-embedded (FFPE) samples.
Main Results:
- The SNaPshot assay successfully detected four frequent PIK3CA hotspot mutations with high concordance and no false positives.
- The assay demonstrated sensitivity in detecting mutant DNA at 5-10% levels.
- The method was successfully applied to DNA from FFPE samples.
Conclusions:
- The developed SNaPshot assay is a fast, sensitive, inexpensive, and specific method for analyzing common PIK3CA mutations.
- This assay is suitable for both fresh and archival patient samples, offering a valuable diagnostic tool.
