qBiomarker Somatic Mutation PCR Assays
For detecting the presence of specific DNA sequence mutations in cancer and oncogenesis
qBiomarker Somatic Mutation PCR Assays identify the presence of individual specific sequence mutations present in cell lines or research samples that are critical for toxicological, drug development, and cancer studies. The mutations are selected from comprehensive curated databases (e.g., COSMIC) and literature reviews based on their clinical or functional relevance and frequency in patient populations.
Principle
Real-time PCR is the most sensitive and reliable method for the detection of DNA mutations. By combining allele-specific amplification and hydrolysis probe detection, qBiomarker Somatic Mutation real-time PCR assays have been developed which can detect as few as 1% somatic mutations in the background of wild-type genomic DNA. Allele-specific amplification is achieved by Amplification Refractory Mutation System (ARMS) technology, which is based on the discrimination by Taq polymerase between a match and a mismatch at the 3’ end of the PCR primer (see figure "Principle of ARMS technology").
Procedure
After the isolation of genomic DNA from fresh, frozen, or fixed samples, add an aliquot of each sample to a separate real-time PCR tube containing the appropriate master mix (included) and the qBiomarker Somatic Mutation PCR Assay. Then, add an aliquot of each sample to a separate real-time PCR tube containing the appropriate master mix (included) and the corresponding reference gene copy assay. Finally, run the recommended cycling program. Determine the CT values for each mutation-specific assay and the corresponding reference gene copy assay for each sample using your instrument's software. Then, paste the values into the correct Excel-based data analysis template to determine which samples contain the tested mutation.
Applications
qBiomarker Somatic Mutation PCR Assays are highly suited for the rapid and accurate identification of individual specific sequence mutations present in fresh, frozen, or fixed samples.
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