Sample Type Compatibility

The assays can be used with gDNA extracted from FFPE tissues, fresh frozen tissues, and cell line samples.

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DNA Isolation Kits

Mutant Allele Assays

Mutant allele assays target key somatic mutations in oncogenes and tumor suppressor genes. All mutation targets are from the comprehensive Sanger COSMIC database (www.sanger.ac.uk/genetics/CGP/cosmic/). Target selection was based on frequency of occurrence and input from leading cancer researchers.

Each mutant allele assay (Figure 1) detects specific or multiple mutant alleles. Each assay contains:

• an allele-specific primer that detects the mutant allele
• a MGB oligonucleotide blocker suppresses the wild type allele
• a locus-specific primer
• a locus specific TaqMan® FAM™ dye-labeled MGB probe

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Figure 1: Mutant allele assay using castPCR™ technology

Gene Reference Assays

Gene reference assays detect the genes that the target mutations reside in. They are designed to amplify a mutation-free and polymorphism-free region of the target gene. Each assay contains:

• a locus-specific pair of forward and reverse primers
• a locus specific TaqMan® FAM™ dye-labeled MGB probe

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Figure 2: Gene reference assay

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For each real-time PCR reaction, the gDNA is combined with:

• A TaqMan® Mutation Detection Assay—to detect a mutant allele or reference gene target
• TaqMan® Genotyping Master Mix—contains AmpliTaq Gold® DNA Polymerase, UP (Ultra Pure), dNTPs, and buffer
• Optional TaqMan® Mutation Detection IPC Reagent Kit—add IPC reagents to any mutant allele assay reaction to distinguish true target negatives from PCR failure or inhibition (see next step for more information)

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TaqMan® Genotyping Master Mix
The TaqMan® Genotyping Master Mix is optimized for somatic mutation detection using TaqMan® Mutation Detection Assays.

Optional Use of Internal Positive Control (IPC)
You can duplex IPC reagents with any TaqMan® Mutation Detection Assay to distinguish true target negatives from PCR failure or inhibition.

Perform Amplification

Reactions are run on an Applied Biosystems® real-time PCR system using a universal mutation detection thermal cycling protocol. After amplification, the Ct values are determined by the real-time PCR system analysis software.

Instrument Compatibility

TaqMan® Mutation Detection Assays are compatible with the following instruments: ViiA™ 7, QuantStudio™ 12K Flex, 7900HT, 7500, 7500 Fast, and StepOnePlus® Real-Time PCR Systems.

Analyze

Data files containing the sample Ct values can be exported from instrument software and imported into Mutation Detector™ Software for post-PCR data analysis of mutation detection experiments. In mutation analysis calculations, the difference between the Ct for each mutant allele assay and the Ct for the gene reference assay is calculated. This ΔCt value represents the quantity of the specific mutant allele detected within the sample.

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