How do the assays work?

Somatic Mutation Detection Workflow

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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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
Mutant allele assayEnlarge Image
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
Mutant allele assayEnlarge Image
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)

Recommended Products

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: QuantStudio™ 3D, 3, 5, 6 Flex, 7 Flex, & 12K Flex, ViiA™ 7, 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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Mutation Detector™ Software
Step 1 : Sample Preparation

Sample Type Compatibility

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

Recommended Products for DNA Isolation
DNA Isolation Kits

Step 2 : Select the Assay

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
Mutant allele assayEnlarge Image
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
Mutant allele assayEnlarge Image
Figure 2: Gene reference assay



Order Products

Order products using our Assay search tool
Order Custom plating services

Step 3 : Prepare the Reaction

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)

Recommended Products

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.

Step 4 : Perform the Reaction

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: QuantStudio™ 3D, 3, 5, 6 Flex, 7 Flex, & 12K Flex, ViiA™ 7, 7900HT, 7500, 7500 Fast, and StepOnePlus® Real-Time PCR Systems.

Step 5 : Analyze

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.

Ordering information

Mutation Detector™ Software

Note

All Applied Biosystems TaqMan Mutation Detection Assays have undergone extensive testing to ensure high sensitivity and specificity. The first set of released assays, covering 14 KRAS, 29 EGFR, and the BRAF V600E mutations, underwent additional testing, including determination of (a) the difference in inherent amplification efficiency between mutant allele assays and corresponding reference assays, to enable quantitative analysis of percent mutation in a sample, and (b) assay detection ΔCt cutoff values using spiked cell line gDNA samples.

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For research use only. Not for use in diagnostic procedures.