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Mutation Detection With High Sensitivity and Specificity

Competitive Allele-Specific TaqMan® PCR (castPCR)

Competitive Allele-Specific TaqMan® PCR (castPCR™) technology, developed by Life Technologies, is a highly specific and sensitive method of detecting and quantitating rare mutations in a sample that contains large amounts of normal, wild type genomic DNA (gDNA). castPCR™ technology combines allele-specific TaqMan® qPCR with allele-specific MGB blockers in order to suppress non-specific amplification from wild type alleles, resulting in better specificity than traditional allele-specific PCR. castPCR™ technology not only maintains the wide dynamic range and reproducibility of conventional TaqMan® assays but also provide higher sensitivity and specificity for detecting rare mutant alleles. The TaqMan® Mutation Detection Assays, powered by castPCR™ technology, are a collection of assays that detect and measure somatic mutations in genes that are associated with cancer. Key features of these assays include:
  • High specificity—mutant allele detection is based on a modified allele-specific primer, while the wild-type background is suppressed by the proprietary MGB blocker oligonucleotide
  • High sensitivity—assays can detect  less than 0.1% mutant molecules in a background of wild type gDNA, as demonstrated in spiking experiments
  • Wide dynamic range and excellent PCR efficiency—assays demonstrate 7 logs of dynamic range and an average efficiency of 100% ± 10%
  • Fast, simple workflow—like other TaqMan® Assays, requires typically 3 hours from sample to results, with minimum hands-on time

Three Assay Types
Within the TaqMan® Mutation Detection Assays collection, three assay types are available, as described in the table below: a mutant allele assay, which specifically detects one or more mutant alleles; a wild type allele assay, which detects the wild type allele; and a reference gene assay, which detects a mutation-free region of the gene in which the target mutations reside. The mutant allele assays are designed to mutations in the Catalogue of Somatic Mutations in Cancer (www.sanger.ac.uk/genetics/CGP/cosmic/).

Assay Type Description Schematic
Mutant Allele Assay
  • Detects specific or multiple mutant alleles
  • An allele-specific primer detects the mutant allele
  • An MGB blocker oligonucleotide suppresses the wild type allele.
 
Mutant Allele Assay
Wild Type Allele Assay
  • Detects the wild type allele
  • An allele-specific primer detects  the wild type allele
  • An MGB blocker oligonucleotide suppresses the mutant allele
 
Wild Type Allele Assay
Gene Reference Assay
  • Detects the gene in which the target mutations reside within
  • A locus-specific pair of forward and reverse primers amplify a mutation free region of the target gene.
 
Gene Reference Assay
 
Two Experimental Approaches
The mutation status and percent mutation for a sample can be determined by two different experimental approaches.
  1. Mutant allele assays paired with a gene reference assay.
    A sample is run with one or more assays that target mutant alleles within a gene and the corresponding gene reference assay. Sample replicates are typically not run. This approach uses a minimal number of wells, samples, and reagents and is suitable for determining the presence or absence of a mutated base in a sample.
  2. Mutant allele assays paired with wild type allele assays.
    A sample is run with one or more assays that target the mutant allele of a given mutation and the corresponding wild type allele assay(s). Three sample replicates are run. This approach is recommended for highly accurate quantitation of the percent mutation in a sample.
  3. 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.  
Instrument Compatibility
TaqMan® Mutation Detection Assays are compatible with the Applied Biosystems® ViiA™ 7, 7900HT, 7500, 7500 Fast, and StepOnePlus™ Real-Time PCR Systems.

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

Data Analysis Software
For data analysis, Mutation Detector™ Software v1.0 allows users to determine the mutation status of their samples from TaqMan®Mutation Detection Assay data collected on Applied Biosystems® ViiA™ Real-Time PCR Systems.

TaqMan® Mutation Detection Assays Product Bulletin
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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan® is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.
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