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The detection ∆Ct cutoff tells you how sensitive the assay is, by finding how much background amplification occurs with wild type samples amplified using the mutant assay. It is determined by the following:
Run the mutant allele assay and the corresponding gene reference assay on wild type gDNA samples that are from the same sample type as the test sample (e.g., gDNA from FFPE tissue samples). Run at least three different wild type samples and three technical replicates of each sample. ∆Ct values are calculated for each sample run with a mutant allele assay + gene reference assay pair. The average ∆Ct value for all samples is then calculated and is used to derive the detection ∆Ct cutoff value for the mutant allele assay.
The two experiment types are detection ∆Ct cutoff and mutation detection. The two experiment types can be run either sequentially or at the same time. All wild type gDNA samples used for determination of ∆Ct cutoff values can be run together on one plate or on separate plates; the sample ∆Ct values from multiple plates can be combined to generate an assay ΔCt cutoff value. Once the mutant allele assay detection ∆Ct cutoff values are determined, these can be used for all subsequent mutation detection experiments run with the same sample type.
The calibration ∆Ct value is the inherent Ct difference between a mutant allele assay and a wild type allele assay or a gene reference assay. This difference is determined by amplifying equal amounts of DNA templates containing 100% mutant allele (for the mutant allele assay) and 100% wild type DNA (for the wild type allele assay or gene reference assay). We determined the calibration ∆Ct values for the validated assay set in experiments using 104 copies of synthetic DNA templates.
The Ct value determined for a gene reference assay on a sample should be approximately 18–28 for a 20 µL reaction and 17–27 for a 10 µL reaction; these ranges correspond to a functional gDNA template input range of approximately 100 ng to 1 ng.
In the real-time PCR system software choose experiment type “Absolute Quantitation” or “Quantitation – Standard Curve”.
There are several options for finding a control sample. You can consult the COSMIC database, which may have information about controls for your mutation of interest. Keep in mind that mutation zygosity should be confirmed. Another option is to create a plasmid by cloning the region 200 bases on each side of the mutation. The castPCR™ assay specific to this mutation and its wild type allele will lie entirely within this region. Finally, we can provide the cloning service for you, with our GeneArt® Custom Synthesis service.
Yes, we can design custom assays for you. However, QC is not performed on these assays, and support will be limited for such products. To order a custom assay, see the following information:
Please provide COSMIC ID(s) for the mutation(s) and send your inquiries to email@example.com for more information, including pricing and feasibility. If the mutations are not in the COSMIC database, you will need to provide the 200 bp sequence on each side of the mutation in FASTA file format with wild type and mutant allele in brackets [A/G]. You are responsible for repeat masking and SNP masking on the sequence if necessary (place N instead of A, C, G, or T). If possible a mutant allele assay will be designed (and a wild type allele assay, if there is not a gene reference assay available for this target already).
We have only validated the TaqMan® Genotyping Master Mix with the mutation detection assays. Since we have not tested, we cannot recommend using any other mix with these assays.
No. The mutation detection assays should only be run with the standard protocol. They are not amenable to fast runs.
Yes, these assays can potentially be run on high-quality non–Applied Biosystems® real-time PCR instruments. However, it may be difficult to provide full support because validation was done only on Applied Biosystems® instruments. Results can be imported into Mutation Detector™ Software for analysis if the formatting is properly done. You can use a template file located in the Mutation Detector™ Software folder as a guide.
The average and median amplicon length for the assays is 100 base pairs.
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, and a locus-specific TaqMan® FAM™ dye–labeled MGB probe. A wild type allele assay is matched to a corresponding mutant allele assays using castPCR™ technology (i.e., it target the same base location as the mutant allele assay). Each wild type allele assay contains four oligos: an allele-specific primer that detects the wild type allele, am MGB oligonucleotide blocker that suppresses the mutant allele, a locus-specific primer, and a locus-specific TaqMan® FAM™ dye–labeled MGB probe.
For Research Use Only. Not for use in diagnostic procedures.