myT BRAF

Product Name Catalog No. Price*
myT BRAF BRAF-S1 $1,495.00 USD

*Inquire about quantity discounts.

qPCR primers for detection of BRAF mutations V600E/K

Provides superior sensitivity and high confidence results
  • 1% sensitivity with no breakthrough amplification from wild-type
  • Definitive Yes/No answer
  • Simple – no need to use a delta Ct method to call a result
  • Performs well with either FFPE or fresh frozen samples



Each package includes reagents to perform a total of 60 reactions (30 locus-specific and 30 allele-specific reactions), sufficient for testing up to 28 samples plus positive and negative controls if performed as a single qPCR run.

The detection probe and PCR master mix must be ordered separately from licensed vendors. The purchase price of the probe and shipping costs are included in your purchase of myT BRAF. Instructions on how to easily order the probe from our recommended vendor and a coupon covering the purchase price and shipping cost of the probe will be provided with your order acknowledgement. This will enable you to synchronize delivery of the probe with delivery of myT BRAF. The cost of master mix purchase is not included.


Notice to Purchaser: Limited License
This product is for research use only and is licensed to the user under Swift Biosciences intellectual property only for the purchaser’s internal research. Purchase of this product does not convey to the purchaser any license to perform the Polymerase Chain Reaction “PCR” process under any third party rights. PCR probes can be purchased from a variety of vendors including Applied Biosystems (Life Technologies), Roche Molecular Systems, Inc., F. Hoffman La-Roche Ltd., Integrated DNA Technologies, Biosearch Technologies, Nanogen Inc. and others. The use of certain probes including TaqMan-MGB, FAM-TAMRA, FAM-BHQ, VIC-MGB in connection with ("PCR") process may require a license from one or more of these vendors. Please contact individual vendors for the necessity of obtaining licenses. The purchase of myT BRAF or any other items delivered by Swift Biosciences hereunder does not, either expressly or by implication, provide a license to use any proprietary technology of these vendors.

Mutation detection with myT BRAF consists of two steps:
  1. Locus-specific qPCR
    A non-allele-specific qPCR is performed to assess total (mutant + wild-type) amplifiable BRAF for each sample. This determines the quantity of DNA to be used in step 2 for each sample.

  2. Allele-specific qPCR
    A mutant allele-specific BRAF qPCR is then performed to assess presence of V600E or V600K. This assay DOES distinguish between wild-type and mutant but DOES NOT distinguish between the two mutant alleles. Results are reported as positive or negative for mutant BRAF for each sample with a sensitivity limit of 1%. Samples containing less than 1% mutant BRAF are either negative or below the detection limit of this assay.
myT BRAF Workflow

Isolate genomic DNA from samples


Obtain UV absorbance readings

 

Perform locus-specific qPCR

 

Determine amplifiable copy number from Ct values

 

Perform allele-specific qPCR

 

Determine BRAF V600E/K status

Reagents Included
Reagent Mix Description
BRAF Locus-specific Primers Non-allele-specific primers
BRAF Allele-specific Primers
V600E/K allele-specific myT BRAF primers
Nuclease-free Buffer For DNA sample dilution and NTC reactions
BRAF DNA Standard BRAF V600E control DNA (200 copies/µl)
 

Reagents Not Included
The following reagents are not provided and must be ordered separately from a licensed vendor. The purchase price of the probe and shipping costs are included in your purchase of myT BRAF. Instructions on how to easily order the probe from our recommended vendor and a coupon covering the purchase price and shipping cost of the probe will be provided with your order acknowledgement. This will enable you to synchronize delivery of the probe with delivery of myT BRAF. The cost of master mix purchase is not included.

Reagent Recommended Vendor
qPCR Master Mix (2X) Fermentas Maxima Probe qPCR Master Mix, Catalog # K0261
Detection Probe IDT PrimeTime Dual-Labeled Probe (voucher included with purchase)

Number of Reactions
The product contents are sufficient to perform 60 reactions consisting of 30 locus-specific and 30 allele-specific reactions. This enables testing of up to 28 samples when including a positive control and a no-template control (NTC) when performed as a single qPCR run. If testing is split into multiple batches, total samples tested will be less as a positive control and NTC are required for each run. For example, if testing is batched into 3 qPCR runs, the total number of samples analyzed will be 24, in addition to 3 positive control and 3 NTC reactions.

Scientific Poster on myT BRAF

Sensitive and Specific Detection of Cancer Mutations with myT® Primers Sensitive and Specific Detection of Cancer Mutations with myT® Primers

Product Literature
Material Safety Data Sheets
PDF versions of Material Safety Data Sheets (MSDS) are available for download.
myT BRAF Performance on the ABI 7500
Top: qPCR reactions containing mutant genomic DNA at the specified quantity in a background of 1000 copies of wild-type genomic DNA resulted in BRAF V600E-specific amplification: 50% mutant content (pink; n = 4 replicates), 10% mutant content (purple; n = 4 replicates) and 1% mutant content (green; n = 8 replicates).

Bottom: qPCR reactions containing 1000 copies of wild-type genomic DNA (no mutant DNA) resulted in no amplification (red; n = 95 replicates).





Conclusion. These results demonstrate mismatch discrimination with very high specificity. The result is clear and unambiguous, eliminating the need for ΔCt analysis to distinguish specific from non-specific amplification. This high confidence, “Yes/No” clarity is a feature that is exclusive to myT Primer reagents.

myT BRAF performance on Roche LightCycler 480 & Bio-Rad CFX96 instruments

qPCR was performed using standard cycling conditions in single tube format with myT BRAF primers on a Roche LightCycler 480 (top) and Bio-Rad CFX96 (bottom). Each reaction had 1000 copies of wild-type genomic DNA alone (red) or was spiked with either 100 copies (10% mutant; purple; n = 8 replicates) or 10 copies (1% mutant; green; n = 16 replicates) of BRAF V600E mutant template, as indicated. Non-specific amplification (from wild-type genomic DNA) was not observed (0% mutant; red; n = 72 replicates).





Conclusion. On both instruments, myT BRAF primers demonstrated 100% sensitivity for 10 mutant copies with 100% specificity.

myT BRAF performance on melanoma FFPE

Genomic DNA was isolated from 26 FFPE melanoma specimens and UV absorbance readings were taken. 50-100 ng per sample were tested first for amplifiable total BRAF locus copy number by qPCR followed by allele-specific V600E/K qPCR using myT BRAF primers. All samples had a total BRAF amplifiable copy number ranging from 100-1,000. For allele-specific V600E/K qPCR, 13/26 samples had positive amplification signals ranging from a Ct of 34.0 – 41.9. The remaining 13/26 samples had no allele-specific amplification, where an observed BRAF V600E/K mutation frequency of 50% is consistent with expected results for melanoma.

 myT BRAF assay n value Ct range frequency
positive for V600E/K 13/26 34.0-41.9 50%
negative for V600E/K 13/26 no amplification 50%

Conclusion. These and other data indicate that myT Primers provide sensitive and specific mutation detection in both FFPE and fresh frozen tissue samples*.

*For research use only

Trademarks
ABI 7500 is a product of Applied Biosystems, now part of Life Technologies Corporation
CFX96 is a product of Bio-Rad Laboratories, Inc.
LightCycler 480 is a product of Roche Applied Science