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.
|Product Name||Catalog No.||Price*|
|myT BRAF||BRAF-S1||$1,495.00 USD|
*Inquire about quantity discounts.
Mutation detection with myT BRAF consists of two steps:
Reagents Not Included
Number of Reactions
PDF versions of Material Safety Data Sheets (MSDS) are available for download.
Locus-specific qPCRA 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.
Allele-specific qPCRA 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
|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)
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.
|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
Scientific Poster on myT BRAF
Material Safety Data Sheets
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.