myT BRAF Performance
myT BRAF performance on an ABI 7500 thermocycler
Positive amplification plot. qPCR reactions containing mutant genomic DNA, at the specified quantity in a background of 103 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).
Negative amplification plot. qPCR reactions containing 103 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 (left) and Bio-Rad CFX96 (right). Each reaction had 103 copies of wild-type genomic DNA alone (red) or spiked with either 102 copies (10% mutant, purple) or 10 copies (1% mutant; green) of BRAF V600E mutant template, as indicated. Non-specific amplification (from wild-type genomic DNA) was not observed (n=72 reactions).
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.
Conclusion. These and other data indicate that myT Primers provide sensitive and specific mutation detection in both FFPE and fresh frozen tissue samples*.
