Discriminating BRAF mutations
View Patent ↗Provided herein are methods for detecting and discriminating BRAF V600 mutations. Also provided herein are methods for diagnosis, prognosis, management, and treatment decisions of BRAF V600 mutation-related diseases or conditions.
1. A method for identifying a BRAF mutation in a biological sample, the method comprising:
(a) amplifying a sample DNA from an individual by PCR utilizing a forward primer and a reverse primer, wherein the forward primer and the reverse primer are configured to amplify DNA sequences containing a thymidine (T) to adenosine (A) mutation at position 1799 of the BRAF gene, wherein the primers amplify both a V600E (1799T>A) mutation and a V600K (1798-1799GT>AA) mutation in the BRAF gene and do not amplify the wild type BRAF gene;
(b) subjecting the amplicons to a restriction enzyme digestion; and
(c) identifying a BRAF V600E mutation (1799T>A) in the sample if a digestion product is detected, or identifying a BRAF V600K mutation (1798-1799GT>AA) in the sample if a product of the restriction enzyme digestion is not detected.
2. The method of claim 1 , wherein the amplicon comprises the BRAF V600E mutation (1799T>A).
3. The method of claim 1 , wherein the amplicon comprises the BRAF V600K mutation (1798_1799GT>AA).
4. The method of claim 1 , wherein the restriction enzyme recognizes a 6-base motif that matches the sequence of the BRAF gene immediately upstream of the thymidine (T) to adenosine (A) mutation at position 1799 of the BRAF gene.
5. The method of claim 1 , wherein the sample DNA is extracted from the plasma of an individual.
6. The method of claim 1 , wherein the individual is a melanoma patient.
7. The method of claim 1 , wherein the incubation for the restriction enzyme digestion is for about one hour incubation at about 37° C.
8. The method of claim 1 , wherein the restriction enzyme is SfcI.
9. The method of claim 1 , further comprising (c) subjecting the digested amplicon to size separation.
10. The method of claim 9 , wherein size separation comprises denaturing HPLC.
11. The method of claim 9 , wherein size separation comprises gel electrophoresis or capillary electrophoresis.
12. The method of claim 11 , wherein the capillary electrophoresis comprises utilizing a fluorophore-conjugated primer in the PCR amplification.
13. A method for determining the drug sensitivity of an individual diagnosed with a BRAF-related disease or condition by determining if the individual has a specific mutation in the BRAF gene associated with a BRAF-related disease or condition comprising:
(a) amplifying a sample DNA from an individual by PCR utilizing a forward primer and a reverse primer, wherein the forward primer and the reverse primer are configured to amplify DNA sequences containing a thymidine (T) to adenosine (A) mutation at position 1799 of the BRAF gene, wherein the primers do not amplify the wild type BRAF gene;
(b) subjecting the amplicon to a restriction enzyme digestion with SfcI; and
(c) determining that the individual is likely to respond to trametinib when a restriction enzyme digestion product is detected in the sample, or determining that the individual is likely to respond to vemurafenib or trametinib if a restriction enzyme digestion product is not detected in the sample.