Discriminating BRAF mutations
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 kit for distinguishing a V600K mutation from a V600E mutation in the BRAF gene in a sample DNA from an individual, said kit comprising:
(a) a forward primer and a reverse primer and no more than two primers, wherein the forward primer and the reverse primer selectively generate an amplicon when a thymidine (T) to adenosine (A) mutation at position 1799 of the BRAF gene is present, wherein the primers are configured to generate an amplicon comprising a V600E (1799T>A) mutation and an amplicon comprising a V600K (1798-1799GT>AA) mutation in the BRAF gene, and
(b) a restriction enzyme for distinguishing a V600K mutation from a V600E mutation in the BRAF gene, wherein the restriction enzyme digests the amplicon comprising the BRAF V600E mutation (1799T>A) and does not digest the amplicon comprising the BRAF V600K mutation (1798_1799GT>AA),
wherein the forward and/or reverse primer are labeled with a detectable moiety.
2 . The kit 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.
3 . The kit of claim 1 , wherein the restriction enzyme is SfcI.
4 . The kit of claim 1 , wherein the detectable moiety is a fluorophore.
5 . The kit of claim 1 , wherein the sample DNA is extracted from the plasma of the individual.
6 . The kit of claim 1 , wherein the individual is a melanoma patient.