Methods, primers, probes and kits useful for the detection of BRAF mutations
The present invention relates to methods, primers and probes useful for detecting the presence of mutant BRAF sequences in a sample, specifically for detecting the presence of the BRAF V600E, V600D, V600K, and V600M mutations.
1. A method for detecting the presence of a BRAF mutation in a sample, said method comprising:
(a) isolating a nucleic acid from said sample, wherein the sample is a FFP formalin-fixed paraffin-embedded (FFPE) tissue sample, wherein the sample comprises DNA sequences;
(b) performing an amplification reaction of said DNA sequences of said sample, wherein said amplification reaction comprises a first primer capable of annealing specifically to a BRAF mutant sequence at a first position in a BRAF DNA sequence wherein said first primer consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 and a second primer capable of annealing specifically at a second position in a BRAF DNA sequence, wherein said first and second primers anneal to different strands of double stranded BRAF DNA sequence, wherein the amplification reaction is capable of producing a BRAF mutant specific amplification product when the DNA sequences of the sample comprise a BRAF DNA sequence comprising a mutant sequence at said first position of said BRAF DNA sequence; and
(c) visualizing amplification products produced by said amplification reaction, wherein detection of a BRAF mutant specific amplification product is a positive indicator of a BRAF mutation in said sample.
2. A method for detecting the presence of a BRAF mutation in a sample, said method comprising:
(a) isolating a nucleic acid from said sample wherein the sample comprises RNA sequences wherein the sample is a FFP formalin-fixed paraffin-embedded (FFPE) tissue sample;
(b) performing an amplification reaction of said RNA sequences of said sample, wherein said amplification reaction comprises a first primer capable of annealing specifically to a BRAF mutant sequence at a first position in a BRAF RNA sequence wherein said first primer consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 and a second primer capable of annealing specifically at a second position in a BRAF RNA sequence, wherein said first and second primers anneal to different strands of double stranded BRAF RNA sequence, wherein the amplification reaction is capable of producing a BRAF mutant specific amplification product when the RNA sequences of the sample comprise a BRAF RNA sequence comprising a mutant sequence at said first position of said BRAF RNA sequence; and
(c) visualizing amplification products produced by said amplification reaction, wherein detection of a BRAF mutant specific amplification product is a positive indicator of a BRAF mutation in said sample.
3. The method of claim 1 wherein the presence of the BRAF mutation is a positive indicator of metastatic melanoma in said sample.
4. The method of claim 2 wherein the presence of the BRAF mutation is a positive indicator of metastatic melanoma in said sample.
5. The method of claim 1 wherein the BRAF mutation is selected from the group consisting of BRAF V600E, BRAF V600D, BRAF V600K, BRAF V600M, and BRAF V600A mutations.
6. The method of claim 1 , wherein the sample is selected from the group consisting of blood, tissue, or cells.
7. The method of claim 1 , wherein the tissue sample is formalin fixed paraffin embedded tissue.
8. The method of claim 1 , wherein said second primer capable of annealing specifically at a second position in a BRAF nucleic acid sequence consists of SEQ ID NO: 10.
9. The method of claim 2 wherein the BRAF mutation is selected from the group consisting of BRAF V600E, BRAF V600D, BRAF V600K, BRAF V600M, and BRAF V600A mutations.