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US 10,947,597 B2Granted
Methods for detecting BRAF in cancer
AssigneeBeth Israel Deaconess Medical Center, Inc.
InventorDavid J. Panka
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Abstract
The invention features compositions and methods for detecting and quantifying BRAFV600E in a sample of a subject. In particular embodiments, the invention provides a highly sensitive and precise quantitative method for diagnosis, post-operative surveillance, and monitoring evidence of disease over time in the adjuvant setting (i.e. treatment with BRAF-inhibitors).
Claims14
A method for detecting a BRAFV600E mutation in a biological sample, the method comprising
- providing a cDNA that is reverse transcribed from RNA isolated from a biological sample of a subject;
- amplifying the cDNA with a forward oligonucleotide to obtain a BRAF amplicon, wherein the forward oligonucleotide comprises an exact match to a nucleic acid encoding amino acid residue 600 in a BRAF mutant protein and is a single base mismatch to a nucleic acid encoding amino acid residue 600 in a wild-type BRAF protein;
- digesting the BRAF amplicon with a TspR1 restriction enzyme;
- and
- detecting a level of the BRAF amplicon with a probe, wherein the probe comprises an exact match to a nucleic acid encoding amino acid residue 600 in the BRAF mutant protein and is a single base mismatch to a nucleic acid encoding an amino acid residue 600 in a wild-type BRAF protein, and
- wherein a level greater than about 5 pg of the BRAF amplicon indicates the presence of a BRAFV600E mutation in the biological sample.
3 more elements
A method for detecting a BRAFV600E mutation in a biological sample, the method comprising
- (a) amplifying a cDNA to obtain a first BRAF amplicon, wherein the cDNA is reverse transcribed from RNA isolated from a biological sample of a subject;
- (b) digesting the first BRAF amplicon with a TspR1 restriction enzyme to obtain a first digested material;
- (c) amplifying the first digested material to obtain a second BRAF amplicon;
- (d) digesting the second BRAF amplicon with the TspR1 restriction enzyme to obtain a second digested material;
- (e) amplifying the second digested material with a forward oligonucleotide to obtain a third BRAF amplicon, wherein the forward oligonucleotide comprises an exact match to a nucleic acid encoding amino acid residue 600 in a BRAF mutant protein and is a single base mismatch to a nucleic acid encoding amino acid residue 600 in a wild-type BRAF protein;
- and
- (f) detecting the third BRAF amplicon with a probe, wherein the probe comprises an exact match to a nucleic acid encoding amino acid residue 600 in the BRAF mutant protein and is a single base mismatch to a nucleic acid encoding amino acid residue 600 in the wild-type BRAF protein,
- wherein a level greater than about 5 pg of the third BRAF amplicon detected indicates the presence of a BRAFV600E mutation in the biological sample.
5 more elements
Dependencies are inferred from the claim text.