Methods of detecting mutations in BRAF and epigenetic changes
View Patent ↗Disclosed are methods for assessing the methylation and mutation status of nucleic acid in a sample. The methods provide for methylation-dependent modification of the nucleic acid in a sample, and subsequently nucleic acid amplification processes to distinguish between mutated and non-mutated target sequence.
1. A method for detecting a mutated BRAF target sequence of interest in a DNA-containing sample, the method comprising:
(a) contacting the DNA-containing sample with a modifying reagent which selectively modifies non-methylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues,
(b) amplifying the modified DNA-containing sample to produce an amplified target sequence using at least one primer that hybridizes specifically to the mutated BRAF target sequence in the modified DNA-containing sample, and
(c) detecting the amplified target sequence, thereby detecting the mutated BRAF target sequence.
2. The method of claim 1 , further comprising detecting methylation status of the BRAF target sequence by amplifying the modified DNA-containing sample to produce an amplified target sequence using at least one at least one primer that hybridizes specifically to a methylated BRAF target sequence in the modified DNA-containing sample, and detecting the amplified target sequence, thereby detecting the methylated BRAF target sequence.
3. The method of claim 1 , wherein amplification is performed using a primer that hybridizes specifically to the mutated BRAF target sequence and a primer that hybridizes specifically to methylated BRAF DNA.
4. The method of claim 1 , further comprising detecting a mutated KRAS target sequence of interest in the modified DNA-containing sample, the method further comprising amplifying the modified DNA-containing sample to produce an amplified target sequence using at least one primer that hybridizes specifically to the mutated KRAS target sequence in the modified DNA-containing sample, and detecting the amplified target sequence, thereby detecting the mutated KRAS target sequence.
5. The method of claim 1 , wherein the modifying reagent comprises a bisulfite salt.
6. The method of claim 1 , further comprising sequencing the amplified target sequence.
7. A method for detecting a mutated BRAF gene target sequence of interest in a DNA-containing sample, the method comprising:
(a) contacting the DNA-containing sample with a modifying reagent which selectively modifies non-methylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues,
(b) amplifying the modified DNA-containing sample to produce an amplified target sequence using a primer pair comprising SEQ ID NO:3 and SEQ ID NO:2; and
(c) detecting the amplified target sequence, thereby detecting the mutated BRAF gene target sequence.
8. The method of claim 7 , further comprising detecting a mutated KRAS target sequence of interest in the modified DNA-containing sample, the method further comprising amplifying the modified DNA-containing sample to produce an amplified target sequence using at least one primer that hybridizes specifically to the mutated KRAS target sequence in the modified DNA-containing sample, and detecting the amplified target sequence, thereby detecting the mutated KRAS target sequence.
9. The method of claim 7 , wherein the modifying reagent comprises a bisulfite salt.
10. The method of claim 7 , further comprising sequencing the amplified target sequence.
11. A method for detecting at least one mutated BRAF gene target sequence of interest in a DNA-containing sample, the method comprising:
(a) contacting the DNA-containing sample with a modifying reagent which selectively modifies non-methylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues,
(b) amplifying the modified DNA-containing sample to produce an amplified target sequence using a primer pair comprising SEQ ID NO:3; and
(c) detecting the amplified target sequence, thereby detecting the mutated BRAF gene target sequence.
12. The method of claim 11 , further comprising detecting a mutated KRAS target sequence of interest in the modified DNA-containing sample, the method further comprising amplifying the modified DNA-containing sample to produce an amplified target sequence using at least one primer that hybridizes specifically to the mutated KRAS target sequence in the modified DNA-containing sample, and detecting the amplified target sequence, thereby detecting the mutated KRAS target sequence.
13. The method of claim 11 , wherein the modifying reagent comprises a bisulfite salt.
14. The method of claim 11 , further comprising sequencing the amplified target sequence.