Method for methylation analysis
AssigneeClinical Genomics Pty. Ltd.
InventorsSusanne Pedersen, Rohan Baker
Life of this patent
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Abstract
The present invention relates generally to a method for assessing nucleic acid methylation, in particular DNA and RNA methylation. More particularly, the present invention relates to a method of either qualitatively or quantitatively assessing, with improved sensitivity, the cytosine methylation of partially methylated DNA or RNA. The method of the present invention is useful in a range of applications including, but not limited to, the diagnosis of conditions or monitoring of developmental phenotypes which are characterised by DNA or RNA methylation changes.
Claims31
A method for detecting the cytosine methylation of a nucleic acid target of interest, said method comprising:
- (i) contacting a nucleic acid sample with an agent which modifies unmethylated cytosine residues;
- (ii) measuring within a single reaction, regions of partial methylation in a DNA form of the nucleic acid sample of step (i) by contacting the DNA with:
- (a) a primer set comprising forward and reverse primers designed to amplify one or more partially methylated forms of a region of cytosine methylation;
- and
- (b) one or more probes directed to said region of cytosine methylation wherein said one or more probes are capable of collectively hybridising to at least two differing methylation patterns at said region and wherein said probe comprises a detectable moiety;
- (iii) amplifying the sample of step (ii), wherein the extension of said primers along said target of interest effects the detection of said hybridised probe;
- and,
- (iv) qualitatively or quantitatively analysing the detection output of step (iii).
A diagnostic kit for detecting the cytosine methylation of a region of a nucleic acid target of interest, said kit comprising:
- (i) a primer set comprising forward and reverse primers designed to amplify one or more partially methylated forms of a DNA form of said nucleic acid region in which unmethylated cytosine residues have been modified, wherein the primer set includes primers which comprise a sequence as set forth in:
- (a) SEQ ID NO:
- 3 and SEQ ID NO:
- 4 or a sequence exhibiting at least 95% identity;
- (b) SEQ ID NO:
- 47 and SEQ ID NO:
- 48 or a sequence exhibiting at least 95% identity;
- (c) SEQ ID NOs:
- 49-62 and SEQ ID NOs:
- 63-76 or a sequence exhibiting at least 95% identity;
- or
- (d) SEQ ID NO:
- 77 and SEQ ID NO:
- 78 or a sequence exhibiting at least 95% identity;
- and
- (ii) one or more probes directed to the regions of partial cytosine methylation of the DNA of step (i) which probes are capable of collectively hybridising to at least two differing methylation patterns, wherein the one or more probes include probes comprising a sequence as set forth in:
- (a) SEQ ID Nos:
- 5-12 or a sequence exhibiting at least 95% identity;
- (b) SEQ ID NO:
- 19 or a sequence exhibiting at least 95% identity;
- (c) SEQ ID NO:
- 20 or a sequence exhibiting at least 95% identity;
- (d) SEQ ID NO:
- 21 or a sequence exhibiting at least 95% identity;
- (e) SEQ ID NO:
- 22 or a sequence exhibiting at least 95% identity;
- (f) SEQ ID NOs:
- 23-30 or a sequence exhibiting at least 95% identity;
- (g) SEQ ID NOs:
- 31-38 or a sequence exhibiting at least 95% identity;
- or
- (h) SEQ ID NOs:
- 39-46 or a sequence exhibiting at least 95% identity.
Dependencies are inferred from the claim text.