Methods for detecting CpG methylation and for diagnosing cancer
The present invention relates to the field of pharmacogenomics and in particular to detecting the presence or absence of hypermethylated DNA. The detection of CpG methylation in marker DNA is useful for the diagnosis of cancers and the invention provides improved methods for this purpose. These improved methods allow in particular for a more sensitive detection of methylated marker DNA with high backgrounds of unmethylated marker DNA.
1. A method for detecting hypermethylation of a target DNA in a sample comprising genomic DNA, the method comprising the steps of:
(a) obtaining a sample comprising genomic DNA;
(b) converting unmethylated cytosine to another base in the genomic DNA using a converting agent, wherein the genomic DNA is at least partially fragmented;
(c) amplifying the converted genomic DNA using a pair of methylation non-specific primers comprising the nucleic acid sequences of SEQ ID NOs: 121 and 122 and at least one methylation specific blocker, wherein the methylation specific blocker comprises the nucleic acid sequence of SEQ ID NO: 123 or 124, and wherein the hypermethylated target DNA is a region of genomic human SHOX2 DNA; and
(d) detecting the amplicon, wherein detecting the amplicon detects hypermethylation of the target DNA in the sample.
2. The method of claim 1 , wherein the converting agent converts unmethylated cytosine to uracil.
3. The method of claim 1 , wherein the converting agent is a bisulfite.
4. The method of claim 1 , wherein the pair of methylation non-specific primers consist of the nucleic acid sequences of SEQ ID NOs: 121 and 122.
5. The method of claim 1 , wherein the methylation specific blocker comprises the nucleic acid sequence of SEQ ID NO: 124.
6. The method of claim 1 , wherein the amplicon is detected by a method comprising annealing a probe to the amplicon and detecting the annealed probe.
7. The method of claim 6 , wherein the probe is linked to a detectable label.
8. The method of claim 6 , wherein the probe comprises the nucleic acid sequence of SEQ ID NO: 125.
9. The method of claim 8 , wherein the probe consists of the nucleic acid sequence of SEQ ID NO: 125.
10. The method of claim 1 , wherein the sample is selected from the group consisting of blood, blood serum, and blood plasma.
11. The method of claim 1 , wherein the genomic DNA sequence comprises SEQ ID NO: 116.
12. The method of claim 1 , wherein the methylation specific blocker consists of the nucleic acid sequence of SEQ ID NO: 124.
13. The method of claim 1 , wherein the methylation specific blocker comprises the nucleic acid sequence of SEQ ID NO: 123.
14. The method of claim 1 , wherein the methylation specific blocker consists of the nucleic acid sequence of SEQ ID NO: 123.