Methylation analysis to determine the presence of cancer cells

1. A method for determination of presence or absence of cancer cells in a tissue obtained from a colon of a subject comprising the steps of:

extracting DNA from the tissue;

carrying out bisulfite treatment to the DNA obtained from the step of extracting;

amplifying DNA of a promoter region of COL4A2 by using the DNA from the bisulfite treatment step as a template, a first primer comprising the sequence of SEQ ID NO: 15 and a second primer comprising the sequence of SEQ ID NO: 16;

obtaining a peak from a methylated DNA fragment and a peak from a non-methylated DNA fragment of the promoter region of COL4A2 by analyzing the amplified DNA by mass spectrometry;

calculating a methylation rate of CpG sites in the promoter region of COL4A2 from an area ratio between a peak from the methylated DNA fragment and a peak from the non-methylated DNA fragment; and

determining presence or absence of cancer cells in the tissue based on a comparison of the rate obtained from the step of calculating to a pre-determined cut-off value, and cancer cells are determined to be present in the tissue when the rate is higher than the cut-off value.

2. The method according to claim 1 , wherein the methylation rate of CpG sites is calculated from the formula:

area of the peak derived from the methylated DNA fragment/(area of the peak derived from the non-methylated DNA fragment+area of the peak derived from the methylated DNA fragment).

3. The method according to claim 1 , further comprising fragmenting the DNA obtained from the step of extracting after the step of extracting.

4. The method according to claim 1 , wherein the first primer has a tag sequence: 5′-AGGAAGAGAG-3′ (SEQ ID NO: 133) at the 5′ terminal of the first primer, and the second primer has a T7 promoter sequence: 5′-CAGTAATACGACTCACTATAGGGAGAAGGCT-3′ (SEQ ID NO: 134) at the 5′ terminal of the second primer.