Thermodynamics-Guided Strand-Displacement-Based DNA Probe for Determination of the Average Methylation Levels of Multiple CpG Sites.

Determination of the methylation levels of genes of interest is fundamental for biological and medical research that involves DNA methylation. Using the average methylation levels of multiple CpG sites to represent the methylation levels of the whole gene is much more accurate than using that of only one CpG site. However, current methods that can provide the average methylation levels of several CpG sites are all expensive, time-consuming (several weeks), and labor-intensive. Herein, guided by the unique thermodynamics of the DNA strand-displacement process, we constructed a DNA fluorescent probe for determination of the average methylation levels of multiple CpG sites. Theoretical calculations of the reaction process and proof-of-concepts experiments on two to three CpG sites of synthesized DNA validated the basic principles of our probe. Taking two CpG sites in the promotor regions of SF-1 (steroidogenic factor 1) gene and VDR (vitamin D receptor) gene as the targets, we successfully measured their average methylation levels in nine endometrial cancer patients and two healthy persons. We believe our probe will be a very useful tool in the field, and we anticipate it being widely adopted by biological and medical investigators.