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 interests 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 labour intensive. Herein, guided by the unique thermodynamics of DNA strand displacement process, we constructed a DNA fluorescent probe for determination of the average methylation levels of multiple CpG sites. Theoretical calcula-tions of the reaction process and proof-of-concepts experiments on 2-3 CpG sites of synthesized DNA validated the basic principles of our probe. Taking two CpG sites in the promotor regions of SF-1 gene and VDR gene as the targets, we successfully measured their average methylation levels in 9 endometrial cancer patients ...