Label-Free and Sequence-Independent Isothermal Amplification Strategy for the Simultaneous Detection of Genomic 5-Methylcytosine and 5-Hydroxymethylcytosine

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are crucial epigenetic modifications in eukaryotic genomic DNA that regulate gene expression and are associated with the occurrence of various cancers. Here, we combined bisulfite conversion with 4-acetamido-2,2,6,6-tetramethyl-1-oxopiperridinium tetrafluoroborate (ACT⁺BF₄^–, TCI) oxidation to develop a label-free and sequence-independent isothermal amplification (BTIA) assay for a genome-wide 5mC and 5hmC analysis. The BTIA strategy can distinguish 5mC and 5hmC signatures from other bases with high sensitivity and good specificity, avoiding sophisticated chemical modifications and expensive protein labeling. Moreover, the utilization of terminal deoxynucleotidyl transferase (TdT) enables the proposed strategy to detect global 5mC and 5hmC without sequence dependence. With only 78 ng of input of genomic DNA, global 5mC and 5hmC levels were accurately quantified in cells (including cancer cells of A549, T47D, and K562 and normal cells of HEK-293T, CHO, and NRK-52E) and clinical whole blood samples (including healthy control, precancerous cervical cancer, and confirmed cervical cancer) within 18 h. The detection results suggested that 5mC was highly expressed in cancer cells. More importantly, a significant increase in 5mC was observed in precancerous cervical cancer and further upregulation in confirmed cervical cancer, suggesting a correlation between 5mC and cancer occurrence and development. However, 5hmC showed the reverse result in these tested cells and clinical samples. Collectively, the BTIA strategy can be easily performed on the ordinary heating apparatus in almost all research and medical laboratories, showing a significant application in the early screening of cervical cancer in the clinic.