Accurate identification and quantification of 5-hydroxymethylcytosine (5hmC) can help elucidate its function in gene expression and disease pathogenesis. Current 5hmC analysis methods still present challenges, especially for clinical applications, such as having a risk of false-positive results and a lack of sufficient sensitivity. Herein, a 5hmC quantification method for fragment-specific DNA sequences with extreme specificity, high sensitivity, and clinical applicability was established using a quantitative real-time PCR (qPCR)-based workflow through the combination of enzymatic digestion and biological deamination strategy (EDD-5hmC assay). The EDD-5hmC approach enriched glycosylated 5hmC via enzyme digestion and then APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-mediated deamination to efficiently differentiate between various cytosine(C) modification states, resulting in 5hmC quantification with extreme specificity such that nonspecific amplification is reduced over eight million-fold. Moreover, the nondestructive biological treatment process of the EDD-5hmC assay exhibits high sensitivity, yielding the limit of detection of 30 aM. For the first time, we measured 5hmC levels in colorectal cancer tissues and matched paracancerous tissues to evaluate the ability to differentiate colorectal cancer, with the area under the receiver operating characteristic curve of up to 82.8% for the single gene of Septin9 and 83.6% for the combinations of Septin9 and Syndecan-2 (SDC2), demonstrating the EDD-5hmC assay is a promising method with clinical applicability for accurately quantifying the 5hmC level.

Fragment-specific Quantification of 5hmC by qPCR via a Combination of Enzymatic Digestion and Deamination: Extreme Specificity, High Sensitivity, and Clinical Applicability