The
aberrant methylation of many genes has been reported to be
associated with various carcinomas. Accurate detection of the methylation
level could provide critical insights into the diagnostic analysis
of diseases. Here, a sensitive HpaII-edited absolute droplet loop-mediated
isothermal amplification (HEADLAMP) method based on methylation-sensitive
restriction enzyme (MSRE) HpaII was developed for the digital quantification
of DNA methylation. Methylation levels of the death-associated protein
kinase 1 (DAPK1) gene that is associated with many cancers were studied
using β-actin as an internal reference. DAPK1 (2.5 pM) with
0.01% methylation (250 aM) can be detected with the conventional HpaII-edited
LAMP assay. Using HEADLAMP, as low as 1% methylation level can be
distinguished with an estimated limit of detection of 5 aM (ca. 3
copies/μL). Moreover, HEADLAMP can detect low levels of methylated
DAPK1 in normal L-02 cells, while the conventional assay cannot. Finally,
HEADLAMP was applied to the detection of DAPK1 methylation in 20 clinical
tissue samples, which revealed hypermethylated DAPK1 in cervical cancer
patients. We envisage potential applications of this robust, specific,
and sensitive HEADLAMP assay in epigenetic studies and early clinical
diagnosis.