Digital Loop-Mediated Isothermal Amplification-Based Absolute Methylation Quantification Revealed Hypermethylated DAPK1 in Cervical Cancer Patients

Abstract

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.

What the Microscale Systems “See” In Biological Assemblies: Cells and Viruses?

“…LAMP is considered a desirable alternative point-of-care (POC) detection method for PCR. LAMP involves four to six primers and a strand-displacing DNA polymerase to promote the amplification process at a constant temperature. − LAMP provides a quick and selective technology for detecting foodborne microorganisms . In addition to controlling microbial contamination in food, it is important to explore fast and sensitive methods for detecting bacteria in water samples. − To avoid the application of expensive and complicated equipment, Fu et al investigated a rapid on-chip most probable number (MPN)–LAMP approach to determine the target genes of fecal indicator bacteria (FIB) in water samples .…”

Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation

“…Nucleic acid amplification is a promising strategy for target signal amplification. Among the numerous amplification techniques, isothermal amplification, such as rolling circle amplification, exponential amplification reaction, and recombinase polymerase amplification (RPA), have attracted increasing attention. Compared with conventional PCR-based detection which requires a thermal cycle process, isothermal amplification can be performed under isothermal conditions with a high amplification efficiency .…”

Agarose-Droplet-Based Digital LAMP Assay for Counting Virus DNA in Single-Particle ICP-MS

“…By counting the partitions with a positive fluorescent signal, the number of targets could be obtained without the need for a calibration curve. A variety of digital isothermal amplification techniques were developed for fast amplifying the target nucleic acids at a simple constant temperature and avoiding the complex process of thermal cycling of dPCR, such as digital loop-mediated isothermal amplification (LAMP) − or recombinase polymerase amplification (RPA). , By virtue of high sensitivity, high accuracy, satisfactory resistance to inhibitors, less amplification bias, and sample contamination, these “digital” techniques have flourished in the fields of nucleic acid detection at extremely low concentrations for analysis of pathogens, ,, cancer biomarkers, , rare mutations, methylation, gene expression, sequencing assistance, , etc. Hitherto, those digital assays are limited to fluorescence detection, mainly including fluorescence microscopy and flow cytometry, and few other techniques have been introduced to digital assay.…”