Elimination of carryover contamination for DNA methylation analysis based on isothermal amplification and lateral flow assay.

BACKGROUND: Aberrant DNA methylation is a significant prognostic of tumorigenesis, making it a promising biomarker for early diagnosis of cancer. Although bisulfite conversion combined with nucleic acid amplification can provide high sensitivity to detect trace of tumorous DNA, carryover contamination is inevitable and conflicts with sensitivity. Besides, uracil-DNA glycosylase-based strategy is inapplicable to aerosol elimination with bisulfite-converted DNA as template. False positive result caused by carryover contamination still exists in bisulfite conversion-based method for DNA methylation analysis. Hence, the deficiencies highlight the need to adopt another strategy for sequence analysis in bisulfite conversion-based methods.

RESULTS: In this study, a novel strategy for eliminating carryover contamination is developed based on the mechanism of DNA damage repair, applicable to bisulfite-converted DNA analysis. Loop-mediated isothermal amplification (LAMP) is employed for DNA methylation detection, ignoring the tendency of carryover contamination caused by the high sensitivity and numerous amplicons. By introducing deoxyinosine triphosphate during amplification, amplicon is labeled with hypoxanthine. The hypoxanthine-labeled amplicon as aerosol can be recognized and excised by endonuclease V subsequently, while bisulfite-converted DNA as template lacking hypoxanthine is insusceptible. Besides, a lateral flow device (LFD) is assembled as a fast and convenient strategy to interpret result visually, ignoring aerosol dispersion caused by the open vessel. The status of DNA methylation located on the promoter of miR-34a is identified using 10 real-world specimens and the result from this method is consistent with that from pyrosequencing.

SIGNIFICANCE: A novel strategy to eliminate carryover contamination is developed and integrated into a LAMP-based lateral flow biosensor for DNA methylation analysis. The conflict between carryover contamination and sensitivity is resolved, facilitating trace of DNA methylation detection. LFD can be employed for visual result interpretation, facilitating point-of-care test. As a universal strategy, this method can be easily expanded to other biomarkers, providing a great candidate for DNA methylation analysis to assist early diagnosis of cancer.