A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

Yueying Sun,Yuanyuan Sun,Kejian Gao,Chenghui Liu,Weimin Tian,Zhengping Li

doi:10.1039/c7sc04975g

Abstract

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

Highlights

DNA methylation at the C5 position of cytosine in CpG dinucleotides is the most frequent and important epigenetic modi cation mechanism in mammalian cells, which plays critical roles in the regulation of gene expression, genomic imprinting, and X-chromosome inactivation.[1,2] Aberrant CpG methylation states, especially those that occur in the gene promoter regions, have been proved to be closely associated with the development of many human diseases including cancers.[3]
By integrating the unique features of GlaI for highly specific methylation discrimination and exponential amplification reaction (EXPAR) for rapid and powerful signal amplification, the elegant GlaI–EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy
Because this methylation site is well-recognized to be closely associated with the tumorigenesis of human colon cancer.[24]

Summary

Introduction

DNA methylation at the C5 position of cytosine in CpG dinucleotides is the most frequent and important epigenetic modi cation mechanism in mammalian cells, which plays critical roles in the regulation of gene expression, genomic imprinting, and X-chromosome inactivation.[1,2] Aberrant CpG methylation states, especially those that occur in the gene promoter regions, have been proved to be closely associated with the development of many human diseases including cancers.[3].

Results

Conclusion