The methods for DNA MTase activity assay involve

The methods for DNA MTase activity assay involve radioactive labelling [15], gel electrophoresis [16], high-performance liquid chromatography [17], [18], bisulfite conversion [19], [20], and affinity enrichment [21], [22]. In these methods, the methylated bases under the action of active MTase could change the DNA's migration rate, hybridization efficiency, or affinity to antibodies, so as to generate the signal different from that under the action of inactive MTase. Recently, the methods based on the MTase-dependent DNA probe cleavage have been proposed [23]. In these methods, the methylated bases can facilitate or inhibit the probe cleavage by the restriction endonuclease that results in the release of the oligonucleotide fragment. The oligonucleotide fragment can be readily modified, flexibly designed and further adopted for the downstream reaction, offering more effective alternatives for signal transduction and amplification. Initially, the released oligonucleotide fragment directly generates the signal [24], [25], [26], [27], [28], [29], such as the fluorescence caused by fluorescence resonance energy transfer change [24], [25], [26], [27] or the electrochemical current caused by electron transfer efficiency change [28], [29]. It achieves a simple DNA MTase activity detection but the sensitivity is compromised due to the limited amount of released oligonucleotide fragments under the action of MTase. Subsequently, the released oligonucleotide fragment is utilized as trigger for the cascade amplification [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], such as acting as DNAzymes for the catalysis of substrate hydrolysis [30], [31], templates for strand displacement amplification [32], [33] or auxiliary strands for nuclease-mediated cyclic adenosine kinase of reporter DNA [34], [35]. Although the sensitivity is improved, in most of these reported methods, only one trigger for cascade amplification is released from the DNA probe under the action of MTase. It means that for each DNA probe, the MTase recognition event is converted to merely single trigger-induced signal transduction, still restricting the further increase of sensitivity. In the view of that more sensitive MTase activity detection is important to the MTase-based early diagnosis [43], new detection strategies are still needed to be developed.
Experimental section
Results and discussion
Conclusions
Epigenetic regulation of tumor is the advancing spot in the present international biochemistry research []. Recent studies have indicated that epigenetic alterations play an essential role in this type of cancer []. DNA methyltransferases (DNMTs) are the important representations of epigenetic alterations. There are numerous of reports have shown that the aberrant expressions of DNMTs in breast cancer [, , ], liver cancer [], lung cancer [] and gastric cancer []. DNA methylation is mainly formed by the addition of 1-methyl at the 5′-position of cytosine to 5-methylcytosine catalyzed by methyltransferase []. One of 5-methylcytosine synthesis pathways is DNA methyltransferase 1 (DNMT1) catalyzes complete methylation of the unmethylated chain in the semi-methylated DNA and maintains methylation consistent with the protocell. In other words, the activity and concentration of DNMT1 directly affect the degree of DNA methylation. Overexpression of DNMT1 may cause aberrant methylation of tumor suppressor gene or proto-oncogene promoter, further leading to tumorigenesis. Many methods for detecting DNMT1 activity were well established which include radioactive labeling strategy [], high performance liquid chromatography [], gel electrophoresis [], and immune-based assay [, , ]. However, they are usually used for the DNMT1 activity detection, hardly applied for the quantitative analysis. The detection of enzyme activity must be based on the measurement of enzyme content, so the determination of enzyme concentration is of great concern.