Fluorescence-based Polymerase Amplification for the Sensitive Detection of DNA Methyltransferase Activity

• Published in: Analytical Sciences 2018/08/10, Vol.34(8), pp.959-964
• Main Authors: ZHANG, Xiao, ZHONG, Xiao-Li, JIANG, Wen-Wen, ZENG, Si-Hao, PI, Ting, ZHENG, Xiang-Juan, LI, Zhi-Mei
• Format: Article
• Language: English
• Subjects: Adenine; Amplification; amplification strategy; Analytical Chemistry; Biological activity; Chemistry; Dam MTase activity; Deoxyribonucleic acid; Diagnosis; DNA; DNA methylation; DNA methyltransferase; Drug development; Endonuclease; Fluorescence; fluorescence system; Nucleotide sequence; polymerization reactions; Site-specific DNA methyltransferase (adenine-specific); Transcription
• ISSN: 0910-6340; 1348-2246
• DOI: 10.2116/analsci.18P080

DNA methyltransferase (MTase) is related to transcriptional repressor activity in biological functions. It is an essential for cancer diagnosis and therapeutics to detect DNA MTase activity sensitively. Here, a fluorescent system based on polymerase amplification has been developed to detect DNA adenine MTase (Dam) activity sensitively. The amplification is triggered by the probe DNA regions a, which are the primes of a polymerase-induced replicated reaction. They come from methylation and a digestion reaction of DNA S1-S1, including a 5′-GATC-3′ sequence recognized by Dam MTase and methylation sensitive restriction endonuclease Dpn I. The intensities of fluorescence are dependent on the Dam MTase activity. The method shows fine sensitivity with a detection limit of 3.2 × 10−4 U mL−1 and specificity for Dam MTase. In human serum samples, the method has been successfully applied, and it has also been used to screen the inhibitors, which means that the developed method can be a powerful and potential tool for drug development and clinical diagnosis in the future.