Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation

Xue, Qingwang; Lv, Yanqin; Xu, Shuling; Zhang, Yuanfu; Wang, Lei; Li, Rui; Yue, Qiaoli; Li, Haibo; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

doi:10.1016/j.bios.2014.12.017

Cited by 44 publications

(35 citation statements)

References 25 publications

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“…18 Furthermore, in order to simplify the process, shorten the analysis time, improve the sensitivity, and lower the cost, many new techniques and methods such as fluorescence methods based on hairpin DNA probe, [19][20][21] enzyme immunoassay based on gold nanoparticles, 22,23 surface-enhanced Raman spectroscopy, 24,25 chemiluminescence assay, 26,27 electrochemical methods, [28][29][30][31] and so on have been designed to evaluate the activity of DNA MTase.…”

Section: Yu Et Almentioning

confidence: 99%

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

Yu¹,

Xiong²,

Yu³

et al. 2018

IJN

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Background DNA methyltransferase 1 (DNMT1), a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase) detection have focused on the prokaryote MTase and its activity. Methods A magnetic fluorescence-linked immunosorbent assay (FLISA) using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized. Results Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1–1,500 ng/mL, the recovery was 91.67%–106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%–11.29% and 7.03%–11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA) kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r =0.866, p =0.001). The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter than ELISA kits. Conclusion These indicated that the proposed FLISA method was sensitive and high throughput and can quickly screen the level of DNMT1 in serum samples.

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Section: Yu Et Almentioning

confidence: 99%

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

Yu¹,

Xiong²,

Yu³

et al. 2018

IJN

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“…[41][42][43][44][45][46][47][48] EXPAR nutzt zur exponentiellen Amplifikation der Zielsequenz ein DNA-Te mplat, Desoxynukleotidtriphosphate (dNTPs) und zwei enzymatische Reaktionen (Abbildung 1A). Seit ihrer ersten Beschreibung im Jahr 2003 durch Galas und Mitarbeiter wurde EXPAR bereits zur Detektion von microRNA( miRNA), DNA, Proteinen, Enzymaktivitätu nd Metallionen verwendet.…”

Section: Das Prinzip Von Exparunclassified

“…[47] Das System enthielt eine EXPAR-Trigger-Sequenz, die in der Stammregion einer Haarnadelstruktur enthalten war.D er Stamm der Haarnadelstruktur wurde durch die MTase methyliert und anschließend durch ein methylierungssensitives Restriktionsenzym gespalten. [47] Das System enthielt eine EXPAR-Trigger-Sequenz, die in der Stammregion einer Haarnadelstruktur enthalten war.D er Stamm der Haarnadelstruktur wurde durch die MTase methyliert und anschließend durch ein methylierungssensitives Restriktionsenzym gespalten.…”

Section: Messungen Der Enzymaktivitätunclassified

Die exponentielle isotherme Amplifikation von Nukleinsäuren und Assays zur Detektion von Proteinen, Zellen, kleinen Molekülen und Enzymaktivitäten: Anwendungen für EXPAR

Reid¹,

Zhang

2018

Angewandte Chemie

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Isotherme exponentielle Amplifikationstechniken, wie die Strangverdrängungsamplifikation (SDA), die Rolling‐Circle‐Amplifikation (RCA), die Loop‐vermittelte isotherme Amplifikation (LAMP), die Nukleinsäuresequenz‐basierte Amplifikation (NASBA), die Helikase‐abhängige Amplifikation (HDA) und die Rekombinase‐Polymerase‐Amplifikation (RPA) besitzen großes Anwendungspotential für Vor‐Ort‐Analysen, patientennahe Labordiagnostik und In‐situ‐Assays. Diese Amplifikationsmethoden benötigen im Gegensatz zur Polymerasekettenreaktion (PCR) keine Temperaturzyklen, liefern aber vergleichbare Ausbeuten. In diesem Kurzaufsatz stellen wir die neuesten Entwicklungen der exponentiellen Amplifikationsreaktion (EXPAR) zur Detektion von Nukleinsäuren, Proteinen, Enzymaktivitäten, Zellen und Metallionen vor. Mithilfe der Verwendung von Fluoreszenz, Kolorimetrie, Chemilumineszenz, Raman und elektrochemischer Verfahren konnte bereits eine hoch sensitive Detektion einer Vielzahl verschiedener Ziele (Targets) realisiert werden. Dennoch bleiben offene Fragen, wie insbesondere die der unerwünschten Hintergrundamplifikation aus unspezifischen Templat‐Interaktionen, die es zu lösen gilt, um die Entwicklung der isothermen und exponentiellen Amplifikationstechniken weiter voranzutreiben.

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“…Isothermal exponential amplification reaction (EXPAR), as one of the most important nucleic acid amplification strategies, has been successfully applied in the analysis of DNAs (14,15), miRNAs (16–18), polymorphic sites in genomic DNA (19,20) and even proteins (21–23). Standard EXPAR is a circuiting-feedback process that combines polymerase strand extension and single strand nicking on a symmetrical template (24).…”

Section: Introductionmentioning

confidence: 99%

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

Zhou

Lin

et al. 2016

Nucleic Acids Res

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The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5′ terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.

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Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation

Cited by 44 publications

References 25 publications

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

Die exponentielle isotherme Amplifikation von Nukleinsäuren und Assays zur Detektion von Proteinen, Zellen, kleinen Molekülen und Enzymaktivitäten: Anwendungen für EXPAR

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

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