A column method for determination of DNA cytosine-C5-methyltransferase activity

Kim, Bo Yeon; Kwon, Oh Sung; Joo, Sung Ah; Park, Jung A; Heo, Keon Young; Kim, Min Soo; Ahn, Jong Seog

doi:10.1016/j.ab.2003.11.009

Cited by 42 publications

(21 citation statements)

References 6 publications

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“…One is quantification of the incorporation of labeled methyl groups in intact DNA by incubation with w 3 Hx-methyl-S-adenosylmethionine (7)(8)(9)(10). The disadvantage of this approach is that the amount of DNA must be exactly quantified, as the result is expressed per mg DNA.…”

Section: Introductionmentioning

confidence: 99%

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects

Kok¹,

Smith²,

Barto³

et al. 2007

Clinical Chemical Laboratory Medicine

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Section: Introductionmentioning

confidence: 99%

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects

Kok¹,

Smith²,

Barto³

et al. 2007

Clinical Chemical Laboratory Medicine

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“…Therefore, a universal method capable of sensitively detecting both bacteria and human methyltransferases is highly desirable. The conventional methods include radioactive labeling method, Western blotting, and high performance liquid chromatography (HPLC) . However, they involve radioisotope-labeled substrates, expensive antibodies, and time-consuming and laborious procedures .…”

mentioning

confidence: 99%

Construction of a Universal and Label-Free Chemiluminescent Sensor for Accurate Quantification of Both Bacteria and Human Methyltransferases

Wang

Cui

et al. 2020

Anal. Chem.

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DNA methylation plays important roles in various biological processes, and the alteration of DNA methyltransferase activity can induce the aberrant DNA methylation patterns. Despite the progress in methyltransferase activity assays, few methods enable the detection of both bacteria and human methyltransferases. Herein, we construct a universal and label-free chemiluminescent sensor for accurate quantification of both bacteria methyltransferases (e.g., M. SssI methyltransferase (M.SssI MTase)) and human methyltransferases (e.g., DNA (cytosine-5)-methyltransferase 1, (Dnmt1)) by integrating a dumbbell probe with BssHII endonuclease-mediated rolling circle amplification (RCA). We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition, BssHII endonuclease-cleavage, RCA amplification and signal transduction in one probe for the detection of both M.SssI MTase and Dnmt1. Moreover, the introduction of two BssHII endonuclease recognition sites in a dumbbell probe can greatly reduce the false positivity resulting from the incomplete cleavage of dumbbell probe by BssHII, because once one of two recognition sites is identified by BssHII, the dumbbell probe can be completely digested by Exonuclease III (Exo III) and Exonuclease I (Exo I) to prevent the nonspecific RCA. This chemiluminescent sensor can accurately quantify M.SssI MTase in both 10% serum and various cell lysis buffers, and even sensitively detect Dnmt1 activity in MCF-7 cells. Furthermore, this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.SssI MTase, with promising applications in disease diagnosis and drug discovery.

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“…The traditional standard approach of detecting DNA MTase activity is a radioactive assay based on [methyl- 3 H]-labeled SAM . To avoid radioactive hazards, many efforts have been devoted to develop new detection methods (e.g., fluorometry, − surface-enhance Raman scattering (SERS) assays, colorimetry, − electrochemical assays , ) for sensitive and specific discrimination of MTase activity.…”

mentioning

confidence: 99%

Dual-Amplification Strategy-Based SERS Chip for Sensitive and Reproducible Detection of DNA Methyltransferase Activity in Human Serum

et al. 2019

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Herein, we present a dual-amplification sensing strategy-based surface-enhanced Raman scattering (SERS) chip, which combines rolling circle amplification (RCA) and polyadenine (PolyA) assembly for sensitive and reproducible determination of the activity of M.SssI, a cytosine-guanine dinucleotide (CpG) methyltransferase (MTase). Typically, in the presence of M.SssI, RCA process is triggered, resulting in long, single-stranded DNA (ssDNA) fragments that are hybridized with thousands of Raman reporters of Cy3. Afterward, the resultant ssDNA fragments are conjugated to SERS-active substrates made of silver core-gold satellite nanocomposites-modified silicon wafer (Ag–Au NPs@Si), with the SERS enhancement factor of ∼5 × 106. The core–satellite nanostructures are assembled relied on the strong affinity of PolyA toward gold/silver surface. Of particular significance, the developed SERS chip displays an ultrahigh sensitivity with a low limit of detection (LOD) of 2.8 × 10–3 U/mL, which is around 2 orders of magnitude higher than most reported methods. In addition, the constructed chip features a broad detection range covering from 0.05 to 50 U/mL. Besides for the ultrahigh sensitivity and broad dynamic range, the chip also features good reproducibility (e.g., the relative standard deviation (RSD) is less than ∼12%). Taking advantages of these merits, the developed chip is feasible for accurate discrimination of M.SssI with various concentrations spiked in human serum samples with good recoveries ranging from 99.6% to 107%.

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A column method for determination of DNA cytosine-C5-methyltransferase activity

Cited by 42 publications

References 6 publications

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects

Construction of a Universal and Label-Free Chemiluminescent Sensor for Accurate Quantification of Both Bacteria and Human Methyltransferases

Dual-Amplification Strategy-Based SERS Chip for Sensitive and Reproducible Detection of DNA Methyltransferase Activity in Human Serum

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