A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, its detection method and application

A chemiluminescence detection and hydroxymethylcytosine technology, which is applied in the direction of chemiluminescence/bioluminescence, and analysis by making materials undergo chemical reactions, can solve the problems of non-specific amplification and false positives, and achieve high reproducibility and Accuracy, simple operation, and the effect of improving detection sensitivity

Active Publication Date: 2022-07-19
SHANDONG NORMAL UNIV
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Problems solved by technology

However, they inevitably suffer from non-specific amplification and even false positives, and require careful design of primers, labeled nucleic acid probes, and specific templates
Furthermore, these methods only allow detection of 5hmC at specific positions and specific fragments

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  • A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, its detection method and application
  • A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, its detection method and application
  • A chemiluminescent biosensor for detecting 5-hydroxymethylcytosine, its detection method and application

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Embodiment

[0049] Preparation of biotinylated 5hmC-DNA products: The preparation of biotinylated 5hmC-DNA products involved three sequential steps, including glucosylation, iodic acid oxidation, and biotinylation. Perform the glucosylation reaction in 30 μl of reaction solution containing double-stranded DNA (dsDNAs), NEBuffer 4 (50 mM potassium acetate, 20 mM Tris-acetic acid, 10 mM magnesium acetate, 1 mM DTT, pH 7.9), 2 mM UDP-Glc and 4 U T4β-GT were reacted in a 37 °C water bath for 2 h, followed by a QIAquick nucleotide excision purification column to remove unreacted nucleotides and ions. The purified DNA was subjected to periodate oxidation in a 50 μl reaction system containing 20 mmol of sodium periodate and 100 mmol of sodium phosphate (pH 7.0) at 22°C for 16 h, and then reacted with 40 mmol of sodium periodate. Molar sodium sulfite was reacted at room temperature for 10 minutes, and then salt ions were removed using a QIAquick nucleotide excision cleanup column. The biotinylat...

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Abstract

The invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, a detection method and application thereof, and belongs to the technical field of molecular detection. The chemiluminescent biosensor of 5-hydroxymethylcytosine includes T4 bacteriophage β-glucosyltransferase, uridine diphosphate glucose, sodium periodate, aldehyde group reaction probe ARP, terminal transferase, hemoglobin and luminol solution. The hydroxymethylcytosine-glycosylation, periodate oxidation, biotinylation-isothermal signal amplification strategy designed in the present invention does not need to change the reaction temperature, nor does it require a special labeled nucleic acid probe or a specific template for signal amplification , which can detect 5hmC of any sequence in genomic DNA and does not involve isotopic labels or specific antibodies, eliminating radioactive hazards and misinterpretation of genome-wide mapping data in antibody-based experiments. This enables genome-wide analysis of 5hmC in a limited number of biological and clinical samples.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, in particular to a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, a detection method and application thereof. Background technique [0002] The disclosure of information in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. [0003] Epigenetic modifications are heritable changes in gene expression that occur without altering the DNA sequence. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two major epigenetic modifications found in mammalian genomes. 5hmC is an oxidative intermediate in the oxidation of 5mC mediated by 10-11 translocase (TET). 5hmC is involved in many key cellular functions, including gene regulat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 张春阳胡娟申大忠李琛琛董月红
Owner SHANDONG NORMAL UNIV
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