Fluorescence chemical sensor for detecting DNA adenine transmethylase, and detection method thereof

A fluorescent chemical sensing and methyltransferase technology, applied in the field of biological analysis, can solve problems such as the complexity of experimental operations that increase the difficulty of program design, the limited application of electrode surface modification, and the fast response speed of electrochemical methods, to prevent The effect of non-specific reaction, reduced experimental cost, simple preparation and detection method

Active Publication Date: 2018-03-06
SHANDONG NORMAL UNIV
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Problems solved by technology

However, they generally require tedious nanoparticle preparation, rely on external labeling with fluorophores and quenchers, have long analysis times, involve complex designs, and are expensive
The electrochemical method has fast response and low design cost, but the complex electrode surface modification limits its application
The introduction of nucl

Method used

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  • Fluorescence chemical sensor for detecting DNA adenine transmethylase, and detection method thereof
  • Fluorescence chemical sensor for detecting DNA adenine transmethylase, and detection method thereof
  • Fluorescence chemical sensor for detecting DNA adenine transmethylase, and detection method thereof

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Experimental program
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Embodiment

[0069] Experimental method steps

[0070] 1. Detection of Dam methyltransferase: in the hairpin probe containing 0.5 micromole per liter, 160 micromole per liter SAM (S-adenosylmethionine), 10 units of restriction endonuclease DpnI, 1 × 200 μl reactions of DNADam buffer (50 mmol / L Tris-HCl buffer, 10 mmol / L EDTA, 5 mmol / L 2-mercaptoethanol, pH 7.5) and different amounts of Dam methyltransferase Methylation and cleavage of the hairpin probes are performed in the mixture. The mixture was incubated at 37°C for 2 hours and then inactivated at 80°C for 20 minutes. In 30 μl of 1× terminal transferase buffer (50 mmol / L potassium acetate, 20 mmol / L Tris-Ac, 10 mmol / L magnesium acetate, pH 7.9), 0.25 mmol / L dichloride Cobalt, 1×SYBR Gold, 0.13 micromole per liter of auxiliary probe reaction solution for hyperbranched amplification, 1 millimole per liter of dATPs (deoxyadenosine triphosphate), 4 units of EndoⅣ (endonuclease IV), 8 Unit TDT (terminal transferase) and 4 microliters of ...

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Abstract

The invention discloses a fluorescence chemical sensor for detecting Dam transmethylase. The fluorescence chemical sensor for detecting the Dam transmethylase comprise a Dam transmethylase detection probe and an auxiliary probe; the Dam transmethylase detection probe is a hairpin DNA probe with stem-loop structures; the two stem structures are complemented to form double-chain DNA; the double-chain DNA has an identification site of the Dam transmethylase; the 3' terminal of the hairpin DNA probe is modified by amino; the identification site of the Dam transmethylase is a palindromic sequence of 5'-G-A-T-C-3'; and the auxiliary probe is a T-enriched single-chain DNA sequence with the near 3' terminal with a apurinic pyridine site (AP site). The preparation and detection method of the fluorescence chemical sensor is simple, the detection sensitivity is greatly improved, nonspecific reaction is effectively avoided, and the Dam transmethylase can be detected accurately in a complex biological sample.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a fluorescent chemical sensor based on a hyperbranched amplification zero-background detection of DNA adenine methyltransferase and a detection method thereof. Background technique [0002] DNA methylation is the most prominent form of epigenetic gene regulation and plays a key role in cell proliferation, gene transcription and aging. Covalent addition of methyl groups to targeted adenine / cytosine residues in the recognition sequence, catalyzed by DNA methyltransferase, using S-adenosyl-L-methionine (SAM) as a methyl donor . DNA methylation usually occurs at the C-5 / N-4 position of cytosine and the N-6 position of adenine. Hypermethylation and hypomethylation have been found in various types of cancers such as breast, ovarian and lung cancers. The abnormality of DNA methylation is related to the abnormal expression and activity of DNA methyltransferase. Therefore,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/682C12Q1/48
CPCC12Q1/682C12Q1/6825C12Q2525/301C12Q2537/162C12Q2563/107C12Q2521/125C12Q2521/301C12Q2521/131C12Q2521/331
Inventor 张春阳张艳王新燕
Owner SHANDONG NORMAL UNIV
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