Sensor based on TdT-RCA and application thereof in DNA methyltransferase detection

A methyltransferase and fluorescent sensor technology, applied in the field of biological analysis, can solve problems such as difficulty in solution design, cumbersome operation process, complex sequence, etc., and achieve the effect of reducing experimental cost, improving fluorescent signal, and simple operation

Active Publication Date: 2019-05-14
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a fluorescent sensor for detecting DNA adenine methyltransferase and its preparation and

Method used

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  • Sensor based on TdT-RCA and application thereof in DNA methyltransferase detection
  • Sensor based on TdT-RCA and application thereof in DNA methyltransferase detection
  • Sensor based on TdT-RCA and application thereof in DNA methyltransferase detection

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1 Preparation of a fluorescent sensor for detecting Dam methyltransferase

[0061] 1. Materials and methods

[0062] 1.1 Materials

[0063] The DNA probe sequences purified by HPLC were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Dam methyltransferase and its reaction buffer (Cat. No.: #M0222S, in the purchased reagents, SAM is provided with the enzyme), restriction endonuclease DpnI and its reaction buffer (Cat. No.: #R0176S), AluI methyl transfer Enzyme (Cat. No.: #M0220S) and its reaction buffer, Hhal methyltransferase and its reaction buffer (Cat. No.: #M0217S), terminal transferase (TdT) and its reaction buffer (Cat. No.: M0315S) were purchased from New England Biolabs. Phi29 DNA polymerase and its reaction buffer (Cat. No.: B600060), T4 DNA ligase and its reaction buffer (Cat. No.: B600511), deoxyadenosine triphosphate dATPs (Cat. No.: A620046), dNTPs (Cat. No.: A610056), Thioflavin T (ThT) (Product No.: A606360) were purchased from Shangha...

Embodiment 2

[0078] Example 2 Verifies the feasibility of a fluorescent sensor for detecting Dam methyltransferase

[0079] 1. In this embodiment, the feasibility of detecting the sensor of Dam methyltransferase is firstly set up to verify the control experiment through fluorescence measurement.

[0080] Such as figure 2 As shown, curves a-d refer to adding: (a) 40U / mL Dam methyltransferase, 160μMSAM, 4UDpnI to 1×Dam methyltransferase reaction buffer containing 1μM methyltransferase detection probe; (b) 40 U / mL Dam methyltransferase, 160 μM SAM; (c) 40 U / mL Dam methyltransferase, 4UDpnI; (d) 160 μM SAM, 4UDpnI. The fluorescence signal of the control group without restriction enzyme DpnI or Dam methyltransferase (curves b and d) was very weak, and the low background signal was achieved due to the use of NH 2 The 3' end of the Dam methyltransferase detection probe was modified to effectively prevent non-specific extension caused by terminal transferase (TdT). Moreover, the fluorescence int...

Embodiment 3

[0083] Fluorescent sensor for detecting Dam methyltransferase prepared in Example 3 and research on its experimental conditions

[0084] In order to obtain the best analytical performance of the constructed fluorescent sensor, the corresponding experimental conditions were explored, such as the amount of restriction endonuclease DpnI, terminal transferase (TdT), phi29 DNA polymerase and the concentration of SAM. F-F 0 Value (F is the fluorescence signal when Dam methyltransferase exists, and F 0 is the fluorescent signal in the absence of Dam methyltransferase) was used to evaluate the performance of this fluorescent sensor.

[0085] 1. In order to investigate the influence of DpnI concentration on the prepared fluorescent sensor for detecting Dam methyltransferase, this experiment adopts different DpnI concentrations (the total amount of DpnI added in the system is 1U, 2U, 3U, 4U, 5U, 6U, respectively). ) reaction system, and then detect the fluorescence intensity, the resu...

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Abstract

The invention provides a sensor based on TdT-RCA and application thereof in DNA methyltransferase detection, the sensor comprises a Dam methyltransferase detection probe and a dumbbell-shaped rollingring template. According to the sensor based on TdT-RCA and application thereof in DNA methyltransferase detection, the non-specific amplification of terminal transferase (TdT) activation is effectively prevented by carrying out amination modification on the 3' end of a substrate hairpin probe acting on methyltransferase, so that a background signal is reduced, and the specificity of the detectionmethod is greatly improved; the primer extension catalyzed by TdT is combined with a dumbbell-shaped template-mediated rolling circle amplification (RCA) reaction, thionein T (ThT) is taken as fluorescent dye (thionein T is specifically combined with a reaction product G triple chain as a signal output), the fluorescent label is not needed, the operation is simple, and the experimental cost is reduced; the method can better distinguish the Dam methyltransferase from other methyltransferase and has better selectivity.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a TdT-RCA-based sensor and its application in the detection of DNA methyltransferase. Background technique [0002] DNA methylation is a well-known epigenetic event that plays a crucial role in regulating gene expression, cell differentiation, and genome stability. Typically, the DNA methylation process is carried out by DNA methyltransferases, which catalyze the covalent addition of methyl groups from S-adenosylmethionine (SAM) to target adenine or cytosine residues in the recognition sequence . Abnormal DNA methyltransferase activity may lead to abnormal DNA methylation patterns, which are closely related to various genetic diseases and human malignancies. DNA methyltransferases have emerged as potential biomarkers and therapeutic targets for the diagnosis and treatment of various cancers. Therefore, sensitive and accurate assessment of DNA methyltransferase acti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/682
Inventor 颜玉蓉丁世家程伟晏小玉马洪敏阙海英王通刘萍甘秀锋
Owner CHONGQING MEDICAL UNIVERSITY
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