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Fluorescence biosensor for detecting UDG (Uracil-DNA Glycosylase) and preparation method thereof

A biosensor and glycosylase technology, applied in the field of biosensors, can solve the problems of long detection period, low specificity and sensitivity, etc., and achieve the effect of fast detection speed, rapid detection and high specificity

Active Publication Date: 2019-03-08
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of relatively low specificity and sensitivity and long detection period of the method for detecting UDG in the prior art, the present invention provides a method based on polymerase-assisted feedback rolling circle amplification with high specificity and sensitivity and fast detection speed. A biosensor for detecting DNA glycosylase UDG with endonuclease amplification fluorescence method, and also provides its preparation method

Method used

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  • Fluorescence biosensor for detecting UDG (Uracil-DNA Glycosylase) and preparation method thereof
  • Fluorescence biosensor for detecting UDG (Uracil-DNA Glycosylase) and preparation method thereof
  • Fluorescence biosensor for detecting UDG (Uracil-DNA Glycosylase) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Preparation of Circular Template and Composite Probe

[0065] Prepared with 50 mM Tris-HCl, 10 mM MgCl 2 , T4 DNA Ligase Reaction Buffer with 10 mM DTT and 1 mM ATP. Formulated with 10 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 , 140 mM NaCl, 1 mM KCl, 1 mM MgCl 2 , 1mM CaCl 2 , PBS buffer solution of pH=7.4.

[0066] (1) Mix 42 μL sterile water, 6 μL linear template (10 μM), 6 μL ligation probe (10 μM) and 6 μL 10× T4 DNA ligase buffer, denature at 95°C for 5 min, then Slowly cool down to room temperature to complete the hybridization, then add 3 μL of T4 DNA ligase (60 U / μL) to the reaction system, and react at 16°C for 20 hours; after that, the reaction system is placed in a water bath at 65°C for 15 minutes , inactivate the T4 DNA ligase in the system.

[0067] (2) Add 3 μL of exonuclease Ⅰ (20 U / μL) and 3 μL of exonuclease Ⅲ (100 U / μL) to the above reaction system and react at 37°C for 2 h; then put the reaction system at 85°C Heated in a water bath for 1...

Embodiment 2

[0069] Embodiment 2 Fluorescence intensity changes with the concentration of composite probe I

[0070] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:

[0071] (1) Mix 2 μL compound probe I (concentrations are 50 nM, 100 nM, 500 nM, 1 μM, 5 μM), 2 μL dNTP (1 mM), 2 μL phi29 DNA polymerase (1 U / μL), 2 μL endonuclease IV (1 U / μL) in 2 μL buffer (50 mMTris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5), add 2 μLUDG enzyme solution (1 U / mL) respectively, mix well and react at 37°C for 60 min;

[0072] (2) Add 2 μL of Composite Probe II (1 μM) to the solution in step (1), mix well and react at a constant temperature of 37°C for 60 min;

[0073] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 486 nm, the emission wavelength is 518 nm, and the detection range is 450 nm-530 nm, and the fluorescence signal changes a...

Embodiment 3

[0077] Embodiment 3 Fluorescence intensity changes with the concentration of compound probe II

[0078] A method for preparing a fluorescent biosensor of the present invention, comprising the following steps:

[0079] (1) Mix 2 μL compound probe I (1 μM), 2 μL dNTP (1 mM), 2 μL phi29 DNA polymerase (1 U / μL), 2 μL endonuclease IV (1 U / μL) in 2 μL buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5), add 2 μL UDG enzyme solution (1 U / mL) respectively, mix well and react at a constant temperature of 37°C for 60 min;

[0080] (2) Add 2 μL of compound probe II (concentrations are 50 nM, 100 nM, 500 nM, 1 μM, 5 μM) to the solution in step (1), mix well and react at 37°C for 60 min;

[0081] (3) Dilute the solution obtained in step (2) with water to 100 μL, and then perform fluorescence detection; the excitation wavelength is set to 486 nm, the emission wavelength is 518 nm, and the detection range is 450 nm-530 nm, and the fluorescence signal changes ...

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Abstract

The invention relates to the technical field of a biosensor, and particularly relates to a fluorescence biosensor for detecting UDG (Uracil-DNA Glycosylase) on the basis of a polymerase-assisted feedback rolling circle amplification and endonuclease amplification fluorescence method. The invention aims to solve problems of both low specificity and low sensitivity of a method for detecting UDG in the prior art. The biosensor for detecting the UDG on the basis of a feedback rolling circle amplification technology achieves a rolling circle amplification effect on matching of phi29 polymerase andendonuclease IV, implements fluorescence resonance energy transfer of a fluorophore and a Quenching group and performs a homogeneous reaction on mixed liquid. A preparation method comprises: constructing a circular template and a composite probe; feeding back a rolling circle amplification signal and carrying out fluorescence detection. Specific hydrolysis of the UDG on a basic group U is utilized, and by utilizing such specific reaction, the UDG can be accurately measured and meanwhile, interference can also be avoided; by utilizing endonuclease IV circle amplification, a signal amplificationeffect is achieved.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor based on feedback rolling circle amplification and endonuclease signal amplification, and a preparation method thereof. Background technique [0002] DNA glycosylase UDG is an important base excision repair enzyme responsible for repairing DNA damage caused by uracil and maintaining the integrity of the genome, and the abnormal expression of UDG is also associated with various cancers. Hydrolysis of cytosine to uracil is the most common form of DNA hydrolytic damage, resulting in the conversion of G:C base pairs to A:U base pairs during DNA replication. As the initiator and "patrolman" of the base excision repair pathway, UDG has high specificity for uracil, so it can be used to recognize and catalyze the hydrolysis and splitting of N-glycosidic bonds on single-stranded or double-stranded DNA. Subsequently, the damaged base is released and an apurinic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 黄加栋王敬锋刘素王玉宋晓蕾张雪王海旺赵一菡瞿晓南张儒峰
Owner UNIV OF JINAN
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