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A new method for label-free detection of t4 polynucleotide kinase/phosphatase and its inhibitors based on fluorescent copper nanoparticles

A technology of polynucleotides and nanoparticles, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of low selectivity and sensitivity, high cost, application limitations, etc., and achieve good selectivity , cost reduction, and high sensitivity

Active Publication Date: 2018-06-12
郑州亮点生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these traditional methods are cumbersome, time-consuming and require special instruments, or have low selectivity and sensitivity, and some require isotope labeling, which limits the scope of their application.
In recent years, a series of molecular beacon detection methods for detecting T4 PNKP have been developed. Although these methods have high sensitivity, the design of molecular beacons is relatively complicated, the cost is high, and its application is also limited to a certain extent.

Method used

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  • A new method for label-free detection of t4 polynucleotide kinase/phosphatase and its inhibitors based on fluorescent copper nanoparticles
  • A new method for label-free detection of t4 polynucleotide kinase/phosphatase and its inhibitors based on fluorescent copper nanoparticles
  • A new method for label-free detection of t4 polynucleotide kinase/phosphatase and its inhibitors based on fluorescent copper nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Step 1. Design the probe sequence as follows: 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CGC ACC TAA AG G GTG CG -3' (phosphorylation modification at the 3' end);

[0041] Step 2. 25 μL Tris-HCl buffer system (10 mM Tris-HCl, 10 mM MgCl, 50 mM NaCl, pH7.9) contains 5 μL probe (1 μM), 1 μL KF polymerase (50 U / mL), 2 μL dNTPs (200 μM) and 2 μL of different concentrations of T4PNKP (0U / mL, 0.25U / mL, 0.5U / mL, 1U / mL, 2U / mL, 5U / mL, 10U / mL, 25U / mL), mixed well, and kept at 37℃ Incubate for 80min;

[0042] Step 3. Supplement the above solution into MOPS buffer solution (10 mM MOPS, 150 mM NaCl, pH 7.6) with a final volume of 100 μL, including 10 μL sodium ascorbate (2 mM) and 10 μL copper sulfate (200 μM), mix well, and incubate at room temperature for 10 min ;

[0043] Step 4, the final reaction solution obtains the fluorescence spectrum figure of 520~660nm under the excitation wavelength of 340nm (see Figure 2a ) and the linear correspondence between the fluore...

Embodiment 2

[0045]Step 1. Design the probe sequence as follows: 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CGC ACC TAA AG G GTG CG -3' (phosphorylation modification at the 3' end);

[0046] Step 2, 25 μL Tris-HCl buffer system (10 mM Tris-HCl, 10 mM MgCl, 50 mM NaCl, pH7.9) contains 5 μL probe (1 μM), 1 μL KF polymerase (50 U / mL), 2 μL dNTPs (200 μM), 5 μL of different concentrations of heparin (0 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL) and 2 μL of T4PNKP (25 U / mL), mixed Evenly, incubate at 37°C for 80 minutes;

[0047] Step 3. Supplement the above solution into MOPS buffer solution (10 mM MOPS, 150 mM NaCl, pH 7.6) with a final volume of 100 μL, including 10 μL sodium ascorbate (2 mM) and 10 μL copper sulfate (200 μM), mix well, and incubate at room temperature for 10 min ;

[0048] Step 4, the corresponding relationship between the relative fluorescence intensity of the system and the concentration of heparin is obtained from the final rea...

Embodiment 3

[0050] Step 1. Design the probe sequence as follows: 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CGC ACC TAA AG G GTG CG -3' (phosphorylation modification at the 3' end);

[0051] Step 2, 25 μL 1% lung cancer cell lysate Tris-HCl buffer system (10mM Tris-HCl, 10mM MgCl, 50mM NaCl, pH 7.9) contains 5 μL probe (1 μM), 1 μL KF polymerase (50 U / mL), 2 μL dNTPs (200μM) and 2μL of different concentrations of T4 PNKP (0U / mL, 0.25U / mL, 0.5U / mL, 1U / mL, 2U / mL, 5U / mL, 10U / mL, 25U / mL), mix well, in Incubate at 37°C for 80 minutes;

[0052] Step 3. Supplement the above solution into MOPS buffer solution (10 mM MOPS, 150 mM NaCl, pH 7.6) with a final volume of 100 μL, including 10 μL sodium ascorbate (2 mM) and 10 μL copper sulfate (200 μM), mix well, and incubate at room temperature for 10 min ;

[0053] Step 4, the final reaction solution obtains the fluorescence spectrum figure of 520~660nm under the excitation wavelength of 340nm (see Figure 4a ) and the corresponding relati...

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Abstract

The invention discloses a new method for detecting T4 polynucleotide kinase / phosphatase and its inhibitors based on fluorescent copper nanoparticles without labeling. Hairpin probe, the 5' end is a sequence rich in T bases; 2. Dilute the above probe with Tris-HCl buffer solution to a concentration of 100-1000nM, and add a certain amount of T4 PNKP, Klenow Fragment, and dNTPs to it Mix and react at 37°C for 40-150 minutes; 3. Dilute the above reaction solution into MOPS buffer system, add copper sulfate and sodium ascorbate, and react at room temperature for 1-30 minutes; 4. Use a fluorescence instrument to detect the fluorescence intensity of the final reaction solution. The corresponding relationship between the fluorescence intensity of the system and the concentration of T4 PNKP is obtained. The method of the present invention is simple in operation, low in cost, high in sensitivity and good in selectivity, and can quantitatively detect and screen T4 PNKP and its inhibitors. Establishment and molecular biology diagnostics offer potential applications.

Description

technical field [0001] The invention belongs to the technical field of bioanalysis, and in particular relates to a new method for detecting T4 polynucleotide kinase / phosphatase and its inhibitor based on fluorescent copper nanoparticles without labeling. Background technique [0002] DNA 3′ phosphatases play crucial roles in various activities in cells, such as nucleic acid metabolism, DNA damage repair, DNA replication and DNA recombination. T4 polynucleotide kinase (T4 Polynucleotide Kinase, T4 PNKP) is one of the typical representatives. In 1965, Richards et al. first discovered T4PNKP when studying the process of T4 phages invading Escherichia coli. T4 PNKP has nucleic acid 5'-terminal kinase activity and 3'-terminal phosphatase activity, which can catalyze the 5'-terminal phosphorylation and 3'-terminal dephosphorylation reaction of nucleic acid in biological systems, and is used in the process of nucleic acid damage repair and nucleic acid replication It plays an imp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48C12Q1/44C12Q1/68
Inventor 张琳李朝辉葛佳董真真
Owner 郑州亮点生物技术有限公司
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