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Novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and inhibitor of T4 PNKP/phosphatase on basis of fluorescent copper nanoparticles

A polynucleotide and nanoparticle technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of application limitation, low selectivity and sensitivity, and high cost, and achieve high sensitivity and selectivity. The effect of good performance and cost reduction

Active Publication Date: 2015-09-09
郑州亮点生物技术有限公司
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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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  • Novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and inhibitor of T4 PNKP/phosphatase on basis of fluorescent copper nanoparticles
  • Novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and inhibitor of T4 PNKP/phosphatase on basis of fluorescent copper nanoparticles
  • Novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and inhibitor of T4 PNKP/phosphatase on basis of 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, pH 7.9) contains 5 μL probe (1 μM), 1 μL KF polymerase (50 U / mL), 2 μL dNTPs (200 μM) and 2 μL Different concentrations of T4 PNKP (0U / mL, 0.25U / mL, 0.5U / mL, 1U / mL, 2U / mL, 5U / mL, 10U / mL, 25U / mL) were mixed evenly and kept at a constant temperature of 37°C Incubate for 80min;

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

[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 corresponde...

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, pH 7.9) contains 5 μL probe (1 μM), 1 μL KF polymerase (50 U / mL), 2 μL dNTPs (200 μM), 5 μL Different concentrations of heparin (0μg / mL, 0.2μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, 25μg / mL) and 2μL T4 PNKP (25U / mL), mixed Evenly, incubate at 37°C for 80 minutes;

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

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

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 with a final volume of 100 μL MOPS buffer solution (10 mM MOPS, 150 mM NaCl, pH 7.6), which includes 10 μL sodium ascorbate (2 mM) and 10 μL copper sulfate (200 μM), mix well and incubate at room temperature 10min;

[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 relationship ...

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Abstract

The invention discloses a novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase) / phosphatase and an inhibitor of T4 PNKP / phosphatase on the basis of fluorescent copper nanoparticles. The method is implemented according to steps as follows: 1), a hairpin probe modified by 3'-terminal phosphorylation is designed, and 5' terminal is a sequence containing a base T; 2), the probe is diluted to the concentration being 100-1,000 nM with a Tris-HCl buffer solution, certain quantities of T4 PNKP, Klenow Fragment and dNTPs are mixed with the probe, and the mixture reacts at the temperature of 37 DEG C for 40-150 minutes; 3), the reaction liquid is diluted to form an MOPS buffer system, and copper sulfate and sodium ascorbate are added and react for 1-30 minutes at the room temperature; 4), the fluorescence strength of the finally obtained reaction liquid is detected with a fluorescence instrument, and a corresponding relation between the fluorescence strength of the system and T4 PNKP concentration is obtained. According to the method, the operation is simple, the cost is low, the sensitivity is high, the selectivity is good, T4 PNKP and the inhibitor can be quantitatively detected and screened, and a potential application value is provided for establishment of other terminal modification enzyme detection methods and molecular biological diagnosis.

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. T4 PNKP was first discovered in 1965 when Richards et al. studied 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(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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