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A kit for detecting polynucleotide kinase and its detection method

A polynucleotide kinase and kit technology, applied in the field of biological analysis, can solve the problems of incomplete fluorescence quenching effect, insufficient support for practical applications, lack of signal amplification strategy, etc., to achieve rapid and efficient detection, reduce experimental costs, and detect low limit effect

Active Publication Date: 2020-11-20
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These fluorescence detection methods have shown good performance in the quantitative determination of PNK activity, but most of these methods require expensive fluorescent labels; at the same time, false positive interference may occur due to incomplete fluorescence quenching effects
In addition, due to the lack of effective signal amplification strategies, the sensitivity of these methods is generally low, and the sensitivity of most methods is not enough to support practical applications.

Method used

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  • A kit for detecting polynucleotide kinase and its detection method
  • A kit for detecting polynucleotide kinase and its detection method
  • A kit for detecting polynucleotide kinase and its detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 A kind of test kit for detecting polynucleotide kinase

[0044] The kit for detecting polynucleotide kinase comprises the following components: probe A, probe S, mixed solution A, template DNA, lambda exonuclease, polynucleotide kinase standard product, 1×NEBuffer and 10 mM adenosine triphosphate.

[0045] Said mixed liquid A comprises: 0.05 milligram per milliliter of bovine serum albumin, 0.1 unit per microliter of Nt.BbvCI nicking enzyme, 0.05 unit per microliter of KF polymerase, 200 micromoles per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.

[0046] The nucleotide sequence of the probe A is 5'-GCA CAA AGG ACT GAG GCT GAG GGT TGC GCA TCTGCT ATG AAA CAA GCC AGA TGC TCA AC-3', as shown in SEQ ID NO.1, its 5' end Phosphorylation modification; the nucleotide sequence of probe S is 5'-GTT GAG CAT CTG GCT TGT TTC ATA GCA GAT GTT TTT T-3', as shown in SEQ ID NO.2, and its 5' end is phosphorylated grooming.

[0047] The template D...

Embodiment 2

[0049] Embodiment 2 A kind of test kit for detecting polynucleotide kinase

[0050] The kit for detecting polynucleotide kinase comprises the following components: probe A, probe S, mixed solution A, template DNA, lambda exonuclease, polynucleotide kinase standard product, 1×NEBuffer and 10 mM adenosine triphosphate.

[0051] Said mixed liquid A comprises: 0.15 milligrams per milliliter of bovine serum albumin, 0.3 units per microliter of Nt.BbvCI nicking enzyme, 0.15 units per microliter of KF polymerase, 300 micromoles per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.

[0052] Said mixed liquid A comprises: 0.1 milligram per milliliter of bovine serum albumin, 0.2 unit per microliter of Nt.BbvCI nicking enzyme, 0.1 unit per microliter of KF polymerase, 250 micromole per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.

[0053] The nucleotide sequences of the probe A, probe S and template DNA are similar to Example 1.

[005...

Embodiment 3

[0055] Embodiment 3 A kind of test kit for detecting polynucleotide kinase

[0056] The kit for detecting polynucleotide kinase comprises the following components: probe A, probe S, mixed solution A, template DNA, lambda exonuclease, polynucleotide kinase standard product, 1×NEBuffer and 10 mM adenosine triphosphate.

[0057] Said mixed liquid A comprises: 0.1 milligram per milliliter of bovine serum albumin, 0.2 unit per microliter of Nt.BbvCI nicking enzyme, 0.1 unit per microliter of KF polymerase, 250 micromole per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.

[0058] The nucleotide sequences of the probe A, probe S and template DNA are similar to Example 1.

[0059] The composition and concentration contained in the 1×NEBuffer are similar to those in Example 1.

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Abstract

The invention belongs to the technical field of bioanalysis and particularly relates to a kit for detecting polynucleotide kinase (PNK) and a detection method for the kit. The kit disclosed by the invention comprises a probe A, a probe S, a mixed solution A, a template DNA (Deoxyribonucleic Acid), lambda exonucleautomotive service engineers, a polynucleotide kinase standard substance and 1xNEBuffer and 10mM triphosadenine. The detection method for the kit disclosed by the invention has the advantages of simple operation, reduction of detection cost, high sensitivity and high specificity, and can be applied to detection of complex samples. In addition, the kit disclosed by the invention is also applied to PNK activity inhibitory detection.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a kit for detecting polynucleotide kinase and a detection method thereof. Background technique [0002] Polynucleotide kinase (PNK) is a DNA end-processing enzyme that catalyzes the transfer of a phosphate group from the γ-position of ATP to the 5'-hydroxyl terminus of DNA to generate a 5'-phosphate terminus. The phosphorylation reaction catalyzed by PNK plays an important role in many cellular processes such as DNA recombination, DNA replication and DNA damage repair; abnormal expression and activity of PNK are closely related to a variety of human diseases; currently, PNK is considered to be a A potential target for cancer therapy. [0003] In the prior art, the fluorescence detection method has become the most commonly used method for PNK activity detection due to its convenience and sensitivity. At present, a variety of fluorescence detection methods...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48C12Q1/6844
CPCC12Q1/485C12Q1/6844G01N2333/9126C12Q2531/119C12Q2563/107
Inventor 张春阳马飞刘萌
Owner SHANDONG NORMAL UNIV
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