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DNA probe, reagent kit and method for detecting deoxyribonuclease

A deoxyribonucleic acid and DNA probe technology, applied in the field of enzyme detection, can solve the problems of large influence and difficult detection technology, and achieve the effects of high sensitivity, small reaction volume and low cost

Pending Publication Date: 2016-06-08
广州锐博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can achieve a lower detection limit, its results are greatly affected by electrode modification and operating environment, so it is difficult to be used as a conventional detection technology.

Method used

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  • DNA probe, reagent kit and method for detecting deoxyribonuclease
  • DNA probe, reagent kit and method for detecting deoxyribonuclease
  • DNA probe, reagent kit and method for detecting deoxyribonuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the preparation of probe

[0043] 1 sequence design

[0044] The design principles are as follows, see figure 1 .

[0045] 1) Palindromic complementary sequences with 5-15 bp at both ends (underlined part)

[0046] 2) In the middle is a 5-20nt DNA sequence (loop region)

[0047] 3) The probe can form a hairpin structure after annealing, and the Tm value of the two palindromic complementary parts is greater than 35°C; its stem structure includes at least one T, and its loop structure includes at least one A, one C, one T and 1G

[0048] 4) There are fluorescent groups and their corresponding quenching groups at both ends. The fluorescent group and its quenching group can be modified at both ends of the sequence or any DNA base in the sequence.

[0049] The sequences of SEQID.NO.1-8 (SEQ1-SEQ8 in the drawings) are as follows.

[0050]

[0051]

[0052] 2 Probe Synthesis

[0053] Entrust Shanghai Jierui Biotechnology Co., Ltd. to synthesize the ...

Embodiment 2

[0055] 1. Reagent synthesis and preparation

[0056] 1.1. DNasetestProbe

[0057] The probes are the 8 probes synthesized in Example 1.

[0058] 1.2. Preparation of DNaseTestProbe solution

[0059] Stock solution preparation (100pmol / ul): add sterilized Mini-Q water to each tube of DNaseTestprobe dry powder to a final concentration of 100pmol / ul, store at -20°C in the dark

[0060] Preparation of working solution (10pmol / ul): take 1.5ml tube without DNase, add 90ulddH 2 O and 10ul DNaseProbe stock solution, mix well, and store at -20°C in the dark.

[0061] 1.3. Reaction buffer

[0062] Formula: 500mMNaCl, 200mMKCl, 500mM Tris-HCl, 100mMMgCl 2 ,pH8.0

[0063] Prepare 10× reaction buffer solution with ultrapure water according to the above formula, sterilize under high temperature and high pressure, and store at -20°C.

[0064] 1.4. DNaseI positive samples

[0065] DNaseI (TAKARA), concentration 5U / ul. Dilute to 0.01U / ul with 1× reaction buffer.

[0066] 2. Methods an...

Embodiment 3

[0075] Example 3 Probe Specific Detection

[0076] 1. Reagent synthesis and preparation

[0077] 1.1. DNasetestProbe

[0078] The probes are the 8 probes synthesized in Example 1.

[0079] 1.2. Preparation of DNaseTestProbe solution

[0080] Preparation method is the same as embodiment two

[0081] 1.3. Reaction buffer

[0082] Preparation method is the same as embodiment two

[0083] 1.4. Samples

[0084] DNaseI (TAKARA), concentration 5U / ul. Dilute to 0.01 U / ul RNaseA (TianGen; #RT405-02; 10 mg / ml) with 1× reaction buffer and dilute to 1 ng / ul with 1× reaction buffer.

[0085] 2. Methods and steps

[0086] 2.1. Preparation of reaction system

[0087]

[0088]Mix the above components except the sample evenly, heat at 95°C for 3min, then 65°C to 25°C, drop 5°C every 30 seconds to anneal the probe. Take a white 8-section PCR tube, place it on a 96-well plate, add 9ul of reaction solution to each well, and then add 1ul of DNaseI (0.01U / ul). DNase-free ddH 2 O repl...

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Abstract

The invention provides a DNA probe, a reagent kit and a method for detecting deoxyribonuclease. The hairpin-shaped DNA probe serves as a substrate of deoxyribonuclease, and a fluorescence group and a corresponding fluorescence quenching group are marked at the two ends or the interior of the probe respectively. When the structure of the probe is complete, the fluorescence group and the fluorescence quenching group are located on the same DNA molecule, excited fluorescence of the fluorescence group is absorbed by the fluorescence quenching group, and the fluorescence group does not emit fluorescence; when the probe is degraded by deoxyribonuclease, the fluorescence group and the fluorescence quenching group are separated, and the fluorescence group can emit fluorescence normally. Whether deoxyribonuclease exists in a reaction system or not can be judged as long as the intensity of fluorescence emitted from the reaction system is detected with a real-time quantitative PCR instrument. The method can be widely applied to quality detection on residues of deoxyribonuclease in biological products or quantitative detection of deoxyribonuclease.

Description

technical field [0001] The invention relates to the field of enzyme detection, in particular to a method for detecting deoxyribonuclease, a DNA probe and a kit for detecting deoxyribonuclease. Background technique [0002] Deoxyribonuclease (DNase) is ubiquitous in cells and participates in various physiological and biochemical processes, such as DNA replication and repair, apoptosis, etc. It can also be used as a marker of diseases, such as in patients with arthritis and nephritis, the expression level of DNase I is low ,39,448–452.]; DNase enzymes can also be used in the treatment of diseases, such as DNaseI can be used in the treatment of cystic fibroma. [0003] Traditional DNase detection methods include high performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA). However, these methods generally have shortcomings such as low sensitivity, time-consuming detection, and high requirements for equipment. In order to solve the above problems...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/34C12N15/11
CPCC12Q1/6876C12Q1/6851
Inventor 张必良刘霭珊曹亮克雷格·梅洛
Owner 广州锐博生物技术有限公司
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