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Cascade enzyme reactor for enzymatically digesting DNA into mononucleoside

An enzyme reactor and DNase technology, applied in the field of DNase digestion, can solve the problems of complex enzymatic hydrolysis system, and achieve the effect of good stability and low back pressure

Inactive Publication Date: 2017-02-15
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(Porebski, P.W.A.; Gyssels, E.; Madder, A.; J.Chromatogr.A 2015,1422,18-26.) The enzymatic digestion process of genomic DNA involves deoxyribonuclease, phosphodiesterase and alkaline A variety of DNA hydrolases, including phosphatase, have complex enzymatic hydrolysis systems. At present, there is no DNA enzyme reactor that can enzymatically hydrolyze genomic DNA into single nucleosides

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  • Cascade enzyme reactor for enzymatically digesting DNA into mononucleoside
  • Cascade enzyme reactor for enzymatically digesting DNA into mononucleoside
  • Cascade enzyme reactor for enzymatically digesting DNA into mononucleoside

Examples

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Effect test

Embodiment 1

[0026] Prepare deoxyribonuclease, phosphodiesterase and alkaline phosphatase immobilized DNA single enzyme reactor respectively, and then these three enzyme reactors are in the order of deoxyribonuclease-phosphodiesterase-alkaline phosphatase Cascaded DNase reactors are obtained in series, such as figure 1 . Among them, deoxyribonuclease in the presence of Mg 2+ When present, it can digest single-stranded or double-stranded DNA to produce single-deoxynucleotides or oligodeoxynucleotides; phosphodiesterase can release 5'-mononucleotides from the 3'-terminus Nucleotides; and alkaline phosphatase can dephosphorylate single nucleotides to obtain single nucleoside products. The preparation method of single-enzyme enzyme reactor is as figure 2 ,Specific steps are as follows:

[0027] 1. Preparation of capillary silica gel monolithic column: several fused silica capillary tubes were cut, washed with water and methanol for 30 minutes each, then rinsed and activated with 1M NaOH s...

Embodiment example 2

[0033] Cascade DNase Reactor Enzymatically digests calf thymus DNA for detection of deoxyguanosine (dG): inject 12 μL of 200 ng / μL calf thymus DNA into the cascade DNase reactor through a low-pressure micro-injection pump, and then use 10 mM Tris- HCl (pH=7.6, 2mM Mg 2+ , 2mM Ca 2+ ) to flush out the enzymatic hydrolysis solution, and digest at room temperature (25° C.) for 15, 30, and 45 minutes respectively until 60 μL of enzymatic hydrolysis samples are collected. LC-MS was used to quantitatively detect dG in the digestion product, and the chromatographic peaks under different digestion times were detected as image 3 shown.

[0034] Main parameter settings of the LC-MS used:

[0035] (1) Chromatographic column: Zorbax Eclipse Plus C18column (2.1×100mm, 1.8μm, Aglient)

[0036] (2) Mobile phase ratio: isocratic separation, 5.0% methanol and 95.0% water (0.1% formic acid)

[0037] (3) Flow rate: 0.3mL / min

[0038] (4) Column temperature: 30°C

[0039] (5) Injection vo...

Embodiment example 3

[0043] Cascade DNase reactor enzymatically digests calf thymus DNA for detection of dC: inject 12 μL of 200 ng / μL calf thymus DNA into the cascade DNase reactor through a low-pressure microsyringe pump, and then use 10 mM Tris-HCl (pH=7.6 , 2mM Mg 2+ , 2mM Ca 2+ ) to flush out the enzymatic hydrolysis solution, and digest at room temperature (25° C.) for 15, 30, and 45 minutes respectively until 60 μL of enzymatic hydrolysis samples are collected. LC-MS was used to quantitatively detect dC in the digestion product, and the chromatographic peaks under different digestion times were detected as Figure 4 shown.

[0044] The main parameter setting of the LC-MS used is the same as that of the implementation case 2, and the quantitative characteristic ion pair is set as: dC m / z 228.1→112.1 (5eV).

[0045] The experimental results show that with the increase of enzymatic hydrolysis time, from 15 minutes to 45 minutes, the content of dC obtained by enzymatic cascade DNase reaction...

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Abstract

The invention relates to a cascade enzyme reactor for enzymatically digesting DNA into mononucleoside. The cascade enzyme reactor is characterized by being formed by serially connecting more than two DNA single enzyme reactors, and genome DNA, by virtue of the reactor, can be enzymatically hydrolyzed into the mononucleoside. The cascade DNA enzyme reactor is rich in porous structure and excellent in mechanical performance, and a DNA sample, which is injected into the enzyme reactor by virtue of a low-voltage micro-injection pump, a hand-push pump and the like, is enzymatically hydrolyzed. The cascade DNA enzyme reactor, which enzymatically hydrolyzes the genome DNA into the mononucleoside, is quite high in enzyme digestion efficiency which can reach 99.0% or above; and the cascade DNA enzyme reactor, which is high in enzyme digestion speed, can shorten an enzyme digestion time to be less than 1h. The cascade DNA enzyme reactor, which is excellent in stability, can be repeatedly used for more than 30 times and can be preserved for more than 2 months. Without the assistance of a thermal denaturating or ultra-filtration enzyme, the sample, which is enzymatically hydrolyzed by virtue of the cascade enzyme reactor, can directly enter a mass spectrum for the qualitative or quantitative analysis of a target nucleoside product; and the sample is applicable to DNA adduct analysis under the exposure of environmental pollutants.

Description

technical field [0001] The invention relates to the technical field of DNA enzymatic cutting, and relates to a cascade enzyme reactor for enzymatically cutting DNA into single nucleosides. It is composed of more than two DNA single-enzyme reactors in series, through which genomic DNA can be enzymatically hydrolyzed into single nucleosides, and mass spectrometry can be used for quantitative and qualitative analysis of common nucleosides and DNA adducts in DNA samples. Background technique [0002] Environmental pollution is closely related to the occurrence of human diseases. The exposure of pollutants to the human body can cause DNA damage and DNA modification. If it cannot be repaired in time, it may cause gene mutation and eventually lead to the occurrence of cancer. In order to evaluate the risk of disease occurrence, it is particularly important to identify DNA damage products and DNA adducts. Liquid chromatography-mass spectrometry (LC-MS) is a common method for detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/40
CPCC12M21/18C12M23/58
Inventor 汪海林尹俊发徐田
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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