CN1493582A - Cleaning convenient, high efficiency degradating method of nucleic acid - Google Patents
Cleaning convenient, high efficiency degradating method of nucleic acid Download PDFInfo
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- CN1493582A CN1493582A CNA031590616A CN03159061A CN1493582A CN 1493582 A CN1493582 A CN 1493582A CN A031590616 A CNA031590616 A CN A031590616A CN 03159061 A CN03159061 A CN 03159061A CN 1493582 A CN1493582 A CN 1493582A
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004140 cleaning Methods 0.000 title 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A process for cleanly, simple and effectively degradating the nucleic acid in the liquid of broken animal, plant or microbe cells, analyzing the DNA sequence and extracting protein and enzyme from cells features that under the existance of hydrogen peroxide and metallic ions, it is irradiated by ultraviolet ray. Its advantages are no damage to protein, not adding new impurities, and high speed and sensitivity.
Description
Technical field the present invention relates to utilize superoxide in the presence of ultraviolet lighting or metal ion, generates hydroxyl radical free radical, macromolecular nucleic acid is degraded into non-specificly the method for small segment.
The background technology nucleic acid molecule is degraded non-specificly or resolved into suitable fragment all is very important in a lot of fields.These fields comprise: 1) dna sequence analysis; 2) DNA footprint technique; 3) the separation and purification process of extraction protein or enzyme from cell; 4) other need be with the field of macromole nucleolysis.
1) dna sequence analysis
For macromole nucleic acid such as genomic dna sequencing,, generally need earlier big segmental dna degradation to be become less fragment in view of the limited length system of each sequencing reaction to DNA.Ordinary method is to handle with nuclease, perhaps the ultrasonic wave section of beating nucleic acid fragment, electrophoretic separation afterwards.Nuclease costs an arm and a leg, and needs thorough removal before order-checking; And the operating process of the intermittent high frequency section of beating of supersonic method nucleic acid molecule is wayward, and therefore the method for suitable degraded nucleic acid still is worth exploring.
2) DNA footprint technique
The DNA footprint technique is mainly used in and detects and identify the sequence-specific binding ability of transcription factor to DNA, if carry out the order-checking of DNA chemistry simultaneously, can the section of declaring goes out the accurate order of land.This technology remains the most popular method of at present the most frequently used researching DNA-protein interaction.Usually, the DNA with protein bound does not separate with DNaseI to fall, but nuclease often molecular dimension can not obtain high resolving power (Chinese ScienceBulletin too greatly, 2000,45 (2): 2017-2028), therefore research and develop small molecule DNA cutting agent to substitute the macromole nuclease will be very meaningful (Nature, 1988,332 (6165): 663).
3) preparation of protein or enzyme
From cell, extract in the protein molecule process such as enzyme, at first will pass through cytoclasis.After the cell wall breaking, thereby the existence of the interior nucleic acid of its born of the same parents can increase the separation and purification of the viscosity interferases of system, and this phenomenon is more outstanding when particularly carrying out ultrafiltration.Except some organism self contains high reactivity nucleic acid enzyme, do not exist beyond this problem, nucleic acid must be by adding the nuclease degradation of sinking agent sedimentation or adding external source generally speaking.In addition, exist if the target of purifying is a form with inclusion body, because of inclusion body can bind nucleic acid in forming process, so its pre-treatment also is to need to remove nucleic acid.
Public use at present multiple more single-minded precipitation agent, the i.e. material that can form mixture with electronegative phosphatase nucleic acid residue of some positively chargeds arranged.By the descending order of its efficient, these compounds are: polymine, cats product cetrimonium bromide, Vetstrep and protamine sulfate.Similarly, they also can with some enzyme form we do not wish the mixture that exists.Ammonium sulfate precipitation method can remove nucleic acid very effectively, but also some protein removals can be influenced the yield of target protein matter simultaneously.
Have a bit significantly: the reagent that is used to reduce nucleic acid can not injury protein matter, and can be easy to they are removed from purification step.Usually people come really to be removed at the stage of back these enzymes of detection validation then except that nucleic acid with the nucleolysis enzyme treatment stage of giving before the separation and purification.But these enzymes are all very expensive.Therefore, the method for new degraded nucleic acid will be very promising.
Sometimes, nucleic acid need pass through the efficient removal of anion-exchange chromatography (as: anionites such as DEAE-and Q-), contains nucleic acid to guarantee protein articles anything but especially for the pharmaceutical grade protein of human injection's purpose.In this case, the degraded of also need going ahead of the rest before upper prop of the nucleic acid in the cytoclasis liquid reducing viscosity, thereby avoids nucleic acid molecule and separating medium or protein bound to damage the efficient of separation and purification.
In sum, no matter be from conventional application point of view or from the separation and purification angle, nucleic acid removal method is crucial in protein or enzyme making processes efficiently safely and fast.
4) other
In other need the field with the macromole nucleolysis, comprise and be convenient to nutritive nucleic acid product that human body or animal absorb etc.
Purpose in invention provides the novel method that a kind of efficient macromole of degraded fast nucleic acid generates small segment and removes cytoclasis liquid amplifying nucleic acid.
Summary of the invention the invention provides a kind of superoxide that utilizes is having ultraviolet lighting, metal ion or them to generate the principle of hydroxyl radical free radical OH, the novel method of the broken liquid of the animal of can efficiently degrading apace, plant or microorganism cells or its genomic dna amplifying nucleic acid simultaneously.
The chemical structure of general formula of the superoxide that the present invention is used is:
R-O-O-H
R=H in the formula, R ', COR ' or OOR ';
R '=CH
3, CH
2H
5Or C
nH
2n+1, n＜15;
C
6H
mX
5-m，m＝0-5；C
10H
mX
8-m，m＝0-8；C
10H
mX
6-mO
2，m＝0-6；
C
14H
mX
10-m，m＝0-10；C
14H
mX
8-mO
2，m＝0-8；
X＝CH
3，C
2H
5，NO
2，N(CH
3)
2，N(C
2H
5)
2，COOCH
3，COOC
2H
5，F，Cl，Br
Or I;
The used metal ion of the present invention is: Fe
2+, Cu
2+, Cd
2+, Co
2+, Hg
2+, Sn
2+, Zn
2+, Mg
2+, Ca
2+Or other transition metal ion;
The used zooblast of the present invention is software kinetoplast or vertebrate cells;
The used vegetable cell of the present invention is gymnosperm or dicotyledons cell;
The microorganism cells that the present invention did is bacterium or virus;
Present method is that the superoxide with said structure directly is added in broken liquid of biological cell or the genomic dna, and making its final concentration is 0.0001-1M, adds quadrol base tetraacethyl-Fe simultaneously
2+Complex compound, to its final concentration be 0.1-10mM, under the room temperature reaction 1-60 minute, the nucleic acid in the cytoclasis liquid is all degraded; Use metal cations Fe
2+, Cu
2+, Cd
2+, Co
2+, Hg
2+, Sn
2+, Zn
2+, Mg
2+, Ca
2+Or other transition metal ion is replaced Fe
2+, all can be with nucleolysis, and with Fe
2+, Cu
2+, Cd
2+, Co
2+Metal ion degradation effect the best; When replacing its genomic dna, do not add or add less metal ion and also can reach the ideal degradation effect with the broken liquid of biological cell; Above-mentioned superoxide directly is added in the organism genomic dna, to its final concentration be 0.01M, use simultaneously 400nm illumination and (or) add quadrol base tetraacethyl-Fe
2+Complex compound was handled 30 minutes, nucleic acid all can be degraded.
The purposes of present method is the dna sequence analysis that is used to comprise except that the DNA footprint technique, extracts the degraded of separation and purification process nucleic acid of protein or enzyme and other need be with the field of macromole nucleolysis from cell.
Description of drawings Fig. 1 is hydrogen peroxide degradation bacillus coli gene group DNA
1% agarose gel electrophoresis is analyzed the degradation efficiency of the hydrogen peroxide of different concns to genomic dna.Hurdle 1 to 5 is represented the different concns 0.3M that adds hydrogen peroxide, 0.1M, 0.01M, 0.001M, 0.0001M respectively.
Fig. 2 is that the existence of metal ion influences the degraded of hydrogen peroxide to the genome e. coli dna
1% agarose gel electrophoresis analysis adds the influence of different metal ion pair hydrogen peroxide degradation genomic dna.Hurdle 1 representative does not add the blank of metal ion; Hurdle 2 to 10 is represented respectively and is added Cu
2+, Hg
2+, Cd
2+, Co
2+, Sn
2+, Zn
2+, Mg
2+, Ca
2+, Fe
2+
Fig. 3 is that the adding of metal ion influences the degraded of hydrogen peroxide to the broken liquid amplifying nucleic acid of Bacillus coli cells
1% agarose gel electrophoresis analysis adds the influence of the broken liquid amplifying nucleic acid of different metal ion pair hydrogen peroxide degradation Bacillus coli cells.Hurdle 1 to 9 is represented respectively and is added Cu
2+, Hg
2+, Cd
2+, Co
2+, Sn
2+, Zn
2+, Mg
2+, Ca
2+, Fe
2+Hurdle 10 representatives do not add the blank of metal ion.
Fig. 4 be under the ultraviolet lighting hydrogen peroxide to the damaging action of genomic dna
Hurdle 1 adds EDTA-Fe
2+, ultraviolet lighting; Hurdle 2 only adds EDTA-Fe
2+Hurdle 3 adds 0.01M hydrogen peroxide and EDTA-Fe
2+Hurdle 4 adds 0.01M hydrogen peroxide and EDTA-Fe
2+, ultraviolet lighting; Hurdle 5 adds the 0.01M hydrogen peroxide, ultraviolet lighting; Hurdle 6 adds the 0.01M hydrogen peroxide; Hurdle 7, DNA is as blank for the bacillus coli gene group.
Fig. 5 is the damaging action of hydrogen peroxide to plant genome DNA
Fig. 6 is the Degradations of other superoxide to bacillus coli gene group DNA
Hurdle 1 does not add superoxide; Hurdle 2 adds hydrogen peroxide; Hurdle 3 adds Peracetic Acid; Hurdle 4, peroxyformic acid.
There is the damaging action of pair cell internal protein in Fig. 7 for hydrogen peroxide
The existence of the hydrogen peroxide of 10%SDS-PAGE analysis different concns is to the damaging action of Bacillus coli cells internal protein.Protein dyes with Xylene Brilliant Cyanine G R-250.Hurdle 1-9 represents the hydrogen peroxide of different concns respectively; 0.3M, 0.1M, 0.01M, 0.001M, 0.0001M, 0%.
Fig. 8 is the Degradation of hydrogen peroxide to the mouse liver cell genomic dna
Method provided by the invention has following characteristics: 1) peroxide at work concentration 0.01M does not have protein Damaging action; 2) when using hydrogen peroxide, it only resolves into moisture in the presence of ultraviolet lighting or metal ion, Can not introduce new impurity; 3) this is quick on the draw fast, and is simple to operate, extremely is fit to be applied to prepare on a large scale protein Or the degraded of the nucleic acid in the enzyme process.
Embodiment
The fragmentation of embodiment 1 Bacillus coli cells and the extraction of genomic dna thereof
Picking list bacterium colony in the Bechtop is inoculated in the LB liquid nutrient medium (Amp of 10ml
+) in, 30 ℃, 180rpm is vibration activation culture spend the night (12-14h) down.Inoculate 2% thalline next day in the 250ml fresh medium, 37 ℃, 180rpm cultivates, to OD
600Absorption value is 0.6, adds sec.-propyl-β-D-sulfydryl semi-lactosi IPTG to final concentration 1mM, continues abduction delivering 3 hours, and centrifugal (6,500rpm 10min) collects thalline.(20mM Tris-HCl, pH8.0), the 5 fens kinds that suspend add 5g N,O-Diacetylmuramidase (Amresco company) afterwards, obtain cytoclasis liquid to add ice-cold buffer A by weight in wet base 1: 5 (W/V).Get 1000ml bacterium liquid centrifugal 5min under 4500-5000rpm.Abandon supernatant, keep precipitation.In precipitation, add solution I (50mM glucose, 25mM Tris-HCl, pH8.0,10mM quadrol base tetraacethyl EDTA) 100ml, light shaking, and then to wherein adding solution II (0.2M NaOH, 1% sodium laurylsulfonate SDS) 20ml, at this moment mixing floss occurs in the solution gently.To wherein adding solution III (5M sodium acetate, 60.0ml, glacial acetic acid 11.5ml, H
2O 28.5ml) 20ml, frozen 20-30min.The centrifugal 20min of 10000rpm.Get supernatant, use filtered through gauze.Filtrate and equal-volume (or 0.6 times of volume) Virahol mixing is placed 10min.Under 4 ℃, the centrifugal 10min of 10000rpm.Abandon supernatant, add 70% ethanol then and shake, precipitation is dissolved again.Under 4 ℃, the centrifugal 10min of 10000rpm.Be deposited in kept dry under the condition of nature.
Hydrogen peroxide is directly joined bacillus coli gene group DNA (5.2 * 10
6Bp) in to its final concentration be 0.3M, 0.1M, 0.01M, 0.001M, 0.0001M adds EDTA-Fe simultaneously
2+To its final concentration be 10mM, the reaction 5min.Degradation results shows that the 0.01M hydroperoxidation can all be degraded genomic nucleic acids in 5 minutes, determines that 0.01M is its working concentration (Fig. 1).
Replace Fe with the different metal ion
2+, investigate its degradation efficiency to genomic dna, show Fe
2+, Cu
2+, Cd
2+, Co
2+Deng degradation effect (Fig. 2) with the best
Replace the genomic dna experiment with the broken liquid of Bacillus coli cells, the result shows that the influence that the different metal ionic adds degradation efficiency does not have significant difference (Fig. 3), because of having had metal ion in the colibacillus tenuigenin, so can not add or add on a small quantity metal ion, also can reach the ideal degradation effect.
With hydrogen peroxide directly join among the bacillus coli gene group DNA to its final concentration be 0.01M, 400nm photo-irradiation treatment 30min observes degradation effect (Fig. 4) simultaneously.
Hydrogen peroxide is directly joined corn gene group DNA (1 * 10
11Bp) and soybean gene group DNA (1.7 * 10
9Bp) in to its final concentration be 0.01M, add EDTA-Fe simultaneously
2+To its final concentration be 10mM, observe degradation effect (Fig. 5).
With Peracetic Acid, peroxyformic acid replace hydrogen peroxide directly join in the cytoclasis liquid to its final concentration be 0.01M, add EDTA-Fe simultaneously
2+To its final concentration be 10mM, observe degradation effect (Fig. 6).Compare with the hydrogen peroxide with concentration, both degradation effects are also very remarkable.
The 10%SDS-polyacrylamide gel electrophoresis detects different concns H
2O
2To the influence of target protein, reaction 30min, 0.001M-0.3M H
2O
2All not having obvious impairment influence (Fig. 7), is that the hydrogen peroxide of 0.01M can not damage target protein matter molecule when the degraded nucleic acid molecule for working concentration therefore.
With hydrogen peroxide directly join in the mouse liver cell genomic dna to its final concentration be 0.01M, add EDTA-Fe simultaneously
2+To its final concentration be 10mM, observe degradation effect (Fig. 8), find that the mouse liver cell genomic dna all degrades.
Claims (3)
1, a kind of method with peroxide degradation nucleic acid, it is characterized in that present method be utilize superoxide ultraviolet lighting, metal ion or they simultaneously in the presence of, the nucleic acid in efficient degradation biological body animal and plant fast or the microorganism fine crushing liquid; Be about to have the superoxide of the following chemical structure general formula:
R-O-O-H
R=H in the formula, R ', COR ' or OOR ';
R '=CH
3, CH
2H
5Or C
nH
2n+1, n＜15;
C
6H
mX
5-m，m＝0-5；C
10H
mX
8-m，m＝0-8；C
10H
mX
6-mO
2，m＝0-6；
C
14H
mX
10-m，m＝0-10；C
14H
mX
8-mO
2，m＝0-8；
X＝CH
3，C
2H
5，NO
2，N(CH
3)
2，N(C
2H
5)
2，COOCH
3，COOC
2H
5，F，Cl，Br
Or I;
Directly be added in broken liquid of biological cell or the genomic dna, making its final concentration is 0.0001-1M, adds quadrol base tetraacethyl-Fe simultaneously
2+Complex compound, to its final concentration be 0.1-10mM, under the room temperature reaction 1-60 minute, the nucleic acid in the cytoclasis liquid is all degraded; Use metal cations Fe
2+, Cu
2+, Cd
2+, Co
2+, Hg
2+, Sn
2+, Zn
2+, Mg
2+, Ca
2+Or other transition metal ion is replaced Fe
2+, all can be with nucleolysis, and with Fe
2+, Cu
2+, Cd
2+, Dd
2+, Co
2+Metal ion degradation effect the best; When replacing its genomic dna, do not add or add less metal ion and also can reach the ideal degradation effect with the broken liquid of biological cell; Above-mentioned superoxide directly is added in the organism genomic dna, to its final concentration be 0.01M, use simultaneously 400nm illumination and (or) add quadrol base tetraacethyl-Fe
2+Complex compound was handled 30 minutes, nucleic acid all can be degraded.
2,, it is characterized in that being applicable to that the zooblast of the biological cell of present method is mollusk or vertebrate cells according to the method for the described degraded nucleic acid of claim 1; Vegetable cell is gymnosperm or dicotyledons cell; Microorganism cells is bacterium or virus.
3,, it is characterized in that being used for comprising dna sequence analysis except that the DNA footprint technique, extract the degraded of separation and purification process nucleic acid of protein or enzyme from cell and other need be with the field of macromole nucleolysis according to the purposes of the described method of claim 1.
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| CNA031590616A CN1493582A (en) | 2003-09-12 | 2003-09-12 | Cleaning convenient, high efficiency degradating method of nucleic acid |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684605A (en) * | 2019-11-18 | 2020-01-14 | 青岛立见诊断技术发展中心 | Nucleic acid remover |
| CN111662957A (en) * | 2020-06-08 | 2020-09-15 | 河北迪纳兴科生物科技有限公司 | Reagent for digesting nucleic acid pollution and preparation method and application thereof |
| CN112812901A (en) * | 2021-01-29 | 2021-05-18 | 北京华瑞康源生物科技发展有限公司 | Reagent capable of efficiently removing nucleic acid pollution in normal-temperature and low-temperature environments and application |
-
2003
- 2003-09-12 CN CNA031590616A patent/CN1493582A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684605A (en) * | 2019-11-18 | 2020-01-14 | 青岛立见诊断技术发展中心 | Nucleic acid remover |
| CN110684605B (en) * | 2019-11-18 | 2021-08-10 | 青岛立见生物科技有限公司 | Nucleic acid remover |
| CN111662957A (en) * | 2020-06-08 | 2020-09-15 | 河北迪纳兴科生物科技有限公司 | Reagent for digesting nucleic acid pollution and preparation method and application thereof |
| CN111662957B (en) * | 2020-06-08 | 2021-12-07 | 湖北擎科生物科技有限公司 | Reagent for digesting nucleic acid pollution and preparation method and application thereof |
| CN112812901A (en) * | 2021-01-29 | 2021-05-18 | 北京华瑞康源生物科技发展有限公司 | Reagent capable of efficiently removing nucleic acid pollution in normal-temperature and low-temperature environments and application |
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