CN1239512C - High efficiency quick degradation genome DNA of light sensitive aromatic heterocyclic compound - Google Patents
High efficiency quick degradation genome DNA of light sensitive aromatic heterocyclic compound Download PDFInfo
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Abstract
The present invention provides a novel method for effectively and quickly degrading and cutting macromolecular nucleic acid in cytoclasis liquid, plasmid DNA or genome DNA of animals, plants or microbes into small segments by utilizing photosensitive heteroaromatic compounds as DNA cutting agents which generate active free radical or lead electron transfer under light irradiation. The method has the characteristics that the compounds which have work concentration do not have damage effect on protein, and the compounds can generate free radical or lead electron transfer under the condition of light irradiation; metal ions which possibly cause pollution do not need to be led in reaction, the reaction is sensitive and rapid, the operation is simple, etc. The present invention can be used for DNA sequence analysis, DNA footprint techniques, separation and purifying processes in which protein or enzyme is extracted from cells and other fields which require nucleic acid degradation.
Description
Technical field the present invention relates to utilize photosensitive heteroaromatic compound to cut off the method that agent is degraded into macromole nucleic acid small segment non-specificly.
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, and perhaps ultrasonic wave interrupts nucleic acid fragment electrophoretic separation afterwards.Nuclease costs an arm and a leg, and needs thorough removal before order-checking; And the operating process that the intermittent high frequency of supersonic method interrupts 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 detection and identifies the sequence-specific binding ability of transcription factor to DNA, if carry out the order-checking of DNA chemistry simultaneously, can judge 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 (Nature too greatly, 1988,332 (6165): 663), therefore research and develop small molecule DNA cutting agent to substitute the macromole nuclease will be very meaningful (Chinese Science Bulletin, 2000,45 (2): 2017-2028).
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 the pretreatment stage of people before separation and purification removes nucleic acid with the nucleolysis enzyme, really is removed at the stage of back these enzymes of detection validation then.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.
The purpose of this invention is to provide a kind of light-sensitive compound and cut off the method that the efficient degraded fast of agent macromole nucleic acid generates small segment.
Summary of the invention the invention provides and a kind ofly can cut off agent as DNA to the photosensitive heteroaromatic compound that luminous energy has a strong absorption, under illumination, produce active free radical or cause transfer transport, the efficient novel method that fast macromole nucleolysis in organism animal and plant or the broken liquid of microorganism cells, plasmid DNA or the genomic dna is cut into small segment.
The photosensitive heteroaromatic compound that present method is used as DNA cut-out agent has: naphthalimide, oxygen or Thiaxanthene, anthraquinone, xanthene, rhodamine, cyanine dyes, metal phthalocyanine, biphenyl, dinaphthalene, Bi ， perylene ， perylene diimide or coumarin derivatives.
It is mollusk or vertebrate cells that the used biological cell of present method has zooblast; Vegetable cell is gymnosperm or dicotyledons cell; Microorganism cells is bacterium or virus.
Present method is that above-mentioned photosensitive heteroaromatic compound directly is added in the broken liquid of biological cell, plasmid DNA or the genomic dna, to its final concentration be 10 μ M-1.0M, under UV300-600nm illumination, reacted 0.5-10 hour simultaneously, macromole nucleic acid is cut off being degraded into small segment.
Present method is applied to dna sequence analysis, 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.
The method of photosensitive heteroaromatic compound degraded nucleic acid provided by the invention has following characteristics:
(1) compound when working concentration does not have damaging action to protein;
(2) it can produce free radical or cause transfer transport under illumination condition;
(3) reaction does not need to introduce the metal ion that may pollute;
(4) this method is sensitive quick, simple to operate.
Description of drawings Fig. 1 is that 1% agarose gel electrophoresis is analyzed the degradation efficiency of synthetics to genomic dna
1-hurdle, hurdle 9, add synthetics N-butyl benzo Thiaxanthene-1 respectively, 3-imide (R-1), N dimethylamine base ethyl benzo Thiaxanthene-1,3-imide (R-2), N-piperazinyl ethyl benzo Thiaxanthene-1,3-imide (R-3), N dimethylamine base propyl group benzo Thiaxanthene-1,3-imide (R-4), N-(fluorine is to the chlorobenzene acyloxy) sulfo-naphthalimide (C-I), N-(furans acyloxy) sulfo-naphthalimide (C-II), N-(m-dimethoxybenzene acyloxy) sulfo-naphthalimide (C-III), N-(pyridine acyloxy between adjacent chlorine) sulfo-naphthalimide (C-IV), behind N-dibutyl naphthalene imide and the thiadiazoles (S), the degraded situation of plasmid pBR322DNA; Hurdle 10, the pBR322DNA blank.
There is the degraded situation of e. coli dna down in Fig. 2 for synthetics
Hurdle 1 adds synthetics N dimethylamine base ethyl benzo Thiaxanthene-1,3-imide (R-2); Hurdle 2, genome NDA blank; Hurdle 3 adds synthetics N dimethylamine base propyl group benzo Thiaxanthene-1,3-imide (R-4); Hurdle 4 adds synthetics N-dibutyl naphthalene imide and thiadiazoles (S).
Fig. 3 is synthetics N-dibutyl naphthalene imide and thiadiazoles (S) damaging action to plant genome DNA
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 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 (0.2MNaOH, 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.
1) Dui Zhi degraded
Investigation is dissolved in damping fluid 10mM Tris/HCl, and 20mM NaCl, pH7.5, final concentration are the different compound Rs-1 of 50 μ M, R2, R3, S, R-4; C-I, C-II, C-III, C-IV, under UV400nm illumination, reaction system is 10 μ l, applied sample amount 1 μ l, the situation that 1% agarose electrophoresis trace analysis cuts off for pBR322 (500ng/ μ l) is found all to show high DNA and is cut off active (Fig. 1).Compound R-1 is degraded into small segment with I type plasmid pBR322; Compound R-2 is converted into the II type with plasmid pBR322 by the I type, is converted into III type DNA at last; Compound R-3 is converted into the II type with plasmid pBR322 by the I type, is degraded into small segment at last; Compound R-4 mainly is converted into the II type with plasmid pBR322 by the I type, is converted into III type DNA at last; Compound S is converted into II type and III type DNA with plasmid pBR322 by the I type; Compound C-series all has the function that I type plasmid pBR322 is degraded into small segment.
2) to the degraded of bacillus coli gene group DNA
The R series compound is directly joined bacillus coli gene group DNA (5.2 * 10
6Bp) in to its final concentration be 50 μ M, simultaneously at UV400nm illumination reaction 2hr, observe degradation effect (Fig. 2).
3) synthetics is to the degraded of genomic dna in the broken liquid of vegetable cell
Synthetics R-4 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 0.0005M, simultaneously illumination 2hr observes degradation effect (Fig. 3).
Claims (3)
1, a kind of method with organic compound degraded nucleic acid is characterized in that it is photosensitive heteroaromatic compound naphthalimide or Thiaxanthene derivative that present method is used as the organic synthesis thing that nucleic acid cuts off agent; This method is that above-mentioned photosensitive heteroaromatic compound directly is added in the broken liquid of biological cell, plasmid DNA or the genomic dna, to its final concentration be 10 μ M-1.0M, under UV300-600nm illumination, reacted 0.5-10 hour simultaneously, macromole nucleic acid is cut off being degraded into small segment.
2,, it is characterized in that it is mollusk or vertebrate cells that the used biological cell of present method has zooblast 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, according to the purposes of the described degraded nucleic acid of claim 1 method, it is characterized in that being applied in dna sequence analysis, DNA footprint technique, from cell, extract the degraded of separation and purification process nucleic acid of protein or enzyme and other need be with the field of macromole nucleolysis.
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| CN 03159532 CN1239512C (en) | 2003-09-17 | 2003-09-17 | High efficiency quick degradation genome DNA of light sensitive aromatic heterocyclic compound |
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| CN 03159532 CN1239512C (en) | 2003-09-17 | 2003-09-17 | High efficiency quick degradation genome DNA of light sensitive aromatic heterocyclic compound |
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| CN1491954A CN1491954A (en) | 2004-04-28 |
| CN1239512C true CN1239512C (en) | 2006-02-01 |
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| CN100406461C (en) * | 2003-04-30 | 2008-07-30 | 华东理工大学 | Sulphur containing heterocyclic and naphthalimides compound and use thereof |
| CN111333667B (en) * | 2020-04-14 | 2021-04-27 | 浙江工业大学 | Selenium-containing heterocyclic naphthalimide derivative and preparation method and antiviral application thereof |
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