CN107916263A - The preservation particulate and long-term preservation method of inhereditary material - Google Patents

The preservation particulate and long-term preservation method of inhereditary material Download PDF

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CN107916263A
CN107916263A CN201711103478.7A CN201711103478A CN107916263A CN 107916263 A CN107916263 A CN 107916263A CN 201711103478 A CN201711103478 A CN 201711103478A CN 107916263 A CN107916263 A CN 107916263A
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inhereditary material
inhereditary
particulate
nanoparticle
dna
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刁晓荃
丁士友
窦本银
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

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Abstract

The present invention relates to the preservation particulate and long-term preservation method of inhereditary material.The preservation particulate of the inhereditary material includes：A) nanoparticle；B) inhereditary material, it is adsorbed in the nanoparticle；C) inhereditary material confining bed, the nanoparticle and inhereditary material are wrapped in interior by it；D) metal material layer, it is located at outside the inhereditary material confining bed.The present invention also provides the article of the preservation particulate comprising the inhereditary material.The preservation particulate for the inhereditary material being prepared by the method for the present invention almost shields the factor that damage may be caused to the structure of inhereditary material, and can completely discharge, it is achieved thereby that the long-term preservation of inhereditary material.

Description

The preservation particulate and long-term preservation method of inhereditary material
Technical field
The present invention relates to inhereditary material field, and in particular to the preservation particulate and long-term preservation method of inhereditary material.
Background technology
Inhereditary material refers to the material to convey hereditary information between parental generation and filial generation, mainly includes DNA and RNA, it is every One bion including animal, plant and Institute of Micro-biology are peculiar.DNA technique have been widely used for biomedical, agricultural, The fields such as environment, material, criminal investigation, information preservation.RNA is transcribed in special time by organism gene (DNA), for translating Albumen needed for vital movement.The research of RNA also has quite varied prospect in agriculture and animal husbandry, medicine etc., dynamic except studying The development of plant, Physiological effect etc. are outer, can be also used for the diagnose and treat of disease.For example the RNA of a Healthy People is preserved, can For me or the medical diagnosis on disease and treatment directly under relatives for having genetic association.Compare the gene table of an organism different time The function of unknown gene, adjustment effect mechanism of specific gene etc. can be studied up to (RNA sequencings).
The extraction of inhereditary material is a kind of ripe and common method.Extraction inhereditary material is simultaneously preserved, when needed Discharged and purified, and expanded by round pcr, can easily utilize the inhereditary material preserved.Therefore, how Realize that the preservation of inhereditary material, preservation especially steady in a long-term become more and more concerned.
Typically, DNA and RNA molecule can select Cord blood, for example, most biology laboratories all by DNA or RNA molecule is stored in -80 DEG C of low temperature refrigerators or liquid nitrogen, and store method is easy.However, this method also has number of drawbacks：One Aspect, inhereditary material can only be stored in the extraordinary professional institution for preserving equipment, preserve place to it and propose high requirement, It can not realize personal routine preservation；On the other hand, Cord blood faces later maintenance cost costly, if necessary to longer It is costly if phase preservation；Another further aspect, due to being aoxidized, ionising radiation, ultraviolet, temperature rise and nuclease etc. The influence of many factors, the middle or short term that Cord blood is suitable only for inhereditary material preserves, such as monthly, years or decades, more Inhereditary material is it is possible to destroyed during long-term preservation.
In nature, microbial spore (P.Setlow, Trends of the scientist from the amber parcel before 2-3 10,000,000 Microbiol.,2007,15,172.；R.J.Cano and M.K.Borucki, Science, 1995,268,1060.) and Weevil (Cano et al., Amplification and sequencing of DNA from as of the 1.2-1.35 before 100000000 years 120-135-million-year-old weevil, Nature 363 (6429):DNA 536-8) is extracted, it was demonstrated that hereditary thing The super-long-term of matter, which preserves, is possible to what is realized.At present, many scientists are also studying the long-term method for preserving DNA, such as： 1) by DNA absorption in filter paper or porous material (Choy, J.H., Oh, J.M., Park, M., Sohn, K.M.＆Kim, J.W.Inorganicbiomolecular hybrid nanomaterials as a genetic molecular code system.Adv.Mater.16,1181–1184(2004).；Burgoyne,L.A.Solid medium and method for DNA storage.US patent 5,496,562(1988)；Taylor,D.J.,Finston,T.L.&Hebert, P.D.N.The 15%solution for preservation.Trends Ecol.Evol.9,230 (1994)；Yang, H.et al.Adsorption and protection of plasmid DNA on mesoporous silica nanoparticles modified with various amounts of organosilane.J.Colloid Interface Sci.369,317–322(2012).；Kapusuz,D.&Durucan,C.Synthesis of DNA- encapsulated silica elaborated by sol-gel routes.J.Mater.Res.28,175–184 (2013).；Pierre,A.,Bonnet,J.,Vekris,A.&Portier,J.Encapsulation of deoxyribonucleic acid molecules in silica and hybrid organic-silica Gels.J.Mater.Sci.Mater.Med.12,51-55 (2001)), but these methods are not by DNA and external environment point Open, the holding time is limited；2) DNA is wrapped in glass material (Daniela Paunescu, Michela Puddu, Justus O B Soellner,Philipp R Stoessel&Robert N Grass.Reversible DNA encapsulation in silica to produce ROS-resistant and heat-resistant synthetic DNA‘fossils’ .Nature Protocols,2013,8(2)：2440-2448.；D.Paunescu,C.A.Mora,M.Puddu,F.Krumeich And R.N.Grass, DNA protection against ultraviolet irradiation by encapsulation In a multilayered SiO2/TiO2assembly.J.Mater.Chem.B, 2014,2,8504-8509), this method So that the DNA holding times greatly extend.However, DNA still can be ionized or light degradation etc. after being coated using silicon dioxide layer Influence；Nano-titanium dioxide stronger ultraviolet screener effect more than nano silicon dioxide, ultraviolet can be prevented to DNA and The damage of RNA, but it is equally unfavorable for the long-term preservation of DNA and RNA with strongly hydrophilic.
Therefore, demand is still had for the long-term preservation method of inhereditary material.
The content of the invention
The present invention will provide a kind of long-term preservation method of inhereditary material, it can be in several years, many decades, hundreds of The inhereditary material is stablized in year, thousands of years, Shuo Wannian, hundreds thousand of years or even more long time range and is preserved.
For achieving the above object, this invention takes following technical solution：
In a first aspect, the present invention provides a kind of inhereditary material to preserve particulate, it includes：
A) nanoparticle；
B) inhereditary material, it is adsorbed in the nanoparticle；
C) inhereditary material confining bed, the nanoparticle and inhereditary material are wrapped in interior by it；With
D) metal material layer, it is located at outside the inhereditary material confining bed.
In said structure, nanoparticle plays the role of carrying and absorption inhereditary material, it is needed to inhereditary material point Son has adsorptivity, and does not have destruction or degradation to its molecule；When the inhereditary material preserves particulate subsequently if desired When discharging preserved inhereditary material, the nanoparticle must also be able to the heredity with being preserved in the case where dedicated reagent is acted on Material separates.Inhereditary material confining bed separates inhereditary material and the external world, plays primary protective effect, and can reduce the external world The photodegradative possibility that ultraviolet of environment etc. is brought；Metal material layer play prevent electromagnetic radiation, various rays and acid, Alkali, water invade wet destruction.
Preferably, the inhereditary material is DNA and/or RNA, such as mRNA (mRNA).
Preferably, the nanoparticle is by SiO2Or selected from golden (Au), silver-colored (Ag), tungsten (W), copper (Cu) metal or include it Metal ion or its alloy material are made, and the nanoparticle is further preferably by SiO2It is made.
Preferably, the inhereditary material confining bed is SiO2Layer or TiO2Layer, more preferably TiO2Layer, because TiO2 Layer compares SiO2Shielding action of the layer to ultraviolet is stronger, so as to further reduce the light degradation of inhereditary material.
Preferably, the metal material layer is made of metal material chosen from the followings：Golden (Au), platinum (Pt), palladium (Pd), rhodium (Rh), osmium (Os), silver-colored (Ag), lead (Pb), mercury (Hg), polonium (Po), ruthenium (Ru), technetium (Tc), copper (Cu), germanium (Ge), tungsten (W)；It is excellent Choosing selected from golden (Au), platinum (Pt), silver-colored (Ag), copper (Cu) or tungsten (W) metal material by being made；Most preferably it is made of gold (Au).Institute Metal material layer is stated to be used to shield TiO2The strongly hydrophilic of layer and to completely cut off other physical factors, chemical factor and biological factor etc. more The damage that kind ray may bring the structure of the inhereditary material of encapsulation.
Preferably, the particle diameter of the inhereditary material preservation particulate is between 250-350nm, preferably between 280-320nm. Particle size is not limited only to this scope.
In second aspect, present invention also offers a kind of long-term preservation method of inhereditary material, it includes the following steps：
A) nanoparticle is prepared；
B) optionally, the nanoparticle is subjected to surface active so that it can adsorb inhereditary material；
C) inhereditary material is adsorbed onto in the nanoparticle by surface active；
D) inhereditary material confining bed is superscribed in the nano-particle surface, forms inhereditary material parcel particulate；
E) the further coated metal material layer of microparticle surfaces is wrapped up in the inhereditary material, it is micro- forms inhereditary material preservation Grain.
In the method, nanoparticle in itself may have inhereditary material certain suction-operated, but pass through surface active Its adsorption effect to inhereditary material can be further enhanced.
Preferably, the inhereditary material is DNA or RNA, such as mRNA (mRNA).
Preferably, the nanoparticle is by SiO2Or selected from golden (Au), silver-colored (Ag), tungsten (W), copper (Cu) metal or include it Metal ion or its alloy material are made, and the nanoparticle is further preferably by SiO2It is made.
Preferably, the surface active for surface roughening and carries positive charge, to lift the inhereditary material of nanoparticle Adsorbance.
Preferably, the inhereditary material confining bed is SiO2Layer or TiO2Layer, more preferably TiO2Layer, because TiO2 Layer compares SiO2Shielding action of the layer to ultraviolet is stronger, so as to further reduce the light degradation of inhereditary material.
Preferably, the metal material layer is made of metal material chosen from the followings：Golden (Au), platinum (Pt), palladium (Pd), rhodium (Rh), osmium (Os), silver-colored (Ag), lead (Pb), mercury (Hg), polonium (Po), ruthenium (Ru), technetium (Tc), copper (Cu), germanium (Ge), tungsten (W)；It is excellent Choosing is made of the metal material selected from golden (Au), platinum (Pt), silver-colored (Ag), copper (Cu) or tungsten (W)；Most preferably it is made of gold (Au). The metal material layer is used to shield TiO2The strongly hydrophilic of layer simultaneously completely cuts off other physical factors, chemical factor and biological factor pair The damage that the structure of the inhereditary material of encapsulation may be brought.
Preferably, the method further includes from the inhereditary material and preserves the step of particulate discharges the inhereditary material preserved. It is further preferred that the step of inhereditary material of the release preservation, includes：
F) KI/I is used2Solution opens the metal material layer；
G) etching solution NH is used4FHF/NH4F opens the inhereditary material confining bed and discharges the inhereditary material of preservation Out.
In the third aspect, present invention also offers a kind of article for including inhereditary material, it includes heredity as described above Material preserves particulate.Article includes but not limited to souvenir.
By inhereditary material long-term preservation method provided by the invention, the inhereditary material being saved is with that may cause degraded Factor, including chemical factor or biological factor such as aoxidize, ionising radiation, nuclease, ultraviolet, chemical mutagen, acid, alkali Almost isolate, its can be achieved at normal temperatures it is steady in a long-term preserve, for example, at least 1 year, at least 2 years, at least 3 years, at least 4 Year, at least at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 15 years, 20 years, at least 30 Year, at least at least 40 years, at least 50 years, at least 60 years, at least 70 years, at least 80 years, at least 90 years, at least 100 years, 200 years, At least 300 years, at least 400 years, at least 500 years, at least 1000, at least 5000, at least 10000, at least 50000, At least 100000 years, at least 500000 years, at least 1000000 years etc..Also, can be by the hereditary thing of preservation whenever necessary Matter therefrom discharges, and the inhereditary material of preservation is used by technologies such as purifying, PCR amplifications.
Inhereditary material long-term preservation method provided by the invention can help the mankind to solve genetic diversity in bio-diversity Property the problem of running into, can be widely applied for the fields such as biology, agricultural, herding and medicine.Such as：The long-term Techniques of preserving of inhereditary material It can be combined with the disease diagnosis and therapy of I or offspring, in terms of being directly applied to human health；Inhereditary material preserves micro- Grain can the inhereditary material of someone or certain family or certain biology is outstanding or healthy gene or special purpose preserve for a long time, Material wealth more important than money, more valuable can be left to me or offspring；With the fast development of biotechnology, future Inhereditary material can be utilized to preserve in particulate and preserve complete inhereditary material, organism is cloned；Inhereditary material preserves micro- Grain can be associated with the emotion and inner world of the mankind, you can inhereditary material is preserved particulate, or inhereditary material is protected It is associated with some articles (such as indication, souvenir, valuables, jewellery and practical items) to deposit particulate, applied to legacy, Neonatal, love-based marriage, wedding celebration, religion, funeral and among the emotion and inner world of the mankind such as Funeral Reform, pet, and Develop corresponding product.
Brief description of the drawings
Fig. 1 is the schematic diagram for showing the inhereditary material long-term preservation method of the present invention；
Fig. 2 is by TiO2The electron-microscope scanning figure of the DNA parcel particulates of layer parcel, the diameter of the particulate is about 250 nanometers；
Fig. 3 is to wrap up the electron-microscope scanning figure that DNA shown in Fig. 2 wraps up the DNA that is prepared of particulate and preserve particulate, institute with layer gold The diameter for stating particulate is about 300 nanometers；
Fig. 4 shows that DNA preserves DNA gel electrophoresis comparing results of the DNA of particulate release with not being wrapped encapsulation；And
Fig. 5 show the present invention DNA preserve particulate with the prior art do not include metal material layer preservation particulate it Between the preservation effect of DNA is contrasted in terms of to ultra-violet radiation resisting.
Embodiment
In the following, the present invention will be described in more detail with reference to embodiment, but the scope of the present invention is not limited to The embodiment provided.
Unless otherwise defined, all technical and scientific terms used herein have with it is of the art common Technical staff normally understood identical meaning.All publications specifically mentioned herein, are to include describing and openly Institute including chemical substance, instrument, statistical analysis and method that the possibility reported in the publication is used in combination with the present invention Purposefully, it is expressly incorporated herein by reference with entire contents.All bibliography should be considered as quoted in this specification Indicate the technical merit of this area.Any content herein is not necessarily to be construed as recognizing the no right of the present invention earlier than passing through These disclosures formerly invented.
As used herein, term " comprising " and "comprising" are open terms, and should be interpreted to mean " including but not limited to ".These terms cover more restricted term " substantially by ... form " and " by ... structures Into ".It should also be noted that term "comprising" is interchangeable with " comprising ", " being characterized in that " and " having ".
As used herein, include its singular or plural without specific amount of denotion and censure thing, unless context It is not such clearly to describe.In addition, without it is specific amount of censure, term " one or more " and " at least one " can exchange makes With.
Embodiment
1. the preparation of silica nanoparticles
A. the water of 18 milliliters of ethanol, the ammonia solution of 0.8 milliliter of 25 weight % and 0.5ml is mixed in 50 milliliters of conical pipes, And add 0.8 milliliter of TEOS (polycondensation of tetraethoxysilane).Stir for 900 revs/min at room temperature Mix 6 it is small when.
B. 20 minutes, and abandoning supernatant are centrifuged at room temperature in 9000g.
C. precipitation is dissolved in 20 milliliters of isopropanols and (shakes several seconds simultaneously water bath sonicator 5 minutes).
D. repeat step b and c.Centrifuged at room temperature 20 minutes in 9000g.
E. precipitation is dissolved in 4 milliliters of isopropanols (shaking several seconds simultaneously water bath sonicator 5 minutes).The concentration of gained particulate is about 50 Mg/ml, it is sufficient to carry out about 120 package experiments.Silica nanoparticles can safely store at room temperature to It is 6 months few.
2. the surface active of silica nanoparticles
A. the silica nanoparticles (step 1.e, about 50 mg/ml of concentration) of 1 milliliter of above-mentioned preparation are added to 2 In milliliter microcentrifugal tube.
B. water bath sonicator 10 minutes, until mixture becomes homogeneous suspension liquid.
C. 10 microlitres of TMAPS (N-trimethoxylsilylpropyl-N, N, N-trimethylammonium are added Chloride) (50 weight % (methanol solution)).
D. 900 revs/min be stirred at room temperature mixture 12 it is small when.
E. 21500g is centrifuged 4 minutes and is abandoned supernatant at room temperature.
F. precipitation is suspended in 1 milliliter of isopropanol (shaking several seconds simultaneously water bath sonicator 3 minutes).
G. repeat step 2.e and 2.f, at room temperature 21500g centrifugations abandon supernatant in 4 minutes.
H. precipitation is suspended in 1 milliliter of isopropanol (shaking several seconds simultaneously water bath sonicator 3 minutes).The solution so obtained is enough Carry out about 30 package experiments.The silicon dioxide microparticle of surface active can be stored safely 4-6 weeks at room temperature.
The encapsulation of 3.DNA or RNA
A. DNA (or RNA) aqueous solution of 50 mcg/mls is prepared.
B. the size of DNA or RNA fragments is depended on, prepares reaction mixture as described in table 1,
Table 1.DNA or RNA encapsulate solution
C. Loading sequence：First plus water, DNA (or RNA) and activated silica Nanoparticle Solution are followed by.Mix content Thing, reacts that (pH is 3 minutes at room temperature)。
D. 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
E. precipitation is suspended in 1 milliliter of water (shaking several seconds simultaneously water bath sonicator 2 minutes).
F. 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
G. precipitation is suspended in 0.5 milliliter of water (shaking several seconds simultaneously water bath sonicator 2 minutes), until mixture becomes uniform Suspension.Add 0.5 microlitre of 50 weight %TMAPS solution, several seconds of concussion mixing, the TEOS of 0.5 microlitre of addition.
H. when 900 rev/min of room temperature stirring mixture 4 is small, 4 microlitres of TEOS solution is then added, 900 turns of room temperature/ Stirring mixture per minute 4 days.
I. 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
J. precipitation is suspended in 0.5 milliliter of water (shaking several seconds simultaneously water bath sonicator 1 minutes).
K. repeat step 3.i and 3.j, at room temperature 21500g centrifugations 2 minutes.
L. precipitation is suspended in 0.1 milliliter of water (shaking several seconds simultaneously water bath sonicator 2 minutes).SiO2The DNA of encapsulation can be Room temperature lucifuge preserves for a long time.
4. form inhereditary material parcel particulate：Titanium dioxide encapsulated dna (or RNA)/SiO2Nanoparticle
A. by DNA/SiO in step 3.l2Nanoparticle suspensions are in 500 microlitres of ethanol.
B. 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
C. repeat step 4.a and 4.b be twice.
D. precipitation is suspended in 500 microlitres of second of 10 microlitres of water and 2.5 microlitres of TBOT (titanium (IV) butoxide, 95%) In alcohol.
E. 900 rev/min of room temperature stirring mixture is stayed overnight.
F. 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
G. 500 microlitres of ethanol are resuspended in.
H. repeat step 4.f and 4.g be three times.
I. it is resuspended in 1 milliliter of ethanol.Titanium dioxide encapsulated dna (or RNA)/SiO2Nanoparticle can be grown in room temperature Phase preserves, without lucifuge.The electron-microscope scanning figure of the DNA parcel particulates of formation is shown in Fig. 2.
5. forming inhereditary material preserves particulate：Gold parcel DNA (or RNA)/SiO2/TiO2Nanoparticle
A. by DNA (or RNA)/SiO in step 4.i2/TiO221500g is centrifuged 3 minutes and abandoned nanoparticle at room temperature Clearly.
B. 1 milliliter of water of precipitation suspension.
C. about 20 milligrams of DNA (or RNA)/SiO are taken2/TiO2Nanoparticle adds 0.20 milliliter of 25mM sodium citrate solutions Contain 2.5mM gold chlorides (HAuCl with 19.84), 900 rev/min of room temperature stirring mixture 10 minutes overnight.
D. 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
E. 1 milliliter of water of precipitation suspension.
F. repeat step 5.d and 5.e be three times.
G. 100 microlitres of water of precipitation suspension.
After silica, titanium dioxide and gold parcel, vacuum drying, forms inhereditary material and preserves particle, use h.DNA Preserved in long-term.The electron-microscope scanning figure that the DNA of formation preserves particulate is shown in Fig. 3.
6. inhereditary material preserves particulate release and purifying
A. DNA (or RNA)/SiO of 100 microlitres of step 6.g synthesis is taken2/TiO2/ Au nanoparticles, at room temperature 21500g Centrifuge 3 minutes and abandon supernatant.
B. KI/I is used2Solution opens golden shell.KI/I2It is prepared by solution：Weigh 4 grams of KI, 1 gram of I2, add 40 milliliters of water.
C. 300 microlitres of KI/I are added2Solution, jog move.
D. after gold disappears, 21500g is centrifuged 3 minutes and is abandoned supernatant at room temperature.
E. plus 500 microlitres of water are centrifuged, washed 3 times.
F. etching solution (NH is prepared4FHF/NH4F)：In polyethylene, in polypropylene or polytetrafluoroethylcontainer container, by 0.23 gram NH4FHF is dissolved in 5 milliliters of water, by 0.19 gram of NH in another container4F's is dissolved in 5 milliliters of water, and two solution then are mixed in one Rise.
G. the etching solution in 300 microlitres of 6.f is added.
H. after solution becomes clear, with commercial reagents (QIAquick PCR DNA purification kits, or RNA purification kits) Purify DNA (or RNA).
7. palliating degradation degree is tested after the inhereditary material release preserved
DNA will be prepared by step 1-5 and preserve particulate at room temperature, continuously shone by the ultraviolet that wavelength is 0.26 micron Penetrate 24 it is small when, common bronsted lowry acids and bases bronsted lowry processing and a variety of restriction enzymes repeatedly processing after, pass through the chemical method of experimental procedure 6 The DNA that DNA preservation particulates are dissolved and discharged, (i.e. same batch of before processing expands with not being wrapped the DNA encapsulated DNA), PCR amplification and gel electrophoresis, electrophoresis photographs such as Fig. 3 while are carried out again.For Fig. 3 since left, first (CBM3) is negative pair According to, illustrate no non-specific DNA, second be it is unencapsulated before full length DNA positive control, the 3rd is not plus DNA Negative control, the four to six road are that the DNA discharged by the use of after encapsulating is dense as substrate, specific amplification (PCR) segment, Substrate DNA Degree is respectively 100,50,25 nanograms.7th is positive control, by the use of encapsulation before DNA as substrate am-plified fragments.8th Road is negative control, and amplification condition is identical but is not added with DNA, and the 9th is DNA molecular standard sample.
The results show positive control, i.e. DNA preserve particle dissolution and the DNA discharged, the DNA length with not being wrapped encapsulation Unanimously.This contrast experiment illustrates that DNA preserves particulate after the processing such as irradiating by ultraviolet well, passes through the change of step 6 DNA can be preserved the DNA in particulate and intactly extracted by method, and the DNA that the method extracts does not change it Length and size.For inhereditary material after the preservation of the method for the present invention, almost shielding all may be to the structure of inhereditary material Physics, chemistry and the biological factor of damage are caused, is truly realized the long-term preservation of inhereditary material.
Comparative example
Inhereditary material with not including metal material layer in the prior art preserves the preservation effect contrast test of particulate
It is prepared by 1.DNA test specimens
The appropriate DNA of PCR amplification (genome or plasmid), and with commercial reagents (such as：QIAquick PCR DNA purifying examinations Agent box) purifying DNA, prepare label DNA nano particles according to the method for step 3 of the embodiment of the present invention, be divided into label 2,3,4, 5th, totally 20 samples of 6,7,8,9,10,11,13,14,15,16,17,18,19,20,21, No. 22, the DNA mass of each sample About 0.5 microgram.Thereafter, according to the method for the embodiment of the present invention, by 3,7,14, No. 18 sample packaging silica (SiO2), By 4,8,15, No. 19 sample packaging silica (SiO2)+titanium dioxide (TiO2), by 5,9,16,20,21, No. 22 sample packagings Silica (SiO2)+titanium dioxide (TiO2)+golden (Ag), the nano particle of 2,6,10,11,13, No. 17 samples are not added with any bag Dress.
2. irradiate DNA sample with ultraviolet
Use the ultraviolet light box irradiating samples of Fotodyne (254nm ultraviolet lights, 8W).In the ultraviolet light boxes of Fotodyne, shine Penetrate 2-5 samples 12 it is small when, when irradiation 6-9 samples 24 are small, when irradiation 13-16 samples 48 are small, irradiation 17-20 samples are about 72 it is small when.10-11 samples and 21-22 samples are without irradiation, as control.
3. the DNA in sample discharges and carries out detected through gel electrophoresis again
After the ultraviolet irradiation experiment of 17-20 samples is completed, the method for step 6 according to embodiments of the present invention, by institute There is the DNA in sample to discharge and purify again.Then it is divided to two groups of carry out gel electrophoresises (referring to Fig. 5), No. 1 sample is in first group DNA molecular amount standard, No. 12 samples are DNA molecular amount standard in second group.By gel electrophoresis it is concluded that：DNA receives Rice grain packs silica and titanium dioxide and cannot all bear to continue strong ultraviolet irradiation, and 12 can not just detect when small DNA.After its additional gold packaging, DNA at least can quite be stored several at room temperature when storage 48 is small under intense UV rays are irradiated 1000000 years, significantly extend the holding time of DNA.
Although it is to be understood that having combined preferable particular implementation of the invention, invention has been described, above Description and embodiment thereafter be intended to illustrative and not limiting the scope of the present invention.It will be recognized by one of ordinary skill in the art that In the case of without departing from the scope of the invention, various changes can be made and available equivalents replace, and it is in addition, it should also be understood that, other Aspect, advantage and modification will be apparent to those skilled in the art.Except reality as described herein Apply outside mode, the present invention covers and is claimed by the feature of invention cited herein and the cited prior art with reference to text Those inventions caused by the combination for the feature (it supplements the feature of the present invention) offered.Similarly, it is to be understood that any material Material, feature or article can be applied in combination with any other material, feature or article, and these combinations are considered as the present invention's In the range of.

Claims (10)

1. a kind of inhereditary material preserves particulate, it includes：
A) nanoparticle；
B) inhereditary material, it is adsorbed in the nanoparticle；
C) inhereditary material confining bed, the nanoparticle and inhereditary material are wrapped in interior by it；With
D) metal material layer, it is located at outside the inhereditary material confining bed.
2. inhereditary material according to claim 1 preserves particulate, it is characterised in that the inhereditary material for DNA and/or RNA。
3. inhereditary material according to claim 1 or 2 preserves particulate, it is characterised in that the nanoparticle is by SiO2Or choosing It is made from gold, silver, tungsten, copper metal or comprising its metal ion or its alloy material, the nanoparticle is preferably by SiO2It is made； The inhereditary material confining bed is SiO2Layer or TiO2Layer, is preferably TiO2Layer；The metal material layer is by metal chosen from the followings Or it is made comprising its metal ion or its alloy material：Gold, platinum, palladium, rhodium, osmium, silver, lead, mercury, polonium, ruthenium, technetium, copper, germanium, tungsten；It is excellent Choosing selected from gold, platinum, silver, copper or tungsten metal material by being made；Most preferably it is made of gold.
4. inhereditary material according to claim 1 or 2 preserves particulate, it is characterised in that the inhereditary material preserves particulate Particle diameter between 250-350nm, preferably between 280-320nm.
5. a kind of long-term preservation method of inhereditary material, it includes the following steps：
A) nanoparticle is prepared；
B) optionally, the nanoparticle is subjected to surface active；
C) inhereditary material is adsorbed onto in the nanoparticle；
D) inhereditary material confining bed is superscribed in the nano-particle surface, forms inhereditary material parcel particulate；
E) the further coated metal material layer of microparticle surfaces is wrapped up in the inhereditary material, forms inhereditary material and preserve particulate.
6. store method according to claim 5, it is characterised in that the inhereditary material is DNA or RNA.
7. the store method according to claim 5 or 6, it is characterised in that the surface active is surface roughening and band There is positive charge.
8. the store method according to claim 5 or 6, it is characterised in that the nanoparticle is by SiO2Or selected from gold, silver, Tungsten, copper metal are made comprising its metal ion or its alloy material, and the nanoparticle is preferably by SiO2It is made；
The inhereditary material confining bed is SiO2Layer or TiO2Layer, is preferably TiO2Layer；
The metal material layer is made of metal material chosen from the followings：Gold, platinum, palladium, rhodium, osmium, silver, lead, mercury, polonium, ruthenium, technetium, Copper, germanium, tungsten；It is preferred that it is made of the metal material selected from gold, platinum, silver, copper or tungsten；Most preferably it is made of gold.
9. store method according to claim 1, it is characterised in that it is further included releases from inhereditary material preservation particulate The step of putting the inhereditary material of preservation；
Preferably, the step of inhereditary material that the release preserves, includes：
F) KI/I is used2Solution opens the metal material layer；
G) etching solution NH is used4FHF/NH4F opens the inhereditary material confining bed and discharges the inhereditary material of preservation.
10. a kind of article for including inhereditary material, micro- it includes being preserved according to claim 1-4 any one of them inhereditary material Grain.
CN201711103478.7A 2017-11-10 2017-11-10 The preservation particulate and long-term preservation method of inhereditary material Pending CN107916263A (en)

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