CN100335648C - PCR reaction based on nano particles - Google Patents
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- CN100335648C CN100335648C CNB200410018098XA CN200410018098A CN100335648C CN 100335648 C CN100335648 C CN 100335648C CN B200410018098X A CNB200410018098X A CN B200410018098XA CN 200410018098 A CN200410018098 A CN 200410018098A CN 100335648 C CN100335648 C CN 100335648C
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Abstract
The present invention relates to a PCR primer based on nanometer particles. The structure of the PCR primer is Z-(X)n, wherein X represents a single-chain oligonucleotide primer whose length is from 10 to 100 bp, Z represents solid particles whose average particle diameter is from 1 to 100 nm, '-' represents a covalent bond between X and Z, and n is an integer of 1 to 1000. The present invention also relates to a method for preparing the PCR primer based on the nanometer particles and a polymerase chain reaction (PCR) based on the nanometer particles. The present invention can be used to simply, quickly and efficiently separate PCR amplification products and prepare single-chain DNA.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to a kind of polymerase chain reaction (PCR) technology based on nanoparticle.The invention still further relates to preparation based on the primer of nanoparticle PCR.
Background technology
Polymerase chain reaction (PCR) is extremely important a kind of technology in the technical field of molecular biology, it can quantity in the sample is few target sequence circulate by a plurality of " annealing-extensions-sex change " (being generally 20-50 circulation), make the quantity of target sequence present the growth of exponential form, final 1,000,000 orders of magnitude even the more target sequence of obtaining detects and operation thereby be convenient to use.
Yet, for the separation of pcr amplification product, all be to be undertaken at present basically by electrophoresis-rubber tapping, lack the method for simple and effective separation pcr amplification product.
As everyone knows, field of nanometer technology is the most popular current scientific and technical research field, nanotechnology is applied to formed emerge science technology-nanometer biotechnology in the bio-science field, be the science of utilizing nanotechnology research and solving the significant problem in the life science, it is just becoming one of current important advanced scientific research field.
Now, the application prospect of magnetic nano-particle in biological technical field is subjected to people's attention day by day.Magnetic nano-particle is through being often used as the separating medium of biological sample magnetic field separation.Separation method with magnetic nano-particle is easy and simple to handle, required equipment is cheap, and velocity of separation is fast simultaneously, helps keeping the biological activity of sample.At present, utilization more and more widely.
The polymer microsphere that magnetic Nano separator of the prior art adopts letex polymerization to obtain more has wherein coated the magnetic-particle of nano-scale.Yet the structure of polymer microsphere is more loose usually, and the magnetic particle that wherein comprises breaks away from microballoon easily and can produce agglomeration in long-time preservation process, thereby influences the performance of product; On magnetic nano-particle, fixedly during active substance, come off easily with absorption or affinity interaction.
Yet before the present invention, not with nanotechnology and PCR bonded report, and this area lacks the method for separating pcr amplification product simple and effectively.Therefore this area presses for the new technology of separating pcr amplification product of exploitation simple and effectively.
Summary of the invention
The purpose of this invention is to provide a kind of new-type round pcr based on nanoparticle.
Another object of the present invention provides a kind of technology of separating pcr amplification product simple and effectively.
In a first aspect of the present invention, a kind of PCR primer based on nanoparticle is provided, it has the following formula structure:
Z-(X)n (I)
Wherein X represents that length is 10-100bp single stranded oligonucleotide primer, and Z represents that median size is the solids of 1-100nm, and the covalent linkage between "-" expression X and the Z, n is the integer of 1-1000.
In another preference, described solids are golden nanometer particle or magnetic nano-particle.
In another preference, described PCR primer based on magnetic nano-particle has following structure:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle;
(2) the 3-sulfydryl propyl trimethoxy silicane middle layer of coating inner nuclear layer;
(3) the single stranded oligonucleotide primer outer shell in coating middle layer.
In another preference, described single stranded oligonucleotide primer outer shell is by crossing the single stranded DNA that sulfide linkage is connected with the middle layer.
In another preference, described nanoparticle is a magnetic nano-particle.
In another preference, core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell.
In another preference, the interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination.
In another preference, the interior nuclear composition of described inner nuclear layer is the oxide compound of iron.
In another preference, the outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination.
In another preference, the outer shell component of described inner nuclear layer is a silicon-dioxide.
In another preference, nanoparticle is a golden nanometer particle, can directly the single strain oligonucleotide primer be connected on the golden nanometer particle by the Au-S key based on the PCR primer of golden nanometer particle.
In a second aspect of the present invention, a kind of primer collection of polymerase chain reaction is provided, described primer collection comprises:
The Oligonucleolide primers that is used to cause the purpose nucleic acid amplification reaction is right, and wherein at least one primer is the described PCR primer based on nanoparticle of claim 1.
In another preference, the Oligonucleolide primers collection that is used to cause the purpose nucleic acid amplification reaction is 2 PCR primers based on nanoparticle.
In a third aspect of the present invention, a kind of polymerase chain reaction method is provided, it comprises step: under the condition that is fit to amplification, make contain template nucleic acid, primer to or the circulation of 20-50 the annealing-extension-sex change of reaction system experience of primer collection, dNTP, polysaccharase, thereby formation pcr amplification product
Described primer to or primer collection at least one primer is arranged is the described PCR primer based on nanoparticle of claim 1.
In another preference, described primer to or primer collection comprise 2 PCR primers based on nanoparticle.
In another preference, this method also comprises step: pcr amplification product is isolated pcr amplification product by centrifugal or the action of a magnetic field, and described pcr amplification product has nanoparticle.
In a fourth aspect of the present invention, the method for a kind of preparation based on the PCR primer of nanoparticle is provided, it may further comprise the steps:
(1) uses 3-sulfydryl propyl trimethoxy silicane that nanoparticle surface is modified, obtained modifying the magnetic nano-particle of sulfydryl;
(2) use modified the single stranded DNA primer of crossing sulfide linkage to the modification of step (1) surface of nanoparticle of sulfydryl react, form PCR primer based on nanoparticle.
Description of drawings
Fig. 1 has shown SiO
2The TEM figure of the magnetic nano-particle after the coating.
Fig. 2 has shown the TEM figure of the magnetic nano-particle behind the connection PCR product.
Fig. 3 has shown that under different annealing temperatures based on the electrophorogram of nanoparticle PCR product, wherein each swimming lane is as follows: 1: the molecular weight standard thing; 2:52 ℃; 3:56 ℃; 4:60 ℃; 5:65 ℃.
Fig. 1 is the surface-enhanced Raman effects figure of the magnetic nano-particle (FSM) of having modified sulfydryl, shows that sulfydryl is modified at the surface of magnetic nano-particle, and the sulfydryl that is modified at the magnetic nano particle sub-surface has good Raman reinforcing effect in the substrate of silver.
Fig. 2 is the surface-enhanced Raman effects figure of the magnetic nano-particle (FSMD) that connected single stranded DNA, shows that DNA is connected to the surface of magnetic nano-particle, and Raman reinforcing effect is preferably arranged.
Embodiment
The inventor finds that through after the extensive and deep research in order to separate pcr amplification product simple and effectively, only the isolation technique of improving after increasing is not enough, must just improve round pcr before the acquisition pcr amplification product.Before the present invention, this area it has been generally acknowledged that its motor capacity will decline to a great extent after short oligonucleotide fragment has connected solid particulate, therefore is not suitable as the primer of PCR.Yet the inventor but finds, the oligonucleotide fragment that links to each other with nano particle still can be effective as the primer of PCR reaction very much, and developed new PCR thus, thereby easy, the separation fast and effectively of pcr amplification product have been realized based on nanotechnology.
Nanoparticle
As used herein, " nano particle " is used interchangeably with " nanoparticle ", refers to that median size is of a size of the solid particulate of 1nm-100nm.
Can be used for nanoparticle of the present invention and be not particularly limited,, and can link to each other with oligonucleotide and get final product as long as its median size is of a size of 1nm-100nm.
The most frequently used nanoparticle comprises golden nanometer particle and magnetic nano-particle.
Nano level golden nanometer particle is commonly used in this area, and for example in the DNA blast technique, the nucleic acid molecule that will carry foreign gene exactly is connected on the nano level gold particle, bombards host cell then, thereby foreign gene is imported host cell.In the present invention, available this area ordinary method is coupled to the nano level gold particle with DNA.
Preferred nanoparticle is a magnetic nano-particle.The magnetic nano-particle general requirement that is used for biotechnology has following characteristic: (1) particle has superparamagnetism, is easy to absorption and wash-out; (2) particle needs the bigger specific magnetising moment, to guarantee the sensitivity of separation efficiency; (3) particle surface will be easy to carry out chemically modified, to be connected with drug molecule with different biologies; Require to have better biocompatibility when (4) being used in the body.Nanoparticle is generally sandwich structure, inner nuclear layer is generally magnetic Nano material, and the centre is a coating layer, and the existing products great majority adopt macromolecular material as coating layer at present, outermost layer is then modified the functional group that specific effect is arranged with biological tissue, to satisfy the requirement of bioseparation.The magnetic nano-particle that this area is used for isolating nucleic acid all can be used for the present invention.
A kind of particularly preferred magnetic nano-particle is to be disclosed magnetic nano-particle in CN 03142274.81 Chinese patent application (application on August 15th, 2003) of " magnetic nano-particle separate nucleic acid device and method for making thereof and application " in denomination of invention.In brief, this preferred magnetic nano-particle separate nucleic acid device contains following three-decker:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle;
(2) the 3-sulfydryl propyl trimethoxy silicane middle layer of coating inner nuclear layer;
(3) the single stranded oligonucleotide primer outer shell in coating middle layer.
In another preference, core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell.Wherein, the interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination, more preferably interior nuclear composition is the oxide compound of iron.The outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination, and more preferably the outer shell component of described inner nuclear layer is a silicon-dioxide.Described single stranded oligonucleotide primer outer shell is by crossing the single stranded DNA that sulfide linkage is connected with the middle layer.
The interior nuclear composition of core-shell type magnetic nano particle inner nuclear layer also has the alloy of nickel (Ni) or nickel (Ni) and iron (Fe) etc. except the oxide compound of iron.
The outer shell component of core-shell type magnetic nano particle inner nuclear layer also has organic integuments such as agarose, olefin polymer, polyacrylonitrile, epoxy compounds except inorganic integuments such as silicon-dioxide.For selected outer shell component, can select for use suitable inner nuclear layer shell to form agent according to prior art.For example when outer shell component is silicon-dioxide, can select for use tetraethoxy or other suitable inner nuclear layer shell to form agent.
Single stranded DNA as primer
The length that is applicable to single stranded DNA of the present invention (being primer) has no particular limits, can be identical with the PCR primer of routine.Usually mean length is about 10～100bp, preferably is about 15～50bp.
PCR primer based on nanoparticle
Compare with the magnetic nano-particle separate nucleic acid device described in CN 03142274.81 Chinese patent application, the single stranded DNA mixture of replacing in the single stranded oligonucleotide primer outer shell with the PCR strand primer of homogeneous just can make the PCR primer based on nanoparticle of the present invention.
In a preference, the preparation method of the PCR primer based on nanoparticle of the present invention may further comprise the steps:
(1) preparation magnetic nano-particle
Use redistilled water H
2O prepares FeSO respectively
47H
2O and FeCl
36H
2The mixing solutions of O and NaOH solution.Fe in the mixing solutions of molysite
2+Ionic concentration is 0.1～0.2mol/l, Fe
3+Ionic concentration is 0.1～0.3mol/l, and the concentration of NaOH solution is 2～3mol/l.Under vigorous stirring be that half NaOH solution of mixing salt solution volume is added drop-wise in the mixing salt solution lentamente with volume.At 40 ℃～60 ℃ following ageing 12h, with redistilled water with sediment undergoes washing for several times, dry 24h under 40 ℃～80 ℃ condition again after the filtration promptly gets product after grinding in agate mortar with resulting solid precipitation.
(2) silicon-dioxide is at γ-Fe
2O
3The modification on surface
With TritonX-100, n-hexyl alcohol, hexanaphthene in 1: (1～3): the ratio uniform mixing of (4～6) forms the microemulsion system of transparent and stable.Place ultrasonic wave to handle 30-60 minute above-mentioned microemulsion system, again to the γ-Fe that wherein adds 0.01～1g
2O
3(magnetic nano-particle) takes out upper strata liquid with ultrasonication and pours in the three-necked flask after 3 minutes, stir and made it even in 30～60 minutes.Get the certain density strong aqua of 1ml with the dilution of 2ml redistilled water, it is slowly joined in the microemulsion of continuous stirring, continue stirring and ammoniacal liquor was dispersed in the microemulsion in 10～60 minutes.After 1 hour, in microemulsion, drip the tetraethoxy (the inner nuclear layer shell forms agent) of 1～5ml, constantly stirred 10 hours simultaneously, and the temperature of system is remained between 15～40 ℃.In system, add acetone and make particle precipitation, perhaps the system standing over night is made the particle natural sedimentation, use the ethanol wash particle.Particle after cleaning placed under 300～700 ℃ the condition calcination 1～4 hour, and collected the core-shell type magnetic nano particle.
(3) with 3-sulfydryl propyl trimethoxy silicane (MPTS) modified magnetic nano particles surface
Get in the acetate and alcoholic acid mixed solution of 15mg magnetic nano-particle adding 2～10ml, use ultrasonication 20～60 minutes; Take by weighing 0.01～1g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～40 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100-300 ℃ of vacuum-drying 2 hours, collects particle (FSM).
(4) single stranded DNA is in the modification of magnetic nano particle sub-surface
Get a certain amount of single stranded DNA primer of crossing sulfide linkage of having modified, join the NaHCO of 500 μ l
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and in a humid environment in reacting 12～36 hours (sulfydryl of crossing on sulfide linkage and the middle layer on the DNA reacts, and makes dna molecular be directly connected in the middle layer by crossing sulfide linkage) under 10～50 ℃ the condition.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain PCR primer based on nanoparticle.
Usually, in the PCR primer complex based on nanoparticle of the present invention, the mol ratio of primer and nanoparticle is generally 1～100: 1, and more preferably be 1～20: 1.In addition, the weight ratio of primer and nanoparticle is generally 1～10: 1, and more preferably be 1～4: 1.
PCR reaction based on nanoparticle
The PCR reaction of using the above-mentioned PCR primer based on nanoparticle of the present invention to be carried out just is based on the PCR reaction of nanoparticle.Wherein, when using two or more primers to carry out the PCR reaction, can only use a kind of PCR primer based on nanoparticle of the present invention, also can use the PCR primer based on nanoparticle of the present invention of two or more, even all primers all are the PCR primers based on nanoparticle of the present invention in the PCR reaction system.
The PCR reaction that the present invention is based on nanoparticle comprises the existing various PCR reaction formations in this area, for example Chang Gui two primer PCRs, nest-type PRC, RT-PCR etc.
The condition that the present invention is based on the PCR reaction of nanoparticle is not particularly limited, can be identical with existing P CR reaction conditions, for example anneal, extension and denaturation temperature conditions such as the ionic strength of reaction system.In brief, the PCR reaction that the present invention is based on nanoparticle can be considered as conventional PCR reaction, unique difference only is that the primer that uses is the primer that is connected with nanoparticle.
After having carried out the PCR reaction, can produce the amplified production that carries nanoparticle.Utilize these nanoparticles that carry just can isolate amplified production easily.For example, when nanoparticle is gold grain, can utilize supercentrifugal process to separate amplified production.When nanoparticle is the magnetic nano particle period of the day from 11 p.m. to 1 a.m, can utilize the magnetic field separation amplified production.
These isolating pcr amplification products can be used for various uses more easily, for example dna molecular operation, dna probe, preparation DNA chip and gene therapy etc.
Major advantage of the present invention is:
(1) compare with conventional P CR, the present invention not only can carry out PCR, and can be extremely easy, fast with separate pcr amplification product effectively; Can make single stranded DNA easily.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The preparation method of the inner nuclear layer of magnetic nano-particle
Adopt improved chemical coprecipitation to prepare the inner nuclear layer of magnetic particle, concrete grammar is as follows: prepare FeSO respectively with redistilled water
47H
2O and FeCl
36H
2The mixing solutions of O and NaOH solution.Fe in the mixing solutions of molysite
2+Ionic concentration is 0.1～0.2mol/l, Fe
3+Ionic concentration is 0.1～0.3mol/l, and the concentration of NaOH solution is 2～3mol/l.Under vigorous stirring be that half NaOH solution of mixing salt solution volume is added drop-wise in the mixing salt solution lentamente with volume.At 40 ℃～60 ℃ ageing 12h, with redistilled water with sediment undergoes washing for several times, dry 24h under 40 ℃～80 ℃ condition again after the filtration promptly gets product after grinding in agate mortar with resulting solid precipitation.
Embodiment 2
The preparation method of hud typed nucleic acid magnetic nano-particle
With TritonX-100, n-hexyl alcohol, hexanaphthene ratio uniform mixing, form the microemulsion system of transparent and stable in 1: 2: 5.Place ultrasonic wave to handle 30～60 minutes above-mentioned microemulsion system, again to the γ-Fe that wherein adds 0.5g
2O
3, take out upper strata liquid after 6 minutes with ultrasonication and pour in the three-necked flask, stir and made it even in 30 minutes.Get the certain density strong aqua of 1ml with the dilution of 2ml redistilled water, after 30 minutes it is slowly joined in the microemulsion of continuous stirring, continue stirring and ammoniacal liquor was dispersed in the microemulsion in 30 minutes.After 1 hour, in microemulsion, drip the tetraethoxy of 1～3ml, constantly stirred 10 hours simultaneously, and the temperature of system is remained between 15～30 ℃.In system, add acetone and make particle precipitation, perhaps the system standing over night is made the particle natural sedimentation, use the ethanol wash particle.Particle after cleaning placed under 400～700 ℃ the condition, particle is collected in calcination 1～4 hour.
Detect through transmission electron microscope (TEM), its median size is approximately 50nm.
Embodiment 3
Preparation based on the PCR primer of nanoparticle
Get the magnetic nano-particle that makes among the 15mg embodiment 2 and add in the acetate and alcoholic acid mixed solution of 2ml, use ultrasonication 20～60 minutes; Take by weighing 0.01g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～30 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100～300 ℃ of vacuum-dryings 2 hours, collects particle (FSM).Show that with the Raman spectrum detection sulfydryl is modified the surface of magnetic nano-particle.
Get modifying of a certain amount of (concentration being 76 μ M) respectively and crossed the single stranded DNA primer of sulfide linkage.Sequence is as follows:
(1)HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5′-TATTGGGCGCTCTTCCGCTTCCT(5′-3′)，
(2)HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5′-TCTTTATAGTCCTGTCGGGTTTCG(5′-3′)，
(3)HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5′-GGGATAA?CGCAGGAAAG(5′-3′)
(4) HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5 '-AGG GT CGGAACAGGAG (5 '-3 ')), join the NaHCO of equivalent
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and reaction 24 hours under 20～40 ℃ condition in a humid environment.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain PCR primer A based on nanoparticle.
Show that with the Raman spectrum detection dna primer has been connected to the magnetic nano particle sub-surface.
Embodiment 4
Preparation based on the PCR primer of nanoparticle
Get the magnetic nano-particle that 15mg embodiment 2 makes and add in the acetate and alcoholic acid mixed solution of 6ml, with ultrasonication 20-60 minute; Take by weighing 0.5g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～30 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100～300 ℃ of vacuum-dryings 2 hours, collects particle (FSM).
Get modifying of a certain amount of (concentration being 76 μ M) respectively and crossed the single stranded DNA primer of sulfide linkage.Sequence is as follows:
(1)HO-(CH
2)
6-S-S-(CH
2)
10-O-(PO
3)-5′-TATTGGGCGCTCTTCCGCTTCC(5′-3′)，
(2)HO-(CH
2)
6-S-S-(CH
2)
10-O-(PO
3)-5′-TCTTTATAGTCCTGTCGGGTTTC(5′-3′)，
(3)HO-(CH
2)
6-S-S-(CH
2)
10-O-(PO
3)-5′-GGG?ATAACG?CAGGAAA(5′-3′)，
(4) HO-(CH
2)
6-S-S-(CH
2)
10-O-(PO
3)-5 '-AG GGTCGG AACAGGA (5 '-3 '), join the NaHCO of equivalent
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and reaction 24 hours under 20～40 ℃ condition in a humid environment.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain PCR primer B based on nanoparticle.
Embodiment 5
Preparation based on the PCR primer of nanoparticle
Get the magnetic nano-particle that 15mg embodiment 2 makes and add in the acetate and alcoholic acid mixed solution of 10ml, with ultrasonication 20～60 minutes; Take by weighing 1g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～30 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100～300 ℃ of vacuum-dryings 2 hours, collects particle (FSM).
Get modifying of a certain amount of (concentration being 76 μ M) respectively and crossed the single stranded DNA primer of sulfide linkage.Sequence is as follows:
(1)HO-(CH
2)
6-S-S-(CH
2)
8-O-(PO
3)-5′-ATTGGGCGCTCTTCCGCTTCCTC(5′-3′)，
(2)HO-(CH
2)
6-S-S-(CH
2)
8-O-(PO
3)-5′-CTTTATAGTCCTGTCGGGTTTCGC(5′-3′)，
(3)HO-(CH
2)
6-S-S-(CH
2)
8-O-(PO
3)-5′-GG?ATAACGCAGGA?AAGA(5′-3′)，
(4)HO-(CH
2)
6-S-S-(CH
2)
8-O-(PO
3)-5′-G?GGTCGGAACAG?GAGA(5′-3′)
Join the NaHCO of equivalent
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and reaction 24 hours under 20～40 ℃ condition in a humid environment.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain PCR primer C based on nanoparticle.
Embodiment 6
Preparation based on the PCR primer of nanoparticle
The surface of getting a certain amount of (40 μ g) respectively is connected with the magnetic nano-particle of sulfhydryl-group activity, adds the Na of a certain amount of (40 μ l)
2CO
3/ NaHCO
3Damping fluid (pH=9.0), ultra-sonic dispersion is even.
Take redistilled water and be diluted to the primer of 76 μ M.Sequence is as follows:
(1)HO-(CH
2)
8-S-S-(CH
2)
6-O-(PO
3)-5′-ATTGGG?CGCTCTTCCGCT-TCCTC(5′-3′)，
(2)HO-(CH
2)
8-S-S-(CH
2)
6-O-(PO
3)-5′-CTTTATAGTC-CTGTCGGGTTTCGC(5′-3′)，
(3)HO-(CH
2)
8-S-S-(CH
2)
6-O-(PO
3)-5′-GG?ATAACGCAGGAAAGA(5′-3′)
(4)HO-(CH
2)
8-S-S-(CH
2)
6-O-(PO
3)-5′-G?GGTCGGAACAGGAGA(5′-3′)
Get 20 μ l primers,, mix to wherein adding the above-mentioned damping fluid of 20 μ l, under the situation of room temperature, in the malaria, reaction 24h.After reaction finishes, with 4000 rev/mins speed centrifugation, abandoning supernatant; After redistilled water washing and precipitating at least twice, to separate with same rotating speed, abandoning supernatant keeps particle, obtains the PCR primer D based on nanoparticle.
Embodiment 7
PCR reaction based on nanoparticle
In the present embodiment, use the primer A-D of embodiment 3-6 preparation to carry out polymerase chain reaction.Condition is as follows:
(1) temperature of reaction and time
I) 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 50s; Annealing temperature is 52 ℃, and the time is made as 50s; 72 ℃ are extended 30s; After reaction finished, 72 ℃ were extended 4min again; 45 circulations.
Ii) 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 50s; Annealing temperature is 56 ℃, and the time is made as 50s; 72 ℃ are extended 30s; After reaction finished, 72 ℃ were extended 4min again; 45 circulations.
Iii) 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 50s; Annealing temperature is 60 ℃, and the time is made as 50s; 72 ℃ are extended 30s; After reaction finished, 72 ℃ were extended 4min again; 45 circulations.
Iv) 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 50s; Annealing temperature is 65 ℃, and the time is made as 50s; 72 ℃ are extended 30s; After reaction finished, 72 ℃ were extended 4min again; 45 circulations.
(2) consumption of reactant
Article two, primer all connects the consumption of the reactant of the magnetic nano particle period of the day from 11 p.m. to 1 a.m.30 μ l systems are selected in this experiment for use.Redistilled water 19 μ l, standard buffer solution 3 μ l, the dNTP solution 2 μ l of normal concentration, the upstream and downstream primer (embodiment 3-6 preparation) that is connected with magnetic nano-particle is respectively got 2 μ l, and the template PSK that the original concentration dilution is 100 times is 1 μ l, Taq enzyme 1 μ l.
Article one, primer all connects the consumption of the reactant of the magnetic nano particle period of the day from 11 p.m. to 1 a.m.30 μ l systems are selected in this experiment for use.Redistilled water 20.75 μ l, standard buffer solution 3 μ l, the dNTP solution 2 μ l of normal concentration, the primer that is connected with magnetic nano-particle is got 2 μ l, the primer that does not connect magnetic nano-particle is got 0.25 μ l, and the template PSK that the original concentration dilution is 100 times is 1 μ l, Taq enzyme 1 μ l.
Result: with transmission electron microscope (TEM) nanoparticle that connects the nanoparticle before the PCR primer and carried out after the PCR reaction is taken pictures, the results are shown in Figure 1 and Fig. 2.Therefrom as can be seen, SiO
2The edge of the magnetic nano-particle after the coating (not connecting primer) is very smooth.And after the PCR reaction, then the edge is very fuzzy to have connected magnetic nano-particle behind the PCR product, and some reunions are arranged.
Embodiment 8
The separation of PCR product
(a) PCR product and magnetic nano-particle separates
To the speed centrifugation (annotating: also can additionally apply magnetic field) of the PCR reaction product of embodiment 7 with 4000 rev/mins, take out magnetic particle, use the second distillation water-dispersion, to wherein adding a certain amount of mercaptoethanol, make its final concentration reach 3%, under 37 ℃ condition, more than the reaction 8h.
(b) agarose electrophoresis detects
With the centrifugation under 4000 rev/mins speed of above-mentioned reacted solution, get supernatant liquor and carry out agarose gel electrophoresis.
The result shows at 3 kinds of reaction conditionss (reaction conditions i among the embodiment 7), ii) and iii) as shown in Figure 3) under, all can carry out PCR.
Embodiment 9
PCR reaction based on nanoparticle
Repeat among the embodiment 7 reaction conditions ii), difference only is with a conventional primer (0.25 μ l) and two original primers based on nanoparticle of a primer based on nanoparticle (2 μ l) replacement.
The result shows, has obtained to be connected with the pcr amplification product of nanoparticle equally.
Under the prerequisite that does not depart from the spirit and scope of the invention, those skilled in the art can carry out various changes or modification to the present invention, and these forms of equal value drop in the application's appended claims institute restricted portion equally.
Claims (8)
1. polymerase chain reaction method, it comprises step: under the condition that is fit to amplification, make contain template nucleic acid, primer to or the circulation of 20-50 the annealing-extension-sex change of reaction system experience of primer collection, dNTP, polysaccharase, thereby the formation pcr amplification product, it is characterized in that
Described primer to or primer collection at least one primer is arranged is the PCR primer based on nanoparticle with following formula structure:
Z-(X)n (I)
Wherein X represents that length is 10-100bp single stranded oligonucleotide primer, and Z represents that median size is the solids of 1-100nm, and the covalent linkage between "-" expression X and the Z, n is the integer of 1-1000;
And described method also comprises step:
Pcr amplification product is isolated pcr amplification product by centrifugal or the action of a magnetic field, and described pcr amplification product has nanoparticle.
2. the method for claim 1 is characterized in that, described primer to or primer collection comprise 2 PCR primers based on nanoparticle.
3. the method for claim 1 is characterized in that, X represents that length is 15-50bp single stranded oligonucleotide primer.
4. the method for claim 1 is characterized in that, described solids are golden nanometer particle or magnetic nano-particle.
5. the method for claim 1 is characterized in that, described PCR primer based on magnetic nano-particle has following structure:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle;
(2) the 3-sulfydryl propyl trimethoxy silicane middle layer of coating inner nuclear layer;
(3) the single stranded oligonucleotide primer outer shell in coating middle layer.
6. method according to claim 5 is characterized in that, described single stranded oligonucleotide primer outer shell is by crossing the single stranded DNA that sulfide linkage is connected with the middle layer.
7. the method for claim 1 is characterized in that, described PCR primer based on nanoparticle is to prepare with the method that may further comprise the steps:
(a) use 3-sulfydryl propyl trimethoxy silicane that nanoparticle surface is modified, obtained modifying the magnetic nano-particle of sulfydryl;
(b) use modified the single stranded DNA primer of crossing sulfide linkage to the modification of step (a) surface of nanoparticle of sulfydryl react, form PCR primer based on nanoparticle.
8. method as claimed in claim 7 is characterized in that, the sequence of the single stranded DNA primer described in the step (b) is as follows:
HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5′-TATTGGGCGCTCTTCCGCTT?CCT-3′)；
HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5′-TCTTTATAGTCCTG?TCGGGTTTCG-3′；
HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5 '-GGGATAA CGCAGGAAAG-3 '; Or
HO-(CH
2)
6-S-S-(CH
2)
6-O-(PO
3)-5′-AGG?GT?CGGAACAGGAG(5′-3′)。
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| CN101153308B (en) * | 2006-09-28 | 2011-11-02 | 天津科技大学 | Optimization method of nucleic acid polymerase chain reaction amplification based on nano metal alloy |
| CN103993005B (en) * | 2014-04-18 | 2016-09-14 | 山东省农业科学院植物保护研究所 | The application in polymerase chain reaction of the sulfhydrylation single stranded DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20020106686A1 (en) * | 2001-01-09 | 2002-08-08 | Mckernan Kevin J. | Methods and reagents for the isolation of nucleic acids |
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| US20020106686A1 (en) * | 2001-01-09 | 2002-08-08 | Mckernan Kevin J. | Methods and reagents for the isolation of nucleic acids |
Non-Patent Citations (2)
| Title |
|---|
| DNA在磁性纳米粒子表面的键合及表面增强拉曼光谱研究 沈鹤柏，汪友宝，杨海峰，姜继森，科学通报，第48卷第21期 2003 * |
| 磁性微粒在核酸研究中的应用 崔亚丽等，西北农业大学学报，第28卷第1期 2000 * |
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