CN100475961C - Asymmetric polymerase chain reaction technology based on nano particles - Google Patents
Asymmetric polymerase chain reaction technology based on nano particles Download PDFInfo
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- CN100475961C CN100475961C CNB2004100528080A CN200410052808A CN100475961C CN 100475961 C CN100475961 C CN 100475961C CN B2004100528080 A CNB2004100528080 A CN B2004100528080A CN 200410052808 A CN200410052808 A CN 200410052808A CN 100475961 C CN100475961 C CN 100475961C
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Abstract
The present invention relates to one kind of asymmetrical PCR technology based on nanometer particles. The nonrestriction primer adopted with PCR primer based on nanometer particle in the structure of Z-(X)n, where X expresses single stranded oligonucleotide primer of 10-100 bp length, Z expresses solid particles with average size of 1-100 nm, '-' expresses the covalent bond between X and Z, and n is integral number of 1-1000. The present invention also relates to PCR based on nanometer particles. The present invention makes it possible to prepare and separate single stranded DNA proliferation product simply, fast and effectively.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to a kind of asymmetric polymerase chain reaction (PCR) technology based on nanoparticle.The invention still further relates to the preparation of the primer collection that is used for this asymmetric 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 one of current the most popular 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 magnetic field separation of biological sample.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.
As everyone knows, when PCR reacts, if the molar concentration rate of two primers is, can obtain ssDNA at 1: 10 to 1: 500 o'clock behind PCR, this technology is referred to as asymmetric PCR.Its principle is: in the process of PCR, after restricted primer (being the primer of comparatively small amt) ran out of less because of amount, non-limiting primer (being a fairly large number of primer) can continue amplification, thereby produces a large amount of ssDNA.Single stranded DNA has a wide range of applications, for example as probe.Especially along with the development of DNA chip technology, this area needs a large amount of highly purified single strand dnas.
Yet before the present invention, not with nanotechnology and asymmetric PCR bonded report, and this area shortage prepares and the method for separating single stranded DNA simple and effectively.Therefore this area presses for the new technology of separating the single stranded DNA amplified production of exploitation simple and effectively.
Summary of the invention
The purpose of this invention is to provide a kind of new-type asymmetric PCR technology based on nanoparticle.
Another object of the present invention provides a kind of the preparation and the technology of separating the single stranded DNA amplified production simple and effectively.
In a first aspect of the present invention, a kind of primer collection that is used for asymmetric polymerase chain reaction is provided, described primer collection comprises:
The single stranded oligonucleotide primer that is used to cause the purpose nucleic acid amplification reaction is right, a right primer of described primer is restricted primer, this restricted primer is that length is the single stranded oligonucleotide of 10-100 base, and another primer is the PCR primer that is based on nanoparticle, and described PCR primer based on nanoparticle has the following formula structure:
Z-(X)n (I)
Wherein X represents that length is the single stranded oligonucleotide primer of 10-100 base, 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 restricted primer is 1: 10～1: 500 with mol ratio based on the PCR primer of nanoparticle.
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 single stranded oligonucleotide primer outer shell that on the core-shell type magnetic nano particle, connects.
In another preference, described single stranded oligonucleotide primer outer shell is by crossing the single stranded DNA that sulfide linkage is connected with inner nuclear layer.
In another preference, described solids are golden nanometer particles, and the single stranded oligonucleotide primer directly is connected on the golden nanometer particle by the Au-S key.
In another preference, described restricted primer is 1: 10～1: 500 with mol ratio based on the PCR primer of nanoparticle.
In another preference, X represents that length is the single stranded oligonucleotide primer of 10-100 base.
In a second aspect of the present invention, a kind of asymmetric polymerase chain reaction method is provided, and it comprises step: under the condition that is fit to amplification, make the circulation of 20-50 the annealing-extension-sex change of reaction system experience that contains template nucleic acid, primer collection, dNTP, polysaccharase, thereby formation pcr amplification product
Wherein said primer collection is the primer collection that is used for asymmetric polymerase chain reaction of the present invention.
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 third aspect of the present invention, a kind of purposes of the PCR primer based on nanoparticle is provided, described PCR primer based on nanoparticle has the following formula structure:
Z-(X)n (I)
Wherein X represents that length is the single stranded oligonucleotide primer of 10-100 base, 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, described primer is used to prepare the single-chain nucleic acid product by asymmetric PCR.
In another preference, described single-chain nucleic acid product is used to make DNA chip and dna probe.
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. 4 has shown under the different mol ratio situation of restricted primer and nanometer primer, the electrophorogram of asymmetric PCR product, and wherein each swimming lane is as follows: the 1st, the molecular weight standard thing; A is 1: 1000, and b is 1: 500, and c is 1: 100, and d is 1: 10.
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 very much, and developed new PCR thus, thereby easy, the separation fast and effectively of pcr amplification product have been realized based on nanotechnology.In addition, the inventor also is used for asymmetric PCR with the primer based on nanoparticle of the present invention, thereby has developed preparation " based on the asymmetric PCR technology of nanoparticle " all extremely easy with separating single stranded DNA first.
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 10～100 bases, preferably is about 15～50 bases.
PCR primer (abbreviating " nanometer primer " as) 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 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, extend and hand over degree warm in nature, 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.
Asymmetric PCR reaction based on nanoparticle
The asymmetric PCR reaction is basic identical with conventional PCR reaction, and unique key distinction is: the comparatively small amt of a primer in the asymmetric PCR, be called as restricted primer, and the quantity of another primer is more, is called as non-limiting primer.Usually, the mol ratio of restricted primer and non-limiting primer is 1: 10 to 1: 500.
Compare with existing asymmetric PCR technology, in the asymmetric PCR reaction of the present invention, unique key distinction is: non-limiting primer must be the primer based on nanoparticle of the present invention.For the rest, as in many conditions such as polysaccharase, dNTP concentration and the prior art and indistinction.
In the present invention, non-limiting primer is covalently bound on the nanoparticle, pass through asymmetric PCR, can obtain to be connected with in a large number the ssDNA of nanoparticle, the existence of nanoparticle will help the separation and the purifying of ssDNA product, and is more simpler than traditional gel electrophoresis or anion-exchange chromatography partition method.The ssDNA that is connected with nanoparticle that obtains can be used to prepare the nucleic acid probe of specific gene, directly carries out the sequential analysis of this gene fragment.The existence of nanoparticle also helps the recovery and reuse of probe.
Major advantage of the present invention is:
(1) compare with conventional P CR, the present invention not only can carry out asymmetric PCR, and can be extremely easy, fast with separate single stranded DNA effectively.
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′-TCTTTATAGTCCTG?CGGGTTTCG(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′-TCTTTATAGTCCTGTCGGTTTC(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.
Embodiment 10
Asymmetric PCR reaction based on nanoparticle
In the present embodiment, with prepare among the embodiment of the invention 3-6 based on any one in the PCR primer of nanoparticle (shown in Figure 4 is the experimental result of the primer of embodiment 3 preparations) as non-limiting primer, will with to another conventional primer in the primer as restricted primer, be that 1 μ l is a template with the template PSK of 100 times of original concentration dilutions
(1) reaction consumption:
A. redistilled water 19.9 μ l, standard buffer solution (500mmol/L KCl; 100mmol/LTris-HCl (pH=8.3); 15mol/L MgCl
20.1% gelatin) 3 μ l, the dNTP solution 2 μ l of 2mol/L are connected with a primer 2 0pmol of magnetic nano-particle, do not connect the restricted primer 0.02pmol of particle, PSK template 1 μ l, Taq enzyme 1 μ l, cumulative volume are 30 μ l.(restricted primer: the mol ratio based on the PCR primer of nanoparticle is 1: 1000)
B. redistilled water 19.85 μ l, standard buffer solution 3 μ l, the dNTP solution 2 μ l of 2mol/L, be connected with a primer 2 0pmol of magnetic nano-particle, the restricted primer 0.04pmol that does not connect particle, PSK template 1 μ l, Taq enzyme 1 μ l, cumulative volume are 30 μ l (restricted primer: the mol ratio based on the PCR primer of nanoparticle is 1: 500).
C. redistilled water 19.8 μ l, standard buffer solution 3 μ l, the dNTP solution 2 μ l of 2mol/L, be connected with a primer 2 0pmol of magnetic nano-particle, the restricted primer 0.2pmol that does not connect particle, PSK template 1 μ l, Taq enzyme 1 μ l, cumulative volume are 30 μ l (restricted primer: the mol ratio based on the PCR primer of nanoparticle is 1: 100).
D. redistilled water 19.75 μ l, standard buffer solution 3 μ l, the dNTP solution 2 μ l of normal concentration, be connected with a primer 2 0pmol of magnetic nano-particle, the restricted primer 2 pmol that does not connect particle, PSK template 1 μ l, Taq enzyme 1 μ l, cumulative volume are 30 μ l (restricted primer: the mol ratio based on the PCR primer of nanoparticle is 1: 10).
(2) PCR reaction conditions
94 ℃ of pre-sex change 1-5min; 94 ℃ of sex change 20-80s, annealing temperature is 58-68 ℃, and the time is 20-80s, and 72 ℃ are extended 20-80s, 20-50 circulation; 72 ℃ are extended 2-6min.
(3) detection of PCR product
According to embodiment 8 the PCR product being separated the back with magnetic nano-particle detects with 3% agarose gel electrophoresis, the result shows, when restricted primer: the mol ratio based on the PCR primer of nanoparticle is 1: 500-1: during 10 scopes, can see the double-stranded and strand (as shown in Figure 4) of target simultaneously on gel electrophoresis figure.
(4) separation of single stranded DNA amplified production
With 95 ℃ of sex change 10min of amplified production, the speed centrifugation of 4000 commentaries on classics/min (annotate: also can additionally apply magnetic field) is removed supernatant liquid and eccentric cleaning repeatedly, thereby is obtained single stranded DNA.This DNA is used as probe and is fixed on the DNA chip.
Embodiment 11
Asymmetric PCR reaction based on nanoparticle
Repeat the process of embodiment 10, difference is to replace magnetic nano-particle with the golden nanometer particle of the about 70nm of median size, and only passes through 5000 commentaries on classics/min centrifugations when separating the single stranded DNA amplified production.
The result shows, when restricted primer: the mol ratio based on the PCR primer of nanoparticle is 1: 500-1: during 10 scopes, can obtain the target strand.The DNA that obtains after the centrifugation is used as probe and is fixed on the DNA chip
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 (10)
1. a primer collection that is used for asymmetric polymerase chain reaction is characterized in that, described primer collection comprises:
The single stranded oligonucleotide primer that is used to cause the purpose nucleic acid amplification reaction is right, a right primer of described primer is restricted primer, this restricted primer is that length is the single stranded oligonucleotide of 10-100 base, and another primer is the PCR primer that is based on nanoparticle, and described PCR primer based on nanoparticle has the following formula structure:
Z-(X)n (I)
Wherein X represents that length is the single stranded oligonucleotide primer of 10-100 base, 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 restricted primer is 1: 10～1: 500 with mol ratio based on the PCR primer of nanoparticle,
Wherein, described solids are golden nanometer particle or magnetic nano-particle.
2. primer collection as claimed in claim 1 is characterized in that described solids are golden nanometer particles.
3. primer collection as claimed in 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 single stranded oligonucleotide primer outer shell that on the core-shell type magnetic nano particle, connects.
4. primer collection according to claim 3 is characterized in that, described single stranded oligonucleotide primer outer shell is by crossing the single stranded DNA that sulfide linkage is connected with inner nuclear layer.
5. primer collection as claimed in claim 1 is characterized in that, the length of described restricted primer is the 15-50 base.
6. primer collection as claimed in claim 1 is characterized in that, X represents that length is the single stranded oligonucleotide primer of 15-50 base.
7. asymmetric polymerase chain reaction method, it comprises step: under the condition that is fit to amplification, make the circulation of 20-50 the annealing-extension-sex change of reaction system experience that contains template nucleic acid, primer collection, dNTP, polysaccharase, thereby form pcr amplification product, it is characterized in that
Described primer collection is the described primer collection of claim 1.
8. method as claimed in claim 7 is characterized in that, 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.
9. purposes based on the PCR primer of nanoparticle, described PCR primer based on nanoparticle has the following formula structure:
Z-(X)n (I)
Wherein X represents that length is the single stranded oligonucleotide primer of 10-100 base, 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, it is characterized in that described primer is used to prepare the single-chain nucleic acid product by asymmetric PCR.
10. as purposes as described in the claim 9, it is characterized in that described single-chain nucleic acid product is used to make DNA chip and dna probe.
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| DNA在磁性纳米粒子表面的键合及表面增强拉曼光谱研究. 沈鹤柏等.科学通报，第2003,48(21)期. 2003 |
| DNA在磁性纳米粒子表面的键合及表面增强拉曼光谱研究. 沈鹤柏等.科学通报，第2003,48(21)期. 2003 * |
| 不对称PCR制备单链探针检测登革Ⅱ型病毒复制型RNA和复制中间体RNA. 郭晓霞等.寄生虫与医学昆虫学报，第2003,10(3)期. 2003 |
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