CN103276074B - A kind of method of synthesizing short single strand deoxyribonucleotide probe - Google Patents
A kind of method of synthesizing short single strand deoxyribonucleotide probe Download PDFInfo
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Abstract
The invention belongs to DNA biosynthesizing field, be specifically related to a kind of short single strand deoxyribonucleotide probe method of synthesis and application thereof, can be used for detecting little non-encoding ribonucleic acid, as micro ribonucleic acid, comprise the steps: that (1) preparation restructuring has the plasmid of template DNA; (2) cut and connect template DNA, from recombinant plasmid, enzyme is cut and is obtained template DNA, makes template DNA head and the tail connect into ring; (3) cut strand, rolling-circle replication, cut off a chain of template DNA with nickase, add polysaccharase and carry out rolling-circle replication with the nicked DNA of ring-type for template, amplify the DNA single chain containing neck ring-probe structure; (4) preparation of the short single stranded DNA of object, adopts II type restriction endonuclease cutting to form object ssDNA probe and by product DNA to the product of step (3), obtains the ssDNA probe without sudden change by PAGE separation and purification.The present invention has extensive use in the Diagnosis and Treat field of little non-coding RNA detection field and little non-coding RNA relative disease.
Description
Technical field
The invention belongs to DNA biosynthesizing field, be specifically related to the short single strand deoxyribonucleotide of a kind of synthesis (short single strand DNA, sssDNA) method of probe, can be used for detecting little non-encoding ribonucleic acid (small non-coding RNAs), as micro ribonucleic acid (microRNA).
Background technology
Short single strand deoxyribonucleotide (sssDNA) can be used as probe for scientific research and medical diagnosis.The nearest more than ten years are very burning hot about the research of little non-coding RNA, especially microRNA, scientist and pharmaceuticals also very good to the prospect researching and developing diagnostic kit and medicine using microRNA as target spot.Because microRNA only has 18-30 base (Bartel usually, 2004), and the microRNA difference of a family may only have 1-2 base (www.mirbase.com), therefore detects microRNA with hybridizing method, high to the purity requirement of probe.If probe has base mutation, detected result can be caused to be false negative or false positive.
The short nucleic acid technique of existing synthesis is mainly by solid-state chemical reaction method (Reese, 2005), although this method is widely used in producing oligonucleotide, it has certain error rate, and error rate increases with the length increase of the Nucleotide of synthesis.This method adds single base gradually from 3 ' to 5 ' end of nucleic acid; because efficiency does not all reach 100% when often adding a base; present common efficiency is about 99%; so the deoxyribonucleotide containing 21 bases synthesized in this way only has purity (Lohmannet al., 2007 of about 79% usually; Pon et al., 1996).Although the purifying (conventional HPLC or PAGE purifying) after synthesis can help the deoxyribonucleotide of enrichment expection object size, the deoxyribonucleotide impurity having onesize sudden change cannot be reduced.Thus the oligonucleotide product of existing solid-state chemical reaction method purified after also can not reach 100% purity.This is a fatal defects to the application of the oligonucleotide of those demand 100% purity (sequence is consistent).
Scientific research person is also seeking the method for producing highly purified short single strand deoxyribonucleotide with biological enzyme synthetic method.PCR(polymerase chain reaction, polymerase chain reaction) be used to short nucleotide probe (the Bertilsson et al. that production length is 30 bases, 2002, Appl Environ Microbiol), but there are two shortcomings in this method: one is that PCR needs to increase with the primer of chemosynthesis, and the primer of synthesis always has sudden change, this just makes the sudden change carried in the product of PCR in primer, product is caused not reach the purity of 100%, and restricted to the product length of amplification; Another shortcoming is the product of PCR synthesis is double-strand, needs extra step double-strand is separated and is purified into object strand.
Van Ness J in 2003 etc. describe a kind of method (VanNess et al., 2003) of constant-temperature amplification single strand oligonucleotide acid fragment.A kind of heat-staple otch restriction endonuclease N.BstNBI is specifically utilized to stamp otch at Double stranded oligonucleotide acid fragment, there is otch oligonucleotide fragment will come off from double-stranded template like this, produce one the 5 ' end given prominence to, archaeal dna polymerase just can produce new double chain oligonucleotide by this end of polishing subsequently, the double chain oligonucleotide of new synthesis can stamp otch by incision restriction endonuclease Nt.BstNBI again, polysaccharase can copy again, so just reaches the object of amplification small segment Nucleotide.This method has significant limitation: the length of constant-temperature amplification to the oligonucleotide of amplification has strict requirement, if the oligonucleotide increased is oversize, Tm value will be too high, and the oligonucleotide fragment that otch restriction endonuclease produces after acting on Double stranded oligonucleotide acid fragment would not come off from template; On the contrary, if the oligonucleotide that will increase is too short, Tm value will be too low, and oligonucleotide fragment is just easy to come off from template.Therefore, this method is only suitable for the oligonucleotide of amplification 8-16 base, just helpless for the amplification of shorter or longer oligonucleotide.
Also the method for report rolling circle amplification is had to produce Nucleotide (Li Yan etc., 2011, a kind of foundation of efficient amplification small pieces segment DNA method; Lohmann et al., 2007).The method of the report rolling circle amplification in 2007 such as Lohmann produces the Nucleotide of 66 bases.The method of the report rolling circle amplifications such as nearest Li Yan produces shorter Nucleotide, they are first linked into ring the single stranded oligonucleotide of chemosynthesis, with this circular oligonucleotide as template, utilize phi29DNA polysaccharase to start to copy with unidirectional or two-way short primer, its product is a very long single stranded DNA (unidirectional primer) or a very long double-stranded DNA (two-way primer) respectively.The shortcoming of this method: one is that the error tape in the masterplate of chemosynthesis and primer has been suffered to product; Two is do not find the method long strand being cut into special short chain; Although three is that long double-stranded DNA can be cut by restriction endonuclease, but require that the target DNA fragment being replicated amplification contains restriction enzyme site, namely can not increase specific not containing the sequence of restriction endonuclease, and product after cutting or double-strand, the experiment and the diagnosis that need single-stranded probe can not be used for.
The present invention is connected in plasmid increases through the DNA of sequence verification without sudden change, then object fragment is cut out with restriction endonuclease, connect into ring, a wherein chain is cut off again with nickase, then amplify the DNA single chain of needs with the method for rolling circle amplification with phi29DNA polysaccharase, this DNA single chain can the secondary structure of spontaneous formation neck ring, thus object single stranded DNA and the by product neck ring DNA of short (10-30bp) can be formed by II pre-designed type restriction endonuclease cutting, the single short single stranded DNA containing concensus sequence (without sudden change) can be obtained by PAGE separation and purification.The advantage of this method: one be template through sequence verification without sudden change; Two is through ingehious design, and the long single stranded DNA of generation can be cut into only containing the short single stranded DNA of aim sequence, does not have sequentiality to limit, and is strand, can be used as probe and use after simple separation purifying; Three is that the error correcting capability of phi29DNA polysaccharase is strong, and error rate is about 1X10
-7, be significantly better than with Taq archaeal dna polymerase that (error rate is 2x10
-4) carry out the single stranded DNA that pcr amplification obtains, more much lower than the error rate of synthesizing by other method.
Summary of the invention
The object of the invention is to overcome the problems referred to above, provide a kind of accuracy high, for the method for the short single strand deoxyribonucleotide probe of synthesis of the accurate detection of little non-coding RNA.
To achieve these goals, the technical solution adopted in the present invention is:
Synthesize a method for short single strand deoxyribonucleotide probe, it is characterized in that, comprise the steps:
(1) preparation restructuring has the plasmid of template DNA
Design template DNA sequence dna: template DNA is double-strand, and identical restriction enzyme site is contained at two ends; Template DNA is provided with at least one neck ring-probe structure; Template DNA is also with nickase site.
Template DNA described in synthesis is also connected in plasmid, through sequence verification without suddenling change and increasing;
(2) enzyme is cut and is connected template DNA
From recombinant plasmid, enzyme is cut and is obtained template DNA, makes template DNA head and the tail connect into ring;
(3) strand is cut, rolling-circle replication
Cut off a chain of template DNA with nickase, add archaeal dna polymerase and carry out rolling-circle replication with the nicked DNA of ring-type for template, amplify the DNA single chain containing neck ring-probe structure;
(4) preparation of the short single stranded DNA of object
Adopt restriction endonuclease cutting to be formed and the object ssDNA probe of microRNA complete complementary and by product DNA to the DNA single chain product of the spontaneous formation neck ring secondary structure of step (3), obtain the ssDNA probe without sudden change containing unique sequence by PAGE separation and purification.
Further technical scheme comprises: neck ring is the become ring structure containing restriction enzyme site, and probe is any one DNA sequence dna; Restriction endonuclease endonuclease site to cut off between neck ring and any one DNA sequence dna.
Further, described probe is the antisense sequences of arbitrary microRNA; Described restriction endonuclease endonuclease site, to cut off between neck ring and the antisense sequences of arbitrary microRNA, cuts off out the antisense sequences with the microRNA of microrna sequences complete complementary.
Further, described restriction endonuclease is BtsCI or BspPI, and described restriction enzyme site is BtsCI site
or BspPI site
restriction endonuclease is corresponding with restriction enzyme site.Or other restriction endonuclease similar with these restriction endonucleases, can cut off between neck ring and the antisense sequences of arbitrary microRNA, and cut off out the antisense sequences with the microRNA of microrna sequences complete complementary.
Further, described nickase site comprises Nt.BstNBI site, and described nickase comprises Nt.BstNBI.
Further, terminal enzyme (DNA) ssDNA probe end is after purification adopted to add marker; Or the amplification substrate of tape label thing is added when polymeric enzymatic amplification.
Marker described further comprises digoxin, fluorescein, vitamin H or isotropic substance.
Further, described object ssDNA probe size is 10-30bp.
Further, described archaeal dna polymerase is phi29DNA polysaccharase.
To sum up, the invention provides a kind of method of synthesizing short single strand deoxyribonucleotide probe, namely with phi29DNA polysaccharase with the ring-type breach DNA checking order correct for template, amplify the long single stranded DNA that can form neck ring structure, after neck ring position cuts off, separable obtain object single stranded DNA through restriction endonuclease, in the Diagnosis and Treat field of little non-coding RNA detection field and little non-coding RNA relative disease, there is extensive use as probe.
Accompanying drawing explanation
Fig. 1 and Fig. 2: the DNA profiling sequence for increasing of design, its 5 ' end is the sequence of the formed neck ring structure containing II type restriction endonuclease BtsCI site, and 3 ' end is the antisense sequences (probe) of a microRNA, or is the sequence (Figure 1A) of any one DNA; Above-mentioned neck ring-microRNA probe sequence superposition, form the sequence of neck ring-probe-neck ring-probe-neck ring, add Nt.BstNBI nickase recognition site GAGTGatat at 5 ' end of this sequence again, finally respectively add EcoRI endonuclease recognized site GAATTC(figure mono-1B and Fig. 2 at two ends); Phi29DNA polysaccharase with the template DNA of nicked ring-type for template, the mode rolling-circle replication replaced with chain and the chain (Fig. 1 C) of complete loop chain complementation;
Fig. 3: the collection of illustrative plates that the agarose gel electrophoresis that the pUC57-miR124 plasmid through restriction endonuclease EcoRI digestion passes through 1.5% is separated; Left side swimming lane is the DNA marker of 100bp, and right lanes is the template DNA fragment of the about 130bp cut out;
The PAGE glue of Fig. 4: 15% is separated the collection of illustrative plates of the single-stranded DNA product after BtsCI cutting, the band gone up most in swimming lane 1 is the probe (i.e. object ssDNA probe) of miR-124, a middle band is neck ring DNA fragmentation, and a bottom band is the DNA fragmentation containing EcoRI and Nt.BstNBI site; Swimming lane 2 is oligonucleotide of 20 bases of chemosynthesis, as marker;
Fig. 5: the collection of illustrative plates that miR-124 expresses in the neurone (mantle zone) of the differentiation of E12.5 mice embryonic midbrain detected with the miR-124 probe (i.e. object ssDNA probe) that the present invention produces.
Embodiment
Below in conjunction with accompanying drawing, with specific embodiment, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.
Synthesize a method for short single strand deoxyribonucleotide probe, comprise the steps:
A, design need the template DNA sequence of amplification, and its 5 ' end is the sequence of formed neck ring structure containing II type restriction endonuclease BtsCI site, and 3 ' end is the antisense sequences (probe structure) of a microRNA, or is the sequence of any one DNA.These two portions are a unit, can superpose such unit, just define the sequence of the neck ring-probe-neck ring-probe-neck ring designed by the present invention.Add Nt.BstNBI nickase recognition site GAGTGatat at 5 ' end of this sequence again, finally respectively add an EcoRI endonuclease recognized site GAATTC at two ends.
The sequence of b, DNA Synesis Company compounding design, is cloned in carrier, and through order-checking without after sudden change, increase in transfection to bacterium plasmid.
After c, extraction plasmid, cut out template DNA fragment with restriction endonuclease EcoRI, then with T4DNA ligase enzyme, template DNA is connected into cyclic DNA, then with nickase Nt.BstNBI, the chain of in DNA double chain is cut.
D, with the nicked template DNA of ring-type for template, the mode rolling-circle replication replaced with chain with phi29DNA polysaccharase and the chain of complete loop chain complementation.
E, the single-stranded DNA product increased with II type restriction endonuclease BtsCI cut-out.
F, PAGE glue separation and purification object single stranded DNA with 15%, can be used as probe for scientific research and medical diagnosis.
Embodiment one: prepare microRNA detecting probe method one
The first step: design needs the DNA sequence dna of amplification, its 5 ' end is the sequence of the formed neck ring structure containing II type restriction endonuclease BtsCI site, and 3 ' end is the antisense sequences (probe) of a microRNA, or is the sequence of any one DNA, sees Figure 1A.In order to optimization production and subsequent experimental, above-mentioned neck ring-microRNA probe sequence superposition, form the sequence of neck ring-probe-neck ring-probe-neck ring, Nt.BstNBI nickase recognition site GAGTGatat is added again at 5 ' end of this sequence, finally respectively add an EcoRI endonuclease recognized site GAATTC at two ends, as Figure 1B.
The required mouse microRNA mmu-miR-124-3p(sequence number MIMAT0000134 detected) sequence be 5 ' UAAGGCACGCGGUGAAUGCC3 ' (seq No.3).The sequence for the production of miR-124 probe of design is in table 1.
Table one is for the production of the template DNA sequence of miR-124 probe
In order to display sequence structure better, forward sequence is 5 ' Gaattc GAGTGatat C
cATCCgCGCtgt gcgc
gGATGGAgGCATTCACCGCGTGCCTTA
cATCCgCGC tgt gcgc
gGATGTAgGCATTCACCGCGTGCCTTA
cATCCgCGC tgt gcgc
gGATGTAgaattc3 ' (Fig. 2).The sequence of the upper and lower chain in Nt.BstNBI site is respectively GAGTGNNNNN, CTCACNNNNNN, point of contact cochain last N (N refer in A, T, C, G any one, lower with) before.The sequence of the upper and lower chain in BtscI site is respectively GGATGNN, CCTACNN, and point of contact is before the end of cochain and latter two N of lower chain.
Second step: allow DNA Synesis Company synthesize the sequence of above-mentioned design, is cloned in pUC57 carrier, through sequence verification synthesis sequence without sudden change after, increase in transfection to bacterium plasmid, plasmid called after pUC57-miR124.
3rd step: after extracting plasmid, with the restriction endonuclease EcoRI(NEB of 1 μ l containing 20 units) digest 1 μ gpUC57-miR124 plasmid, cut out template DNA fragment, with the agarose gel electrophoresis (120V of 1.5%, 30min) isolate the template DNA fragment of about 130bp, see Fig. 3.Rubber tapping, blend compounds reclaims test kit and reclaims template DNA fragment, DNA 20 μ l deionized water wash-outs.
4th step: get the template DNA that 10 μ l reclaim, the T4DNA ligase enzyme (NEB) containing 400 units with 1 μ l connects into cyclic DNA template DNA, total reaction system 20 μ l, and 16 DEG C are reacted 2 hours.Then 65 DEG C of incubations 10 minutes are ligase enzyme inactivation.
5th step: get the above-mentioned connection product of 5 μ l, with 1 μ l containing the nickase Nt.BstNBI(NEB of 10 units) chain is cut, react and in 1X NEBuffer3.1 damping fluid, be placed in 55 DEG C carry out 2 hours.Then 80 DEG C of incubations 30 minutes are nickase inactivation.
6th step: get the product that 5 μ l the 5th step enzymes are cut, the phi29DNA polysaccharase (NEB) containing 10 units with 1 μ l carries out the mode rolling-circle replication (RCA) of chain replacement and the chain (Fig. 1 C is shown in by schematic diagram) of complete loop chain complementation with the template DNA of nicked ring-type, react in 20 μ l systems (containing the phi29DNA polysaccharase of 2 units, 1Xphi29DNA polymerase buffer, 0.1mg/ml BSA, 0.2mM dTTP, 0.2mM dGTP, 0.2mMdATP and 0.2mM dCTP) in carry out 6 hours in 30 DEG C.Then be placed in 65 DEG C and within 10 minutes, allow enzyme deactivation.7th step: with II type restriction endonuclease, namely 1 μ l cuts off containing the BtsCI enzyme of 20 units the single-stranded DNA product increased.Reaction carries out 1 hour in 50 DEG C in 1X CutSmart damping fluid (50mM Potassium ethanoate, 20mM Tris-acetate, 10mM magnesium acetate, 100 μ g/ml BSA, pH7.9).
8th step: adding 6x DNA loading buffer to final concentration in the reaction solution of the 7th step is 1x DNAloading buffer.To the PAGE glue of 15% on sample, with 150V electrophoresis 2 hours separation and purification object single stranded DNAs in 1xTBE.Glue, with after GelRed dyeing, is taken pictures with gel imaging instrument, be the results are shown in Figure 4.
9th step: the glue of the miR-124 probe of cutting containing 20bp, is cut into small shreds glue, shakes spend the night with the 1xTBE wash-out of 3 times of volumes.After brief centrifugation (600g, 1min), solution is transferred in new 1.5ml pipe, adds the isopropanol precipitating DNA of 0.7 times of volume, once, air-dry DNA precipitation, adds 5ul TE buffer dissolving DNA to the washing with alcohol with 70%.
Tenth step: utilize terminal enzyme (DNA) (Terminal Transferase) label probe.Get the DNA probe that 5pmol the 9th step produces, add the terminal enzyme (DNA) (NEB) of 1 μ l containing 20 units at 1X Terminal transferring enzyme reaction buffer, 0.2mM DIG-dUTP, 0.25mM CoCl
2in hatch half an hour in 37 DEG C, then 70 DEG C of heating make enzyme deactivation in 10 minutes.The DNA probe of such 3 ' end mark DIG-dUTP just may be used for detecting miR-124.
Embodiment two: prepare microRNA detecting probe method two
Method two is substantially identical with method one, just in the six-step process of method one, replaces 0.2mM dTTP with 0.1mM DIG-dUTP and 0.1mM dTTP, and the single stranded DNA of such phi29DNA polysaccharase synthesis is just mixed with DIG-dUTP.DIG is a kind of marker, can be digoxin, fluorescein, vitamin H, isotropic substance etc.After the rubber tapping purify DNA completing the 9th step, what obtain has been exactly the DNA probe marked.
Embodiment three: check mouse miR-124 by the object ssDNA probe made (i.e. miR-124 probe)
The first step:
The brain section of fetal development 12.5 days mouse of eight micron thickness, through following step process
1. dimethylbenzene dewaxing twice, each 15 minutes
2.100% ethanol washes twice, each 5 minutes
3.70% ethanol washes 5 minutes
4.30% ethanol washes 5 minutes
5. wash 5 minutes in deionized water
Twice is washed, each 5 minutes in 6.1x PBS
7.4% PFA fixes 20 minutes on ice
Twice is washed, each 5 minutes in 8.1xPBS
9.10 μ g/ml Proteinase Ks process 5 minutes in 1x proteinase K buffer
Twice is washed, each 2 minutes in 10.1xPBS
11.4%PFA fixes 10 minutes on ice
Twice is washed, each 5 minutes in 12.1xPBS
13. containing the 1xTEA[200ml of0 of 600 μ l diacetyl oxides, 1M trolamine-hydrochloric acid (triethanolamine-HCl), pH8] in process 10 minutes
14.2xSSC process twice, each 5 minutes
Second step: prehybridization
Add the hybridization buffer (Ambion) of 100 μ l on the above-mentioned brain sheet handled well, covered, be placed in a moisture preservation box containing 50% methane amide, 5xSSC, prehybridization 1 hour in incubator moisture preservation box being placed on 52 DEG C.
3rd step: hybridization
Get miR-124 probe prepared by 1pmol method one, add the hybridization buffer (Ambion) of 100 μ l, mixing.Cover glass on brain sheet is removed, adds the hybridization buffer of the 100 μ l containing probe, cover a new cover glass, put back in moisture preservation box, put 52 DEG C of hybridized overnight to incubator.
4th step: washing
1.50% methane amide, 2xSSC, 0.1%Tween-20 wash four times in 57 DEG C, each 15 minutes
2.50% methane amide, 0.2xSSC, 0.1%Tween-20 wash four times in 57 DEG C, each 15 minutes
Twice is washed, each 10 minutes in 3.1xPBS
5th step: antibody test signal
1. at room temperature close brain sheet 1 hour with the 1x PBS containing 10% foetal calf serum
2. anti-DIG antibody (Roche) incubated at room that the HRP adding 400 μ l1:2000 dilutions is cross-linked 2 hours
3. damping fluid I(0.1M Tris0.1M NaCl pH7.5) in wash twice, each 15 minutes
4. damping fluid I(0.1M Tris0.1M NaCl pH9.5) in washing 15 minutes
5. by specification 1:50 dilutes NBT/BCIP storage liquid (18.75mg/ml nitroblue tetrazolium(NBT) and the chloro-3-indyl-phosphoric acid salt of the bromo-4-of 9.4mg/ml5-, Roche), add NBT/BCIP substrate that 400 μ l dilute on brain sheet, 37 DEG C of color reactions 3 hours, stop in TE damping fluid
6. take pictures after mounting, the results are shown in Figure 5, by hybridization in situ experiment, miR-124 probe in detecting of the present invention is to miR-124 high expression level specifically in the neurone (mantle zone) of the differentiation of E12.5 mice embryonic midbrain.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (6)
1. synthesize a method for short single strand deoxyribonucleotide probe, it is characterized in that, comprise the steps:
(1) preparation restructuring has the plasmid of template DNA
Design template DNA sequence dna: template DNA is double-strand, and identical restriction enzyme site is contained at two ends; Template DNA is provided with at least one neck ring-probe structure, and described neck ring is the become ring structure containing restriction enzyme site, and described probe is the ssDNA probe with arbitrary microRNA complete complementary; Template DNA is also with nickase site;
Template DNA described in synthesis is also connected in plasmid, through sequence verification without suddenling change and increasing;
(2) enzyme is cut and is connected template DNA
From recombinant plasmid, enzyme is cut and is obtained template DNA, makes template DNA head and the tail connect into ring;
(3) strand is cut, rolling-circle replication
Cut off a chain of template DNA with nickase, add phi29DNA polysaccharase and carry out rolling-circle replication with the nicked DNA of ring-type for template, amplify the DNA single chain containing neck ring-probe structure;
(4) preparation of the short single stranded DNA of object
Restriction endonuclease cutting is adopted to the DNA single chain product of the spontaneous formation neck ring secondary structure of step (3), cut off between neck ring and DNA probe sequence, formed and the object ssDNA probe of microRNA complete complementary and by product DNA, obtain the ssDNA probe without sudden change containing unique sequence by PAGE separation and purification.
2. a kind of method of synthesizing short single strand deoxyribonucleotide probe according to claim 1, it is characterized in that: described restriction endonuclease is BtsCI or BspPI, described restriction enzyme site is BtsCI site or BspPI site, and restriction endonuclease is corresponding with restriction enzyme site.
3. a kind of method of synthesizing short single strand deoxyribonucleotide probe according to claim 1, it is characterized in that: described nickase site is Nt.BstNBI site, described nickase is Nt.BstNBI.
4. a kind of method of synthesizing short single strand deoxyribonucleotide probe according to claim 1, is characterized in that: adopt terminal enzyme (DNA) ssDNA probe end after purification to add marker; Or the amplification substrate of tape label thing is added when polymeric enzymatic amplification.
5. a kind of method of synthesizing short single strand deoxyribonucleotide probe according to claim 4, is characterized in that: described marker comprises digoxin, fluorescein, vitamin H or isotropic substance.
6. a kind of method of synthesizing short single strand deoxyribonucleotide probe according to claim 1, is characterized in that: described object ssDNA probe size is 10-30bp.
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| EP3516055B1 (en) | 2016-09-21 | 2021-12-22 | Technische Universität München | Scalable biotechnological production of dna single strand molecules of defined sequence and length |
| CN109763172A (en) * | 2017-11-09 | 2019-05-17 | 深圳华大生命科学研究院 | A kind of mutant nucleic acid library construction method and obtained mutant nucleic acid library |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213310A (en) * | 2005-04-12 | 2008-07-02 | Novia公司名下的现场Rcp公司 | Methods for production of oligonucleotides |
| CN101213311A (en) * | 2005-04-29 | 2008-07-02 | J·克雷格·文特尔研究院 | Amplification and cloning of single DNA molecules using rolling circle amplification |
| CN101445797A (en) * | 2008-12-08 | 2009-06-03 | 中国科学院广州生物医药与健康研究院 | Reagent used for ex vivo expansion and method thereof |
-
2013
- 2013-05-21 CN CN201310189835.1A patent/CN103276074B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213310A (en) * | 2005-04-12 | 2008-07-02 | Novia公司名下的现场Rcp公司 | Methods for production of oligonucleotides |
| CN101213311A (en) * | 2005-04-29 | 2008-07-02 | J·克雷格·文特尔研究院 | Amplification and cloning of single DNA molecules using rolling circle amplification |
| CN101445797A (en) * | 2008-12-08 | 2009-06-03 | 中国科学院广州生物医药与健康研究院 | Reagent used for ex vivo expansion and method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Jakob S Lohmann, Magnus Stougaard and Jø * |
| rn Koch.Kanpo Seizai no Bunkenteki na Kosatsu.《BMC Biotechnology》.2007,1-9. * |
| 古先祥,李鸿岩,袁毅君.有效扩增小片段DNA的一种方法.《天水师范学院学报》.2011,29-32. * |
| 李岩,李珊珊和张玉祥.一种高效扩增小片段DNA方法的建立.《首都医科大学学报》.2011,121-124. * |
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