CN105297143A - Preparation method of emulsion asymmetric PCR (Polymerase Chain Reaction)-based ssDNA (single-stranded deoxyribonucleic acid) secondary library - Google Patents
Preparation method of emulsion asymmetric PCR (Polymerase Chain Reaction)-based ssDNA (single-stranded deoxyribonucleic acid) secondary library Download PDFInfo
- Publication number
- CN105297143A CN105297143A CN201510604766.5A CN201510604766A CN105297143A CN 105297143 A CN105297143 A CN 105297143A CN 201510604766 A CN201510604766 A CN 201510604766A CN 105297143 A CN105297143 A CN 105297143A
- Authority
- CN
- China
- Prior art keywords
- emulsion
- ssdna
- asymmetric pcr
- preparation
- product
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biomedicine and clinical medicine, in particular to a preparation method of an emulsion asymmetric PCR-based ssDNA secondary library. The method comprises the following steps of synthesizing a primer and an ssDNA library, and preparing an emulsion-asymmetric PCR reaction liquid; preparing the emulsion-asymmetric PCR reaction liquid into an emulsion, and performing emulsion asymmetric PCR amplification to obtain an object product without a by-product, wherein the object product contains biotin labeled dsDNA (double-stranded deoxyribonucleic acid) and non-biotin labeled dsDNA; combining the object product with streptavidin magnespheres, so that a non-biotin labeled product, i.e. the secondary ssDNA library, is separated. The preparation method of the emulsion asymmetric PCR-based ssDNA secondary library is built, the problem of by-product of other method is overcome, and the PCR amplification step is simple and convenient; meanwhile, due to the fact that by-product interference does not exist, the downstream separation step is more simple and effective.
Description
Technical field
The present invention relates to biomedicine and technical field of clinical medicine, particularly a kind of preparation method of ssDNA the level library based on emulsion asymmetric PCR.
Background technology
Aptamer is a kind of ssDNA or RNA molecule, its can high-affinity high specific in conjunction with multiple target molecule, as protein, amino acid, medicine, metal ion, cell etc.Aptamer is similar to antibody, but more easily synthesizes than antibody and preserve, and therefore aptamer has the potentiality replacing antibody for fundamental research and clinic diagnosis.
Aptamer is obtained by SELEX technology screening.The preparation in ssDNA level library is the committed step of SELEX technology.Method prepared by current ssDNA level library mainly contains: asymmetric PCR, biotinstreptatin partition method, exonuclease digestion method etc.
Asymmetric PCR amplified production is containing object ssDNA, dsDNA and by product.Need to be separated by the gel electrophoresis of modacrylic acyl ammonia, object ssDNA and dsDNA and separation of by-products are opened, reclaims a large amount of loss that can cause object ssDNA in purge process at glue; If by product is more simultaneously, easy and object ssDNA merges and forms Coating tape, ssDNA can not be separated, thus can not get purer object ssDNA level library.
Biotinstreptatin partition method be use biotin labeling wherein a primer carry out pcr amplification, amplified production usually containing biotin labeled dsDNA, the by product of biotin labeled by product and lifeless matter element mark.Amplified production is combined with Streptavidin MagneSphere, by biotin labeled product separation out, then by alkaline denaturation, the ssDNA of unmarked vitamin H is separated.The shortcoming of this method is that isolated ssDNA contains a certain amount of by product, and isolated ssDNA is in the alkaline solution of high ph-values simultaneously, reclaims the loss that purifying can cause ssDNA.
Exonuclease digestion method utilizes exonuclease digestion pcr amplification product, makes dsDNA change into ssDNA, and after digestion terminates, be separated with the gel electrophoresis of modacrylic acyl ammonia, purifying reclaims.In the process that purifying reclaims, easily cause object ssDNA to lose in a large number.
Above three kinds of methods also have an important common drawback: a pcr amplification committed step being in these methods, the quality of pcr amplification product directly has influence on the quality in secondary ssDNA library.Not only optimizing process is loaded down with trivial details for the random ssDNA of pcr amplification, and inevitably produces by product, not only makes amplification efficiency decline, and also causes potential high-quality aptamer to lose.
Traditional PCR method inefficiency is because random oligonucleotide library is very rich and varied, and during amplification, storage capacity is 10
9left and right, the hybridization between different product and formed non-specific amplification be in PCR process one be difficult to the major issue avoided.Random library PCR is huge with the Standard PCR difference of a kind of object template that only increases, Musheev etc. find when product reaches peak value, only many amplifications 5 circulations just can make object product change nonspecific products into completely, and main nonspecific products may be the hybridization between product and product, form ssDNA-dsDNA, the hybridization of product and product can make again potential high-affinity, high specific aptamer lose and cause screening unsuccessfully.Reduce the generation of nonspecific products, traditional method only has optimize PCR reaction conditions and designs the fixed sequence program in library, but these methods can only make nonspecific products reduce to some extent, make PCR circulate reach product amount maximum time stop amplification, and fundamentally can not solve non-specific amplification, more can not stop the hybridization between product and product.
The shortcoming of conventional PCR amplification ssDNA pool is in a word: the hybridization 1, between different product and non-specific amplification cannot be avoided.2, the object product that PCR generates can be converted into by product, and increases with the increase of cycle number, until be all converted into by product.3, Optimal reaction conditions complex steps, and can not by product be avoided completely to generate.Therefore normal PCR is used for the inefficiency of the preparation in ssDNA level library and unavoidably causes potential aptamer to be lost.
The emulsion asymmetric PCR method that the present invention sets up thoroughly can solve the shortcoming of normal PCR, emulsion asymmetric PCR by emulsion particle about 10
9individual different form spacing is opened, and increases respectively in different emulsion particles, and therefore their product can not phase mutual cross and produce by product.This method not only increases product amount, solves by product problem, does not also need cycle number optimization, 35-50 circulation of can directly increasing, simply efficiently.
Summary of the invention
The present invention overcomes the defect existed in prior art, a kind of preparation method of ssDNA the level library based on emulsion asymmetric PCR is provided, this preparation method not only improves product amount, solve prior art by product problem, and do not need cycle number optimization, can directly to increase 35-50 circulation, simply efficiently.
For solving the problems of the technologies described above, the invention provides a kind of preparation method of ssDNA the level library based on emulsion asymmetric PCR, comprising the steps:
S1. synthetic primer and ssDNA pool, wherein upstream primer lifeless matter element mark, downstream primer has biotin labeling; Described ssDNA pool length is 90bp, and two ends are respectively the fixed sequence program of long 19bp, and centre is the stochastic sequence of 52bp.SsDNA pool sequence is:
5′-GAACATTGGCGTCCGTGAG-N52-CACTTCCTCAAACGCCCAA-3′；
Upstream primer is 5 '-GAACATTGGCGTCCGTGAG-3 ',
Downstream primer is 5 '-biotin-TTGGGCGTTTGAGGAAGTG-3 '.
S2. emulsion-asymmetric PCR reaction solution is configured:
With ssDNA pool in step S1 and primer for template and primer configuration emulsion-asymmetric PCR reaction solution.
Described emulsion-asymmetric PCR reaction solution 100 μ L system is containing Tris-HCl, 2.5mmol/LMgCl of 50mmol/LKCl, 10mmol/LpH8.6
2, 0.5mmol/LdNTP, 0.6 μm of ol/L draw, draw under 0.03 μm of ol/L, 0.04 μ g/mL template and 100U/mLpfuDNA polysaccharase.
S3. the preparation of emulsion:
With the emulsion in step S2-asymmetric PCR reaction solution configuration emulsion, wherein aqueous phase is emulsion-asymmetric PCR reaction solution, oil phase consists of 4.5% (v/v) Span80,0.4% (v/v) Tween80,0.05% (v/v) TritonX-100, and all the other are mineraloil(mineral oil); Then the making of emulsion is carried out: aqueous phase is at the uniform velocity instilled in the flat freeze pipe of 2mL containing oil phase with the speed of often dripping 8 ~ 10 μ L, be that the magnetic stirring apparatus of 8 × 1.5mm stirs with the speed of 1500rpm with stirrer simultaneously, continue to stir 20min after aqueous phase adds, obtain the emulsion of 0.3mL, be distributed in PCR reaction tubes;
Wherein the volume ratio of aqueous phase and oil phase is 1:2; The described time at the uniform velocity instilled is that the time controling of the aqueous phase instillation 0.2mL oil phase of every 0.1mL was at about 1 minute.
S4. emulsion asymmetric PCR amplification:
The PCR reaction tubes that emulsion is housed of the packing in step S3, insert in PCR instrument and increase; Reaction conditions is 94 DEG C of sex change 30s, then enters 40 circulations, 94 DEG C of sex change 40s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, finally extends 3min.
S5.PCR product purification: the PCR primer obtained by step S4 carries out purifying, obtains the mixture of target ssDNA level library and biotin labeling dsDNA.
(1) by model be 28004 QIAGEN Purification Kit product, amplification in step S4 is terminated PCR reaction tubes, with 13000rpm rotating speed, centrifugal 10 minutes, removes upper oil phase;
(2) in residual reaction liquid, add the PBbuffer of 5 times of volumes in PCR primer, mixing, adds 1 μ L Glacial acetic acid and makes solution become yellow, and be transferred to and reclaim in post, the centrifugal 1min of 13000rpm, abandons liquid;
(3) add 750 μ LPEbuffer to reclaiming in post, the centrifugal 1min of 13000rpm, abandons liquid, recentrifuge 1min;
(4) by the 2mL centrifuge tube that posts transfer is extremely new, 30 ~ 50 μ LEBbuffer are added, after room temperature leaves standstill 1min, the centrifugal 1min of 13000rpm, obtaining purified product is target ssDNA level library and biotin labeling dsDNA mixture;
S6. the target obtained in Streptavidin MagneSphere separating step S5 ssDNA level library and biotin labeled dsDNA:
(1) by model be 65001 invitrogen Beads enrichment, get 6 ~ 7 × 10
7individual Streptavidin MagneSphere suspension, adds 1.5mL centrifuge tube, with containing 2 × B & W buffer solution magnetic bead of 10mMTris-HClpH7.5,1MmEDTA, 2MNaCl once, upper magnetic frame 1 ~ 2min, inhaling and abandoning washings;
(2) the resuspended magnetic bead of 1 × B & W damping fluid containing 5mMTris-HClpH7.5,0.5MmEDTA, 1MNaCl is added;
(3) the PCR purified product obtained in the step S5 of equal-volume purifying is added, under room temperature, shaking table is in conjunction with about 15min, upper magnetic frame 1 ~ 2min, being combined with Streptavidin MagneSphere containing the dsDNA of vitamin H and being adsorbed on tube wall, the ssDNA not containing vitamin H is free in supernatant;
(4) draw supernatant, be ssDNA level library.
S7. result detects:
(1) Concentration Testing, uses nucleic acid-protein quantitative instrument to detect the secondary library concentration obtained;
(2) purity detecting, uses urea-denatured polyacrylamide gel electrophoresis silver dye to detect.
beneficial effect of the present invention:
First the present invention sets up emulsion asymmetric PCR method, applies the object product that this method amplifies no coupling product, the ssDNA that object product marks containing biotin labeled dsDNA and lifeless matter element; Then by being combined with Streptavidin MagneSphere, by the product separation of lifeless matter element mark out, secondary ssDNA library is.
The preparation method in the secondary library based on emulsion asymmetric PCR that the present invention sets up, overcomes the by product problem of other method, makes pcr amplification step simple and convenient, simultaneously owing to not having by product to disturb, makes the separating step in downstream become simple efficient.
The advantage of the inventive method is mainly manifested in the following aspects:
1. emulsion asymmetric PCR amplified production no coupling product, does not need polyacrylamide gel electrophoresis to be separated object ssDNA, thus not easily loses ssDNA;
2. be separated simple and convenient: emulsion asymmetric PCR amplified production only has two kinds: the ssDNA of biotin labeled dsDNA and lifeless matter element mark.Application Streptavidin MagneSphere, in conjunction with biotin labeled dsDNA, single stage method can be isolated ssDNA and is secondary library, and ssDNA reclaims without the need to purifying in damping fluid, can be directly used in the aptamer screening of next round; And biotinstreptatin law of segregation first isolates biotin labeled dsDNA, ssDNA is isolated from biotin labeled dsDNA again with alkaline denaturation, not only use magnetic bead amount many, output is few, and isolated ssDNA is in the alkaline solution of high ph-values, need process further could be used for the screening of next round aptamer;
3. to obtain ssDNA level library purity high for this law, close to primary libraries, as accompanying drawing 2;
4. step is simple, as accompanying drawing 1.
Accompanying drawing explanation
Fig. 1 is preparation method's schema in ssDNA the level library based on emulsion asymmetric PCR of embodiment 1.
Fig. 2 is the secondary library of preparation and the contrast of original synthetic library in embodiment 1; Wherein, 1 is original synthetic library, and 2 is preparation library, and M is marker.
Fig. 3 is emulsion asymmetric PCR amplification procedure figure in the secondary library process of preparation in embodiment 1, from 10 cyclic amplifications to 50 circulations; Wherein, C is primary libraries contrast, and M is marker.
Fig. 4 is conventional asymmetric PCR amplification procedure figure of the prior art, respectively from 10 cyclic amplifications to 50 circulations; Wherein, C is primary libraries contrast, and M is marker.
Embodiment
embodiment 1:
The present embodiment 1 provides a kind of preparation method of ssDNA the level library based on emulsion asymmetric PCR, and its preparation flow, as shown in Figure 1, comprises the steps:
S1. synthetic primer and ssDNA pool:
SsDNA pool length is 90bp, two ends are respectively the fixed sequence program of long 19bp, centre is the stochastic sequence of 52bp: 5 '-GAACATTGGCGTCCGTGAG-N52-CACTTCCTCAAACGCCCAA-3 ', upstream primer is 5 '-GAACATTGGCGTCCGTGAG-3 ', downstream primer is 5 '-biotin-TTGGGCGTTTGAGGAAGTG-3 ', library is synthesized by Integrated Device Technology, Inc., and primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
S2. emulsion-asymmetric PCR reaction solution is configured:
With ssDNA library in step 1 and primer for template and primer configuration emulsion asymmetric PCR reaction solution, 100 μ L systems are containing Tris-HCl, 2.5mmol/LMgCl of 50mmol/LKCl, 10mmol/LpH8.6
2, 0.5mmol/LdNTP, 0.6 μm of ol/L draw, draw under 0.03 μm of ol/L, 0.04 μ g/mL template and 100U/mLpfuDNA polysaccharase.
S3. the preparation of emulsion:
With the reaction solution configuration emulsion in step 2, aqueous phase is PCR reaction solution, and oil phase consists of 4.5% (v/v) Span80,0.4% (v/v) Tween80,0.05% (v/v) TritonX-100, and all the other are mineraloil.The making of water-in-oil emulsion particle (namely emulsion): the PCR reaction solution of 0.1mL, at the uniform velocity instill in the flat freeze pipe of 2mL containing 0.2ml oil phase with the speed of often dripping 8 ~ 10 μ L, instillation time controling was at about 1 minute, be that the magnetic stirring apparatus of 8 × 1.5mm stirs with the speed of 1500rpm with stirrer simultaneously, continue to stir 20min after aqueous phase adds, obtain the emulsion of 0.3ml, be distributed in 3 pipe PCR reaction tubess.
S4. emulsion asymmetric PCR amplification:
The PCR reaction tubes that emulsion is housed of the packing in step 3, insert in PCR instrument and increase; Reaction conditions is 94 DEG C of sex change 30s, then enters 40 circulations, 94 DEG C of sex change 40s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, finally extends 3min.
S5. purified product:
(1) by model be 28004 QIAGEN Purification Kit product, amplification in step 4 is terminated PCR reaction tubes, with 13000rpm rotating speed, centrifugal 10 minutes, removes upper oil phase;
(2) in residual reaction liquid, add the PBbuffer of 5 times of volumes in PCR primer, mixing, adds 1 μ L Glacial acetic acid and makes solution become yellow, and be transferred to and reclaim in post, the centrifugal 1min of 13000rpm, abandons liquid;
(3) add 750 μ LPEbuffer to reclaiming in post, the centrifugal 1min of 13000rpm, abandons liquid, recentrifuge 1min;
(4) by the 2mL centrifuge tube that posts transfer is extremely new, add 30 ~ 50 μ lEBbuffer, after room temperature leaves standstill 1min, the centrifugal 1min of 13000rpm, obtaining purified product is target ssDNA level library and biotin labeling dsDNA mixture.
S6. ssDNA the level library of the target in Streptavidin MagneSphere separating step 5 and biotin labeling dsDNA:
(1) by model be 65001 invitrogen Beads enrichment, get 6 ~ 7 × 10
7individual streptavidin magnesphere suspension, adds 1.5ml centrifuge tube, with containing 2 × B & W buffer solution magnetic bead of 10mMTris-HClpH7.5,1MmEDTA, 2MNaCl once, upper magnetic frame 1 ~ 2min, inhaling and abandoning washings;
(2) the resuspended magnetic bead of 1 × B & W damping fluid containing 5mMTris-HClpH7.5,0.5MmEDTA, 1MNaCl is added;
(3) the PCR purified product in the step 5 of equal-volume purifying is added, under room temperature, shaking table is in conjunction with about 15min, upper magnetic frame 1 ~ 2min, being combined with streptavidin magnesphere containing the dsDNA of vitamin H and being adsorbed on tube wall, the ssDNA not containing vitamin H is free in supernatant;
(4) draw supernatant, be ssDNA level library.
S7. result detects:
(1) Concentration Testing, uses nucleic acid-protein quantitative instrument to detect the secondary library concentration obtained;
(2) purity detecting, uses urea-denatured polyacrylamide gel electrophoresis silver dye to detect.
In the Concentration Testing of above-mentioned steps S7, using nucleic acid-protein quantitative instrument to detect the secondary library concentration obtained is 6.55 μ g/ml, and being converted to original PCR reaction system is 13.1 μ g/mL, i.e. 473.64pmol/mL;
In the purity detecting of above-mentioned steps S7, use urea-denatured polyacrylamide gel electrophoresis silver dye to detect the secondary library purity obtained, its step is as follows:
(1) the urea-denatured PAGE gel of 12%7M is prepared: 40% acrylamide gel 3ml, 5 × TBE2mL, urea 4.2g, 10% Ammonium Persulfate 98.5 33 μ L, deionized water 1.5mL, TEMED7.5 μ L;
(2) application of sample: get PCR primer 4 μ L and electrophoresis sample-loading buffer 4 μ L mixes, adds in gel sample hole;
(3) vertical electrophoresis: voltage sets is in 150V.When indicator arrives lower 1/3 of gel, stop electrophoresis, carefully take off gel;
(4) silver dye: fix 10 ~ 15min, distilled water flushing 3 times with containing in 10% dehydrated alcohol and 0.5% acetic acid stationary liquid; With 0.2% cma staining 10min, distilled water flushing 3 times; Develop the color with containing in 1.5% sodium hydroxide and 0.4% formaldehyde nitrite ion, until occur clearly after DNA band, rinsing 3min in clear water;
(5) take pictures.
Interpretation of result:
Concentration Testing result: the ssDNA concentration obtained in embodiment 1 is 473.64pmol/mL, illustrates that the inventive method can prepare the higher secondary ssDNA library of concentration;
Electrophoretic analysis result: reclaim product no coupling product in embodiment 1 as can be seen from Figure 2, close with primary libraries; Embodiment 1 equal no coupling product in whole emulsion asymmetric PCR amplification cycles occurs as can be seen from Figure 3; Namely conventional asymmetric PCR of the prior art increases 25 to circulate as can be seen from Figure 4 has by product to occur, 30 circulation time by products occur in a large number.Illustrate that the inventive method can obtain the high-quality secondary ssDNA library of high purity.
Claims (8)
1. based on the preparation method in ssDNA level library of emulsion asymmetric PCR, it is characterized in that, carry out according to following steps:
S1. synthetic primer and ssDNA pool: wherein downstream primer has biotin labeling;
S2. emulsion-asymmetric PCR reaction solution is configured: with ssDNA pool in step S1 and primer for template and primer configuration emulsion-asymmetric PCR reaction solution;
S3. the preparation of emulsion: with the emulsion in step S2-asymmetric PCR reaction solution configuration emulsion, the aqueous phase wherein in emulsion is emulsion-asymmetric PCR reaction solution; Oil phase is the mixed solution of Span80, Tween80, TritonX-100 and mineraloil;
S4. emulsion asymmetric PCR amplification: the PCR reaction tubes that emulsion is housed of the packing in step S3, insert in PCR instrument and increase;
S5.PCR product purification: the PCR primer obtained by step S4 carries out purifying, obtains the mixture of target ssDNA level library and biotin labeling dsDNA;
S6. the target obtained in Streptavidin MagneSphere separating step S5 ssDNA level library and biotin labeled dsDNA;
S7. result detects: detect the concentration in the target obtained ssDNA level library and purity.
2. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 1, it is characterized in that, the primer described in step S1 is:
Upstream primer is 5 '-GAACATTGGCGTCCGTGAG-3 ',
Downstream primer is 5 '-biotin-TTGGGCGTTTGAGGAAGTG-3 '.
3. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 1, it is characterized in that, ssDNA pool length described in step S1 is 90bp, and two ends are respectively the fixed sequence program of long 19bp, and centre is the stochastic sequence of 52bp; Wherein ssDNA pool sequence is:
5′-GAACATTGGCGTCCGTGAG-N52-CACTTCCTCAAACGCCCAA-3′。
4. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 1, it is characterized in that, emulsion described in step S2-asymmetric PCR reaction solution 100 μ L system is containing Tris-HCl, 2.5mmol/LMgCl of 50mmol/LKCl, 10mmol/LpH8.6
2, 0.5mmol/LdNTP, 0.6 μm of ol/L draw, draw under 0.03 μm of ol/L, 0.04 μ g/mL template and 100U/mLpfuDNA polysaccharase.
5. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 1, it is characterized in that, oil phase in emulsion described in step S3 consists of 4.5% (v/v) Span80,0.4% (v/v) Tween80,0.05% (v/v) TritonX-100, and all the other are mineraloil.
6. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 1, it is characterized in that, described emulsion is that aqueous phase is at the uniform velocity instilled in the flat freeze pipe containing oil phase with the speed of often dripping 8 ~ 10 μ L, use magnetic stirrer simultaneously, continue to stir 20min after aqueous phase adds, obtain emulsion, be distributed in PCR reaction tubes.
7. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 6, it is characterized in that, in described emulsion, the volume ratio of aqueous phase and oil phase is 1:2; The described time at the uniform velocity instilled is that the time controling of the aqueous phase instillation 0.2mL oil phase of every 0.1mL was at about 1 minute.
8. the preparation method in a kind of ssDNA level library based on emulsion asymmetric PCR according to claim 1, it is characterized in that, the reaction conditions of pcr amplification described in step S4 is 94 DEG C of sex change 30s, then enter 40 circulations, 94 DEG C of sex change 40s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, finally extend 3min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510604766.5A CN105297143A (en) | 2015-09-22 | 2015-09-22 | Preparation method of emulsion asymmetric PCR (Polymerase Chain Reaction)-based ssDNA (single-stranded deoxyribonucleic acid) secondary library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510604766.5A CN105297143A (en) | 2015-09-22 | 2015-09-22 | Preparation method of emulsion asymmetric PCR (Polymerase Chain Reaction)-based ssDNA (single-stranded deoxyribonucleic acid) secondary library |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105297143A true CN105297143A (en) | 2016-02-03 |
Family
ID=55194932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510604766.5A Pending CN105297143A (en) | 2015-09-22 | 2015-09-22 | Preparation method of emulsion asymmetric PCR (Polymerase Chain Reaction)-based ssDNA (single-stranded deoxyribonucleic acid) secondary library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105297143A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312396A (en) * | 2016-04-07 | 2019-02-05 | 伊鲁米那股份有限公司 | For constructing the method and system of standardization nucleic acid library |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101617046A (en) * | 2007-02-23 | 2009-12-30 | 新英格兰生物实验室公司 | Use external compartmentation to select and enrichment protein |
| US20100311602A1 (en) * | 2006-10-13 | 2010-12-09 | J. Craig Venter Institute, Inc. | Sequencing method |
| US20130190191A1 (en) * | 2009-02-25 | 2013-07-25 | Thomas Froehlich | Miniaturized, high-throughput nucleic acid analysis |
| CN104745589A (en) * | 2015-03-11 | 2015-07-01 | 河北大学 | Screening method and application of nucleic acid aptamers for specifically recognizing streptomycin |
| CN104818533A (en) * | 2015-04-24 | 2015-08-05 | 深圳市血液中心 | Preparing method of ssDNA (single-stranded DNA) secondary library |
| CN104830841A (en) * | 2015-06-04 | 2015-08-12 | 北京中科紫鑫科技有限责任公司 | Production method of sequencing template |
-
2015
- 2015-09-22 CN CN201510604766.5A patent/CN105297143A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100311602A1 (en) * | 2006-10-13 | 2010-12-09 | J. Craig Venter Institute, Inc. | Sequencing method |
| CN101617046A (en) * | 2007-02-23 | 2009-12-30 | 新英格兰生物实验室公司 | Use external compartmentation to select and enrichment protein |
| US20130190191A1 (en) * | 2009-02-25 | 2013-07-25 | Thomas Froehlich | Miniaturized, high-throughput nucleic acid analysis |
| CN104745589A (en) * | 2015-03-11 | 2015-07-01 | 河北大学 | Screening method and application of nucleic acid aptamers for specifically recognizing streptomycin |
| CN104818533A (en) * | 2015-04-24 | 2015-08-05 | 深圳市血液中心 | Preparing method of ssDNA (single-stranded DNA) secondary library |
| CN104830841A (en) * | 2015-06-04 | 2015-08-12 | 北京中科紫鑫科技有限责任公司 | Production method of sequencing template |
Non-Patent Citations (1)
| Title |
|---|
| KEKE SHAO等: "Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection", 《PLOS ONE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312396A (en) * | 2016-04-07 | 2019-02-05 | 伊鲁米那股份有限公司 | For constructing the method and system of standardization nucleic acid library |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105177125B (en) | 20 STR composite amplification systems of mankind's X chromosome and application | |
| CN108614102A (en) | Multiple assay based on aptamer | |
| CN107090457A (en) | Detect the multivariate analysis of sample | |
| TR201811157T4 (en) | Quantitative evaluation of messenger rna's 8217# on header efficiency. | |
| CN105463585A (en) | Method for constructing sequencing library based on single-stranded DNA molecule, and applications thereof | |
| WO2016023431A1 (en) | Wash solution and washing method for hybrid-enrichment-capture dna sequencing library | |
| CN105886512B (en) | Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine | |
| CN103013999B (en) | Oligonucleotides aptamer special for distinguishing fumonisin B1 | |
| Lin et al. | Lab in a tube: isolation, extraction, and isothermal amplification detection of exosomal long noncoding RNA of gastric cancer | |
| Jiang et al. | Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence | |
| CN103739664B (en) | Methods for separating and identifying DNA (deoxyribonucleic acid) binding protein through DNA co-immunoprecipitation | |
| CN108613862B (en) | Double-target synchronous aptamer screening method based on capillary electrophoresis | |
| CN102443585A (en) | Zearalenone nucleic acid aptamer and application thereof | |
| CN103614489A (en) | Constant-temperature amplification detection kit for dengue viruses and detection method | |
| Katilius et al. | Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents | |
| CN107354207B (en) | liquid phase hybridization capture kit based on double-stranded probe, washing kit and application thereof | |
| CN104297325A (en) | Method applied to on-line reaction and separation of protein-oligonucleotide compound by capillary electrophoresis | |
| CN105297143A (en) | Preparation method of emulsion asymmetric PCR (Polymerase Chain Reaction)-based ssDNA (single-stranded deoxyribonucleic acid) secondary library | |
| Zhang et al. | A one-pot strategy for the sensitive detection of miRNA by catalyst–oligomer-mediated enzymatic amplification-based fluorescence biosensor | |
| CN107179404B (en) | A kind of furans metabolite derivatization reagent and its rapid detection card | |
| CN114045281A (en) | Screening method of glioma marker and aptamer | |
| CN103789197B (en) | A kind of test kit and extracting method thereof extracting Microrna | |
| Martins et al. | New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA | |
| CN102952848B (en) | A kind of method detecting non-coding tiny RNA | |
| WO2023202030A1 (en) | Method for constructing high-throughput sequencing library of small rna |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160203 |