CN103525820A - Affinity nucleic acid molecule screening method based on sectioned collection - Google Patents
Affinity nucleic acid molecule screening method based on sectioned collection Download PDFInfo
- Publication number
- CN103525820A CN103525820A CN201310521470.8A CN201310521470A CN103525820A CN 103525820 A CN103525820 A CN 103525820A CN 201310521470 A CN201310521470 A CN 201310521470A CN 103525820 A CN103525820 A CN 103525820A
- Authority
- CN
- China
- Prior art keywords
- molecule
- screening
- nucleic acid
- library
- target
- 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
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention established a fast affinity nucleic acid molecule screening method comprising the steps that: target molecules are mixed with library molecules to be screened; and separation is carried out through capillary electrophoresis or chromatography technologies. The method is characterized in that when the composition is collected, collection is carried out in sections, and the plurality of collected samples are respectively subjected to fluorescence quantitative PCR amplification; and the position of the composition formed by the target molecules and the target nucleic acid molecules is determined with a fluorescence quantitative PCR method, such that the target nucleic acid molecules combined with high affinity with the target molecules can be rapidly obtained. The method can be widely applied in aspects such as nucleic acid aptamer screening, promoter screening, and the like, and assists in greatly improving screening efficiency.
Description
Technical field
The invention belongs to a kind of method for screening and separating, particularly from the nucleic acid molecule storehouse of a mixing, screen and isolate the method for the molecule with certain specific character.
Background technology
Aptamer is single stranded DNA or the RNA molecule that a class can be combined with multiple targets such as small molecules, albumen, bacterium and cells, due to the similar antibody of character, and phase antagonist has multiple advantage: target is extensive, molecular weight is little, production is easy, Quality Control is simple, be widely used, storage and convenient transportation etc., is therefore paid attention to widely.The technology that screening obtains aptamers is known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology.But how to obtain the bottleneck that aptamers is aptamers widespread use always.Capillary electrophoresis (Capillary Electrophoresis, CE) technology is because of its efficient separation efficiency and micro-amount of samples, and when screening aptamer, efficiency is obviously far away higher than other technology.Utilize the technology of capillary electrophoresis screening aptamer to be called CE-SELEX technology.Mendonsa in 2004, S.D. (JACS.126,20-21,2004) report capillary electrophoresis technique can obtain aptamers in the screening of 3-4 wheel.2006, Berezovski, the NON-SELEX technology of M. (JACS.128,1410-1411,2006) report, continuous separate, from three times, can filter out the aptamer of high-affinity.The Li SF laboratory of 2012 departments of chemistry of Nian， NUS is also reported and is utilized NON-SELEX technology screening to go out the catalatic aptamers of ox.Although these technology can obtain aptamer fast, up to now, NON-SELEX only has several pieces of bibliographical informations.Although CE-SELEX report is relatively many, in practical study, find, still have many problems, successfully example is obviously less than paramagnetic particle method screening.
In conventional CE-SELEX technology, be generally to grope a separation condition, this condition must be separated target molecule and random DNA library or RNA library well.During actual screening, target molecule is mixed with library to be screened, between may exist the region of mixture to collect.Due to mixture accurately position be unknown, so need to collect wider window while collecting, can cause like this having in gleanings some and uncombined mixtures, but free library molecule, thereby cause screening efficiency to reduce.NON-SELEX technology collects to reduce the probability of collecting non-binding molecule by continuous several times.The storage capacity that the method is used due to reality is very little, and repeatedly collects and easily cause pollution, makes the success ratio of the method extremely low.
Summary of the invention
Object of the present invention is exactly to set up a kind of method of the nucleic acid molecule that separation is combined with target molecule high-affinity quickly and efficiently from random nucleic acid library.
Method of the present invention comprises the steps:
(1), after target molecule is mixed with library to be screened, separated by capillary electrophoresis or chromatographic technique, feature is when collecting mixture, to be divided into multistage to carry out Fractional Collections, then the sample of all collections is carried out respectively to fluorescent quantitative PCR;
(2) according to the result of fluorescent quantitative PCR, can judge different collect template amount in components number, thereby determine the position at place, mixture peak;
(3) by occurring that the collection product in the pipe at mixture peak amplifies amplification, prepare the library of enrichment, can directly carry out avidity test or repeat next round screening, until the avidity recording reaches expection.
The present invention can be for the aptamer of screening high-affinity, also can be for screening promotor binding sequence.
The initial library of using of the present invention, can be the DNA library molecule of strand, double-stranded DNA library molecule, or RNA library molecule.Library molecule type is different, and library molecule after preparation enrichment method used is slightly different: if original sample is single stranded DNA, obtain after PCR product, need to pass through strand preparation process; If primary sample is RNA, also need through transcription step; If original nucleic acid is double-stranded DNA, conventionally only need through purifying;
Above said Fractional Collections, can be divided into more than two sections; Specifically be divided into how many sections and collect, those skilled in the art can determine according to the convenience of separated efficiency and operation with to the controllability of polluting; Conventionally can be chosen in predetermined collecting zone is divided into 5-15 section and collects.
Although directly test does not utilize the isolation technique such as liquid chromatography technology and capillary electrochromatography to screen in conjunction with Fractional Collections in the present invention, but know by analogy analysis, in conjunction with Fractional Collections, can improve equally the efficiency of these technology aspect the affine nucleic acid molecule of screening.
In sum, the invention provides following:
1. the method for the nucleic acid molecule that a sharp separation is combined with target molecule high-affinity, described method comprises: by after described target molecule and library molecular mixing to be screened, separated by capillary electrophoresis or chromatographic technique, feature is when collecting mixture, to be divided into multistage to carry out Fractional Collections, then the multiple sample of Fractional Collections is carried out respectively to fluorescent quantitative PCR; By the method for quantitative fluorescent PCR, determine the position at described target molecule and the formed mixture of target nucleic acid molecule place, thereby obtain the target nucleic acid molecule of being combined with target molecule high-affinity fast.
2. according to the method described in the 1st, wherein said Fractional Collections is to be divided into two sections to collect above.
3. according to the method described in the 1st, selected library molecule is single-stranded DNA banks molecule, double-stranded DNA library molecule or RNA library molecule.
4. according to the method described in the 1st, wherein when using RNA library to screen when separated, described method also comprises the step that the described target molecule of collecting and the formed mixture of target RNA molecule are carried out to reverse transcription, and then by the method for quantitative fluorescent PCR, determines the position at described target molecule and the formed mixture of target RNA molecule place.
The present invention is with the advantage that existing triage techniques is compared:
1. the present invention compared with prior art, has improved the efficiency of enrichment target molecule greatly.The method that reason has been to take multistage to collect, the sample of collecting in each pipe is less, so the more nonspecifically bound molecule of collecting is also few, pollutes also less; Because mixture only can be distributed in several pipes, now target product is compared and is just occupied absolute predominance with pollution molecule;
2. another advantage of this technology is, by the amplification amplification effect of PCR realize the detection to target peak, make us in first round screening, just can determine the position at mixture place; Technology is in the past all to utilize ultraviolet or Laser-Induced Fluorescence Detection Technology judgement mixture position; Because ultraviolet sensitivity is very low, be difficult to detect mixture; Although Laser-Induced Fluorescence Detection Technology sensitivity is very high, but at the initial stage of screening, target molecule number is few, even if laser Induced Fluorescence Technology still can not detect mixture position, and in the present invention, the sample of collecting is first amplified to 1,000,000 times by pcr amplification, be now very easy to detect mixture position.
3. in our comparative experiments, take Streptavidin as target, screen the single stranded DNA aptamer of combination with it, adopt traditional Technology Need just obvious avidity can be detected through three-wheel, and employing present method only needs one to take turns the aptamer that can obtain higher affinity.
4. this programme is compared with NON-SELEX, and the reliability of method is higher, and resistant to pollution ability is stronger; NON-SELEX technology is separated by continuous several times, and the one, target molecule number is few, very easily causes pollution, and the 2nd, operation steps is many, also easily introduces and pollutes; In this programme, whole screening process can be utilized capillary electrophoresis apparatus, automatically completes, and has reduced manual operation and interference, has reduced pollution, has improved stability, the reliability of method, and success ratio also promotes greatly simultaneously.
Accompanying drawing explanation
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious.
Fig. 1: corresponding curent change figure during the inventive method (be called for short fCE-SELEX) Fractional Collections:
Transverse axis represents the time, and scale unit is minute, and the longitudinal axis represents current value, and scale unit is microampere; Be divided into from left to right 12 sections, represent respectively to collect for 12 times; In figure, between two pipe collections, electric current is that the zero time is the time of switching collection tube.Can be according to the variation of electric current, judgement Fractional Collections correctness.
Fig. 2: the fluorescent quantitative PCR result figure of the inventive method fCE-SELEX Fractional Collections sample, the amplification cycles number that transverse axis represents, the longitudinal axis is fluorescent value; The pipe number that in figure, numeral is collected, unlabelled is other pipe sample and contrast; Wherein No. 10 pipes, No. 3 pipes are managed take-off prior to other, illustrate that the number of this two pipes amplifying nucleic acid molecule is managed more than other, are the positions at mixture place.
Fig. 3: the collection of illustrative plates of avidity comparison (first round product of the third round product of conventional CE-SELEX and segmentation screening compares), according to the screening effect of two kinds of methods of avidity comparison.FP1 represents to adopt technology fCE-SELEX mono-of the present invention to take turns the library that segmentation screening obtains; NP3 represents the conventional CE-SELEX library molecule that screening obtains through three-wheel, and in figure, transverse axis represents the time of electrophoresis, and scale unit is minute that the longitudinal axis represents the fluorescent signal detecting.The shared total peak area ratio of mixture peak area and the positive correlation of avidity size.Because both mixture peak proportions are suitable, therefore both avidity approach.
Fig. 4: utilize gel retardation assasy, the situation of being relatively combined with transcription factor through the double chain DNA molecule of two kinds of method screenings
The 1st swimming lane is molecular weight standard; From top to bottom, stripe size is followed successively by 200pb, 100bp and 50bp;
The 2nd swimming lane is the library molecule (nP3) of conventional CE method screening three-wheel;
The 3rd swimming lane is in nP3, to add the protein target molecule of decile subnumber; Top band is mixture band; Below be the band of library molecule;
The 4th swimming lane is in the first round library molecule (fP1) of Fractional Collections screening, to add the protein target molecule of decile subnumber; Top band is mixture band; Below be the band of library molecule;
The 5th swimming lane is the first round library molecule (fP1) of Fractional Collections screening;
The 6th swimming lane is initial library molecule, with comparing;
The 7th swimming lane is, in initial library molecule, to add protein target molecule;
Embodiment
Below with reference to specific embodiment, further describe the present invention, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
One, the aptamer of the screening Streptavidin of the fCE-SELEX method based on Fractional Collections (SA)
1, nucleic acid library to be screened is for being with fluorescently-labeled single strand dna, length is 80 bases, each 20 bases of two ends are guiding region, centre is stochastic sequence, by life work biotechnology (Shanghai), limited-liability company is synthetic, is dissolved in screening damping fluid (PH8.2 borate buffer solution+5mM MgCl
2) in, final concentration is 25uM;
2, protein target molecule is Streptavidin (SA) 0.1ug;
3, in the fluorescent core acid molecule library of 20ul25uM, add 0.1ug Streptavidin, incubated at room 10 minutes;
4, adopt capillary electrophoresis apparatus to carry out separation, separation condition is: 49 centimetres of long capillary tubes, and useful length is 39 centimetres, internal diameter 75um; First with 100mM NaOH, rinse kapillary 30psi3min, then clean 30psi3min with 100mM HCl, finally with screening damping fluid, rinse kapillary 30psi5min; Loading: under 1psi pressure, in sample introduction 5 seconds, electrophoresis under the condition of 200v/cm then.According to the appearance time of independent sample introduction Streptavidin and nucleic acid, at outlet punishment 12 pipes, collect the sample of the 9th to 13.6 minutes; As shown in Figure 1, black curve represents curent change, and current interruptions represents switch sample collection tube, collects altogether 12 pipes.
5, all samples of collection is carried out to fluorescent quantitative PCR, amplification condition be 94 ℃ 10 seconds, 60 ℃ 10 seconds, 72 ℃ 30 seconds, 30 circulations of coamplification; According to the Cq value of amplified production in every pipe, judge, in the 3rd pipe and the 10th pipe, template number, obviously more than other several pipes, should be position, mixture peak; See Fig. 2.Further PCR product is amplified to amplification, prepare single-chain nucleic acid, carry out avidity mensuration; And contrast with the selection result of traditional C E-SELEX method, as shown in Figure 3.Result shows to utilize improved method screening 1 to take turns, and effect and ordinary method screening 3 effects of taking turns approach, and absolutely prove the superiority of the method.
Two, conventional CE-SELEX method screening Streptavidin (SA) and aptamer
1) conventional CE-SELEX screening method in separation with the present invention in method no significant difference, therefore can screen with reference to above-mentioned condition;
2) when conventional CE-SELEX screens, in a pipe, collect the sample between the 9th minute to the 14th minute, then increase; Further PCR product is amplified to amplification, prepare strand, as the library molecule of next round screening use;
3) repeat above-mentioned screening process.After three-wheel screening, prepare the avidity that strand detects gained library molecule.
Three, avidity comparison and detection result
According to avidity detected result, Fractional Collections is taken turns screening through one, and the library molecule (fP1) after the enrichment obtaining is suitable with library molecule (nP3) avidity that conventional CE-SELEX third round obtains.
Get two libraries of same concentrations, add the target molecule of same molecular number, hatch same time, by the size at CE test compound thing peak.According to the size at mixture peak, can compare the difference of both avidity.As seen from Figure 3, both mixture peak sizes are basically identical, show that avidity approaches.Explanation thus, the method for Fractional Collections will obviously be better than conventional screening method, can greatly improve screening efficiency.
Four,, for other target molecule, as long as according to separation case, change the scope of collection and the pipe number of collection.
1) the random library molecule of strand (purchased from life work biotechnology (Shanghai) limited-liability company) is carried out to the amplification of 5 circulations, guarantee that all molecules all change duplex molecule into; Separation and purification obtains the double-stranded library molecule for screening;
2) separation condition is with embodiment 1, by target molecules (SOX2) and double-stranded DNA library molecule, carry out respectively separation, determine the approximate location at mixture place, the interval that then may exist according to mixture (9-18 minute), is divided into 10 sections and collects;
3) will collect sample and carry out fluorescent quantitative PCR, according to amplification, the 5th pipe is mixture position;
4) the 5th pipe product is amplified to amplification, and the PCR product of purifying gained;
5) utilize gel retardation assasy to detect the combination situation of albumen and DNA, take turns screening through one, can obviously can see the mixture band (band that Fig. 4 the 4th swimming lane is top) of double-stranded DNA and albumen formation.Approach with the effect of traditional capillary electrophoresis method screening three-wheel, concrete outcome is shown in Fig. 4 (swimming lane 3), wherein:
A) the 1st swimming lane is molecular weight standard; From top to bottom, stripe size is followed successively by 200pb, 100bp and 50bp;
B) the 2nd swimming lane is the library molecule (nP3) of conventional CE method screening three-wheel;
C) the 3rd swimming lane is in nP3, to add the protein target molecule of decile subnumber; Top band is mixture band; Below be the band of library molecule;
D) the 4th swimming lane is in the first round library molecule (fP1) of Fractional Collections screening, to add the protein target molecule of decile subnumber; Top band is mixture band; Below be the band of library molecule;
E) the 5th swimming lane is the first round library molecule (fP1) of Fractional Collections screening;
F) the 6th swimming lane is initial library molecule, with comparing;
G) the 7th swimming lane is, in initial library molecule, to add protein target molecule;
The screening of RNA aptamers is similar to Example 1, and difference part is cannot be directly used in due to RNA the amplification of DNA, so collect after mixture, must first carry out reverse transcription; Another difference is, obtains behind the double-stranded DNA library of enrichment, need to obtain RNA library by the method for transcribing, and the operational guidance specifically providing with reference to QIAGEN company, obtains after RNA molecule, can be used for the detection of avidity or carries out next round screening.Concrete steps are as follows: 1) the synthetic random DNA library molecule of the preparation in RNA library, in 1ml PCR system, contain the library molecule of 0.1uM, 1uM3 ' and 5 ' end primer, 2mM MgCl2 and 200uM dNTP, (reaction conditions of each circulation is 94 ℃ of 10s to 5 circulations of pcr amplification, 60 ℃ of 10s, 72 ℃ of 20s); The PCR product of a large amount of amplifications is separated through non-sex change PAGE gel electrophoresis, GelRed dyeing, the position of definite double-stranded product in gel imaging instrument, and cut object band, by gel chopping, with binding buffer liquid (50mM Tris-HCl, pH7.4100mM NaCl, 4mM EDTA, 2mM MgCl2,0.05%NP-40,0.4% nucleosides vanadyl complex body, 200U/ml Rnasin, 100ug/ml poly A, 100ug/ml tRNA) soak and boil, centrifugal, get supernatant; Add 1%NaAc and triploid accumulated ice ethanol, place 4h for-80 ℃; In centrifugal 5 minutes of 4 ℃ of 12000rpm, suck supernatant, vacuum-drying is also preserved;
2) use the MAXIscript T7Kit of life work biotechnology (Shanghai) limited-liability company to carry out in-vitro transcription, RNA output is 2-6ug; Transcribe after end, add DNAase digestion template (every 1ug DNA adds 1U DNAase), 37 ℃ of reaction 60min;
3) get about 1ug RNA library molecule, in 100ul binding buffer liquid, add 0.1ug P53 albumen, be placed in 37 ℃ and hatch 1 hour.Utilize capillary electrophoresis to carry out electrophoresis by the condition of embodiment 1 in the sample after hatching, and collect the sample between 10-22 minute; In the sample hose of collecting, need to add single stage method reverse transcription reagent (QIAGEN OneStep RT-PCR Kit), thereby carry out Q-PCR, according to the result of quantitative fluorescent PCR, determine position, mixture peak;
4) by the PCR product of position, mixture peak through amplification after amplification, by step (1), prepare the RNA library after enrichment, for avidity, test or carry out next round screening;
5) through 2, take turns screening, the avidity of the RNA library molecule that present method obtains is better than the result that paramagnetic particle method 8 is taken turns screening.
Although with reference to its exemplary embodiment, the present invention is carried out to description particularly, but will be understood by those skilled in the art that, under the condition not deviating from by the spirit and scope of the present invention as defined in the claims, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (4)
1. the method for the nucleic acid molecule that a sharp separation is combined with target molecule high-affinity, described method comprises: by after described target molecule and library molecular mixing to be screened, separated by capillary electrophoresis or chromatographic technique, feature is when collecting mixture, to be divided into multistage to carry out Fractional Collections, then the multiple sample of Fractional Collections is carried out respectively to fluorescent quantitative PCR; By the method for quantitative fluorescent PCR, determine the position at described target molecule and the formed mixture of target nucleic acid molecule place, thereby obtain the target nucleic acid molecule of being combined with target molecule high-affinity fast.
2. method according to claim 1, wherein said Fractional Collections is to be divided into two sections to collect above.
3. method according to claim 1, selected library molecule is single-stranded DNA banks molecule, double-stranded DNA library molecule or RNA library molecule.
4. method according to claim 1, wherein when using RNA library to screen when separated, described method also comprises the step that the described target molecule of collecting and the formed mixture of target RNA molecule are carried out to reverse transcription, and then by the method for quantitative fluorescent PCR, determines the position at described target molecule and the formed mixture of target RNA molecule place.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310521470.8A CN103525820A (en) | 2013-10-29 | 2013-10-29 | Affinity nucleic acid molecule screening method based on sectioned collection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310521470.8A CN103525820A (en) | 2013-10-29 | 2013-10-29 | Affinity nucleic acid molecule screening method based on sectioned collection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103525820A true CN103525820A (en) | 2014-01-22 |
Family
ID=49928169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310521470.8A Pending CN103525820A (en) | 2013-10-29 | 2013-10-29 | Affinity nucleic acid molecule screening method based on sectioned collection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103525820A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018461A (en) * | 2014-04-29 | 2015-11-04 | 中国科学技术大学 | Method for rapid screening of nucleic acid aptamer |
| CN109920474A (en) * | 2019-03-14 | 2019-06-21 | 深圳市检验检疫科学研究院 | Absolute quantification method, device, computer equipment and storage medium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636547A (en) * | 2012-04-18 | 2012-08-15 | 北京理工大学 | Oligonucleotide library classification and assessment method based on capillary zone electrophoresis |
-
2013
- 2013-10-29 CN CN201310521470.8A patent/CN103525820A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636547A (en) * | 2012-04-18 | 2012-08-15 | 北京理工大学 | Oligonucleotide library classification and assessment method based on capillary zone electrophoresis |
Non-Patent Citations (2)
| Title |
|---|
| NITSCHE ET AL: "ne-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX", 《BMC BIOTECHNOLOGY》, 15 August 2007 (2007-08-15) * |
| 邵宁生等: "SELEX技术及Aptamer研究的新进展", 《生物化学与生物物理进展》, 31 December 2006 (2006-12-31), pages 329 - 335 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018461A (en) * | 2014-04-29 | 2015-11-04 | 中国科学技术大学 | Method for rapid screening of nucleic acid aptamer |
| CN105018461B (en) * | 2014-04-29 | 2018-05-01 | 中国科学技术大学 | A kind of rapid screening method of aptamer |
| CN109920474A (en) * | 2019-03-14 | 2019-06-21 | 深圳市检验检疫科学研究院 | Absolute quantification method, device, computer equipment and storage medium |
| CN109920474B (en) * | 2019-03-14 | 2021-03-02 | 深圳市检验检疫科学研究院 | Absolute quantitative method, device, computer equipment and storage medium |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yeri et al. | Evaluation of commercially available small RNASeq library preparation kits using low input RNA | |
| CN102636547B (en) | Oligonucleotide library classification and assessment method based on capillary zone electrophoresis | |
| Petrovic | Methodological challenges of multi-residue analysis of pharmaceuticals in environmental samples | |
| Fullwood et al. | Chromatin interaction analysis using paired‐end tag sequencing | |
| US10975370B2 (en) | Methods for screening nucleic acid aptamers | |
| Luo et al. | Development of a fraction collection approach in capillary electrophoresis SELEX for aptamer selection | |
| CN103224936B (en) | The aptamer of one group-specific identification staphylococcus aureus toxin A | |
| Datinská et al. | Recent progress in nucleic acids isotachophoresis | |
| CN104975081A (en) | Amplimers, kit and method for detecting PKD1 gene mutation | |
| Turnpenny et al. | Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid–liquid extraction LC–MS/MS analytical approach | |
| CN103525820A (en) | Affinity nucleic acid molecule screening method based on sectioned collection | |
| WO2006079009A3 (en) | Methods for separating short single-stranded nucleic acid from long single- and double-stranded nucleic acid, and associated biomolecular assays | |
| CN103243101B (en) | One group of aptamers for specifically recognizing staphylococcus aureus enterotoxin C1 | |
| CN115101128A (en) | Method for evaluating off-target risk of hybridization capture probe | |
| Thomas et al. | Deployment and detection of a novel barcoded advanced tracers system for the optimization of improved waterflood recovery in hydrocarbon reservoirs | |
| Luo et al. | Simultaneous measurement of surface proteins and gene expression from single cells | |
| CN102980878A (en) | Quantitative plasmid DNA (Deoxyribonucleic Acid) detection kit | |
| CN115948607B (en) | Method and kit for simultaneously detecting multiple pathogen genes | |
| CN102796829A (en) | LAMP detection kit for macrobrachium rosenbergii nudivirus and detection method thereof | |
| Chen et al. | Aptamer‐based thrombin assay on microfluidic platform | |
| US9938567B2 (en) | High-throughput RNA interaction assay | |
| WO2020043174A1 (en) | High-throughput next-generation sequencing method for double-stranded nucleotide sequencing | |
| CN106650313B (en) | A method of it filtering out DNA base in DNase high-flux sequence data and is inclined to sexual deviation | |
| JP2008256636A (en) | Methods of quantitatively determining nitrate nitrogens, anions, and nitrogens in three states | |
| Mifsud | Introduction to ChIP-seq |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140122 |