CN108804872A - The method for eliminating three-dimensional genomics technologies noise and application are combined using excision enzyme - Google Patents
The method for eliminating three-dimensional genomics technologies noise and application are combined using excision enzyme Download PDFInfo
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Abstract
The invention discloses a kind of to combine method and the application for eliminating three-dimensional genomics technologies noise using excision enzyme, this method is that excision enzyme combination is added when connecting by the way that the committed step in three-dimensional genomics technologies is neighbouring to eliminate linear DNA after neighbouring connect, and improves cyclic annular effectively interaction ratio.It is to combine digestion elimination " noise " by excision enzyme after the connection of C- technologies to provide a kind of effective means that the present invention, which is combined by excision enzyme in the superior performance in eliminating linear DNA, it is convenient that the present invention has using the method that excision enzyme combination endonuclease reaction eliminates three-dimensional genomics technologies noise, fast, efficient advantage, the possibility for generating false positive products or signal can be reduced, increase effective interaction cyclic DNA accounting, greatly improve the effective interaction data yield ratio of terminal, save sequencing space, reduce sequencing cost, simplify the resource spent by invalid information in the biological information means elimination big data of later-stage utilization complexity.
Description
Technical field
The present invention relates to genomic sequencing technique fields, particularly belong to three-dimensional genomics, be related to it is a kind of utilize excision enzyme
Method and the application of three-dimensional genomics technologies noise are eliminated in combination.
Background technology
Chromatin conformation capture technique (C- technologies) is the important technology in three-dimensional genomics technologies.C- technologies are answered extensively
For illustrating genome interaction folding mode, genome three dimensional topology is reconstructed, the transcriptional control and cell life of biology are studied
The important biomolecules knowledge topic such as fortune decision.C- technologies are mainly developed by 3C technologies (chromatin conformation capture technique), including
3C, 4C (cyclisation chromatin conformation capture technique), 5C (chromatin conformation captures carbon duplication technology), Hi-C (high-throughput chromatin
Conformation captures sequencing technologies), ChIA-PET (the chromatin interactive analysis technology based on pairing end tag sequencing).Than
Such as, by 3C technologies, researcher, which is found that, influences the sites the H19-Igf2 interaction that flesh generates differentiation；By Hi-C technologies, grind
Study carefully in the relatively conservative TAD (topological correlation domain) and TAD that CTCF/Cohesin albumen on personal appraisal genome is anchored
The relationship of the gene expression of portion's dynamic change and cell-specific.The C- technologies of these continuous developments are exactly based on, researcher can
To probe into the relationship of genome dynamic change and transcriptional control function from the unique perspective of three-dimensional genomics, it is reconstructed eukaryon life
Three-dimensional genome structure in object.
Most of interaction is interaction (including promoter and the enhancing between the genomic DNA that albumen corpusculum participates in genome
Sub- interaction, promoter and promoter interaction, enhancer and enhancer etc.).C- technology cardinal principles is after genomic fragments
Interaction between the DNA that albumen corpusculum is mediated is separated, after the neighbouring connection of albumen corpusculum intramolecular and by PCR or
NGS (next generation) sequencings read out the DNA of interaction (see Fig. 2).So it includes 3C, 4C, 5C, ChIA-PET that neighbouring connection, which is,
With the committed step of all C- technologies including Hi-C.Hi-C technologies are developed to from 3C technologies, researcher can be by primary real
Test the single crawl full-length genome interaction mutually accomplished " entirely to complete " of crawl from " one-to-one ".C- technologies still have very high make an uproar
Sound, and need cumbersome biological information to work and remove the effective interaction of noise excavation.C- technologies are still neighbouring connection at present
PCR detections are directly carried out afterwards or purifying DNA carries out the fixation of subsequent bio element, builds library step.Since genome is endonuclease bamhi
Change, can mix unwanted linear DNA (i.e. " noise "), such as the incomplete DNA (interior segments of digestion after neighbouring connection
It is Internal, reconnection, continuously coupled), the biotinylated DNA (suspension end Dangling end) connected not successfully, not
DNA (intermolecular connection DNA) after being connected between the different albumen corpusculums of interaction is connected to DNA (the random connections of free segment
DNA) (see Fig. 1).After neighbouring connection, the cyclic DNA overwhelming majority that head and the tail connection is formed is that effectively connection is (a small amount of to be removed from connection
Outside), while in system these the unwanted various linear DNAs mixed are exactly the main source of C- technology noises.To 3C, 4C,
For 5C, in the case of various linear voices exist after neighbouring connection, PCR will produce heteroduplex (heteroduplexes), embedding
With the false positive products such as chain (chimeras) and amplification deviation (biases, template DNA topology are inconsistent).For Hi-
C, for ChIA-PET, in the case of various linear voices exist after neighbouring connection, a large amount of sequencing spaces can be occupied so that effectively mutually
It is low to make accounting, waste sequencing resource.For example, reliable effective interaction ratio that classics ChIA-PET technologies obtain only has 0.042%.
About 60% is invalid interaction in the data that classical Hi-C technologies obtain.This crucial Connection Step institute band of attention is not yet had been reported that at present
The high noisy come, removes these linear DNA noises, is to eliminate high noisy to three-dimensional genomics technologies (C- technologies), increase skill
The important impetus of art efficiency and result specificity.
Invention content
In view of the above technical problems, it is made an uproar using the three-dimensional genomics technologies of excision enzyme combination elimination the present invention provides a kind of
The method of sound, wherein addition excision enzyme combination elimination is neighbouring even in the neighbouring connection of the committed step of three-dimensional genomics technologies
Rear linear DNA is connect, cyclic annular effectively interaction ratio is improved.
It is described that the method for eliminating three-dimensional genomics technologies noise is combined using excision enzyme, wherein the excision enzyme combination
Including two excision enzymes Lambda and RecJF.
It is described that the method for eliminating three-dimensional genomics technologies noise is combined using excision enzyme, wherein the excision enzyme combination
Further include Lambda Buffer or the CutSmart Buffer for coordinating two excision enzymes Lambda and RecJF to use.
It is described that the method for eliminating three-dimensional genomics technologies noise is combined using excision enzyme, wherein the three-dimensional genome
Technology is with the neighbouring three-dimensional genomics technologies for being connected as base growth and coming of 3C-
It is described that the method for eliminating three-dimensional genomics technologies noise is combined using excision enzyme, wherein the three-dimensional genome
Technology includes 3C, 4C, 5C, ChIA-PET and Hi-C technology.
The application as described above that three-dimensional genomics technologies noise method is eliminated using excision enzyme combination, wherein utilize
The elimination application of excision enzyme combination noise in the neighbouring connection of three-dimensional genomics technologies committed step.
The present invention by excision enzyme combine the superior performance in eliminating linear DNA be C- technologies connect after by circumscribed
Enzyme combination digestion eliminates " noise " and provides a kind of effective means, is to eliminate height to three-dimensional genomics technologies (C- technologies) to make an uproar
Sound, the important impetus and innovative point for increasing technical efficiency and result specificity.The present invention combines endonuclease reaction using excision enzyme
The method for eliminating three-dimensional genomics technologies noise.Remarkable advantage be it is convenient, fast, efficient, will connection rear pattern plate or library by
Hybrid state becomes the more uniform circular DNA template containing effective interaction or library, reduce generate false positive products or
The possibility of person's signal increases effective interaction cyclic DNA accounting, substantially increases the effective interaction data yield ratio of terminal,
Sequencing space is saved, sequencing cost is reduced, simplifies and believes in vain in the biological information means elimination big data of later-stage utilization complexity
The spent resource of breath.
Description of the drawings
Fig. 1 is the experiment noise in three-dimensional genomics technologies C- technologies.
Fig. 2 is tactful schematic diagram of the excision enzyme combination for eliminating C- technology noises.
Fig. 3 is that DNA principles is eliminated in excision enzyme combination cutting
Fig. 4 is the agarose gel electrophoresis result that linear plasmid mixing species center line DNA is eliminated in excision enzyme combination.
Fig. 5 is that Qubit quantifies excision enzyme combination elimination linear DNA plasmids experimental result.
Fig. 6 is that QPCR quantifies excision enzyme combination elimination linear DNA plasmids experimental result.
Fig. 7 is that 572ng DNA library linear voices eliminate agarose gel electrophoresis result.
Fig. 8 is that 572ng DNA library linear voices eliminate Qubit quantitative ratio results.
Fig. 9 is that 40ng DNA library linear voices eliminate agarose gel electrophoresis result
Figure 10 is that 40ng DNA library linear voices eliminate Qubit quantitative ratio results.
Figure 11 is that 40ng DNA libraries (add with plasmid) linear voice to eliminate agarose gel electrophoresis result.
Figure 12 is that 40ng DNA libraries (add with plasmid) linear voice to eliminate Qubit quantitative ratio results.
Figure 13 is the Hi-C HiCUP software filter results combined by LRL after noise Processing for removing.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, the every other implementation that those skilled in the art are obtained without creative efforts
Example, shall fall within the protection scope of the present invention.
The basic thought of the present invention：As mentioned in the background, three-dimensional genome the relevant technologies (C- skills classical at present
Art：3C, 4C, 5C, ChIA-PET and Hi-C) in, neighbouring connection is the committed step of C- technologies, and still " noise " is very high (see Fig. 1
And Fig. 2).They are based on following principle, if in the same albumen corpusculum wraps up interaction occurs for two DNA elements (segment),
The end of the two DNA fragmentations will be suffered close.The two ends DNA are connected, just " crawl " this interaction pass
System, after the digestion cracking of albumen corpusculum, this interaction (ring) exists always in the DNA of purifying.Then by PCR or next
Quantitative this connecting node of generation sequencing (NGS), so that it may with for detecting and quantify the interaction between genomic elements.Neighbouring connection
After can form interaction DNA circle, this just contains effective interaction information (see Fig. 2).But us is worth to pay attention to, simultaneously also
It forms a large amount of " noise ".These noises are almost linear DNA form (see Fig. 2).Linear DNA is being eliminated in excision enzyme combination
In superior performance be C- technologies connection after by excision enzyme combine digestion eliminate " noise " provide a kind of effective means.Cause
This, it is a kind of high efficiency method to C- technology noise reductions to combine elimination linear voice using excision enzyme and improve cyclic DNA ratio.It should
Strategy can be used for optimizing all three-dimensional genomics technologies developed based on " the neighbouring connections of 3C- " (see Fig. 2).Under
Will in conjunction with specific embodiments and technical scheme of the present invention be described with reference to the attached figures in face.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified, and is certainly
Standard PCR, sequencing build library biochemical reagents shop (Thermo Fisher, NEB, KAPA, Illumina etc.) purchase.
Embodiment 1, the assessment of excision enzyme combined effect and its application in eliminating linear plasmid DNA.
One, the selection of excision enzyme and excision enzyme combination
Following excision enzyme is chosen from NEB (New England Biolabs), such as table 1.
Linearly related excision enzyme and its feature are tested in removal to table 1
Single excision enzyme has a single function, and the excision enzyme that two kinds are had complementary functions is combined, it will than single excision enzyme
There are more comprehensive function and better removal effect.It is reported according to having complementary functions property and its pertinent literature, is used in the present embodiment
Lambda and RecJF excision enzymes combine (see Fig. 3)：LRL is combined, and the merging of Lambda and RecJF excision enzyme groups is allowed in Lambda
It is reacted in buffer；LRC is combined, and the merging of Lambda and RecJF excision enzyme groups is allowed to react in CutSmart buffer.
Two, linear plasmid DNA evaluation tests are eliminated in excision enzyme combination
Plasmid DNA has supercoil annular form, linear forms and open loop form, is a kind of to eliminate the good of linear trial test
Material.Extract using EZNA Endo-free Plasmid Mini Kit II kits and to specifications pGL4.23 plasmids
(4283bp), with restriction enzyme BamHI-HF digested plasmid pGL4.23, the plasmid pGL4.23 linearized, linearisation
Plasmid run 1% agarose gel size and be shown as neat clean about 4kb sizes linear strip (4283bp).For better mould
The case where linear and cyclic DNA mixes after neighbouring connection in quasi- C technologies, by etc. the protoplasm grains of quality mixed with linearization plasmid
Come, obtains a sample mixing (Mixture).Including two kinds of excision enzyme combinations in CutSmart buffer and Lambda buffer
The processing reacted respectively is for eliminating the linear DNA form in sample mixing Mixture.Endonuclease reaction system is respective treated circumscribed
Enzyme combination, corresponding reaction solution (Buffer) and ultra-pure water and digestion substrate Mixture.Enzyme amount used in the present embodiment
It is enough to eliminate whole linear DNA amounts, the digestion time is 1h.After conventional phenol chloroform purification process, agarose gel electrophoresis is used
Detection is linear to be eliminated as a result, electrophoresis result is shown in Fig. 4.The result shows that cyclic annular, open loop and it is linear exist simultaneously in the case of,
LRL, LRC combination can successfully removal be linear in Mixture.
Three, linear plasmid DNA quantitative tests are eliminated in excision enzyme combination
1, Qubit is quantitative
For the effect that better quantitative linearity is eliminated, the DNA concentration of digestion product by Qubit (Qubit 3.0) into
Row measures, and sees Fig. 5.Mixture M ixture linearly remove ratio be 61.36% (LRL combinations), 66.56% (LRC combinations), two
A combination eradicating efficacy is suitable.
2, QPCR is quantitative
For the effect that more accurate quantitative linearity is eliminated, digestion product by XP magnetic beads for purifying, (build in library often by DNA sequencing
With the method for purifying DNA) after, it is quantitative to carry out QPCR in the system of same volume for back dissolving.The BH2 primers used in quantitative are
Across BamHI-HF restriction enzyme sites, the purpose designed in this way is can to expand cyclic annular signal by BH2 primers, but can not expand line
Property signal.See Fig. 6, compared with Mixture mixture groups, two combination eradicating efficacies are suitable.
Embodiment 2, the excision enzyme combination application that noise is eliminated in C- key problem in technology steps.
C- technologies are used for three genomics chromatin conformation crawls, it is important to derived from the committed step of neighbouring connection.
How the ratio of cyclisation connection molecule is improved, it is extremely important for subsequent detection DNA joint efficiencies.C- technologies are adjacent to Connection Step
Before, key step is consistent (see Fig. 2).The excision enzyme combination that the present embodiment is had verified that using the experiment of 1 plasmid of embodiment disappears
Except existing linear " noise " DNA after neighbouring connection in C- technologies, and it is successfully authenticated its effect efficiently eliminated.Due to current
In C- technologies, the cell initial amount used finds that initial amount is 0.1million starting from 0.01~25million, experiment
Cell can obtain about 300ng DNA libraries, and 0.01million cells about obtain 30ng DNA libraries.The present embodiment is selected
570ng and 40ng library DNAs carry out implementation explanation.
1, noise eliminates experiment after the neighbouring connection of 572ng DNA C- technologies
C2C12 cells are fixed into chromatin (genome interaction is fixed up) by 1% formaldehyde crosslinking, then pass through 0.2M
Glycine quenches formaldehyde.After 1x DPBS wash twice, cell is collected into centrifuge tube with cell spatula.The cell that will be fixed
It is distributed into about every pipe about 0.5million cells.Chromatin is released completely by cell cracking and cell karyorhexis.Often
2ul Hind III digestion is added overnight in pipe.It is blunt ends to carry out digestion end mark and polishing according to conventional C- technical steps.
Connection enzyme system is added, carries out neighbouring connection.Then Proteinase K and SDS is added, is allowed to crack digestion in 1~1.5%SDS
At least 1h.DNA library is extracted, obtain at this time include a large amount of " linear voices " library (see Fig. 2).In order to better
The eradicating efficacy for comparing excision enzyme combination needs the starting amount of DNA of homogeneous phase etc. between guarantee group.Therefore, it will often pipe DNA library mix
Unify, and is averagely packed as 572ng/ pipes.Two kinds of excision enzymes combine LRL, and LRC is separately added into each pipe, is removed line
Property digestion.1 is added after digestion：The Ampure XP magnetic beads of 1.2 times of volumes are purified.It is assessed by agarose gel electrophoresis
The topological form in library after processing, this mode can more intuitively reflect " linear voice " removal effect.It is examined by Qubit
It surveys the quality of remaining cyclic DNA and calculates linear voice DNA removal efficiency.Agarose gel electrophoresis is as a result, be shown in Fig. 7.As schemed
Show, digests, compared with control group Co, removed by 1h, LRL by removal, LRC successful purifications cyclic DNA, with embodiment 1
Plasmid test result is consistent.Because the experiment of embodiment 1 confirms, if in all linear DNAs (500ng~600ng) 1
Hour is enough almost all and removes linear DNA.Therefore, mix template under same system by after linear eliminate, remaining is ring
Shape form.It is handled by two kinds of excision enzyme combination linear removals, the cyclic DNA ratio that Qubit after purification is detected, sees Fig. 8.Such as
Shown in figure, cyclic annular effectively library ratio is 18.19% (LRL), 34.19% (LRC) after two kinds of combined treatments.
2, noise eliminates experiment after the neighbouring connection of 40ng DNA C- technologies
In C- technologies, cyclic plasmid DNA can be added after neighbouring connection, inherent reference is used as (to make when running glue and fragmentation
For reference), the present embodiment includes with cyclic plasmid DNA (see Figure 11) and without with cyclic plasmid DNA (see Fig. 9).
For 0.01million cell initial amount C- technologies, the present embodiment devises comparable library DNA amount removal therewith
" noise " is tested.C2C12 cells are passed through into the same processing in the parts embodiment 2-1, crosslinking, cell collection, cell cracking, cell
Karyorhexis, Hind III digestions fragmentation, interaction DNA end mark polishings, neighbouring connection cyclization, purified library.By library etc.
Amount dispenses often pipe 40ng.LRL is combined using excision enzyme, LRC is separately added into each pipe and is removed linear " noise " digestion.Digestion
1 is added later：The Ampure XP magnetic beads of 1.2 times of volumes are purified.Carry out Qubit detections and agarose gel electrophoresis detection.
It is small due to originating total Library Quality, after excision enzyme combined treatment, although Qubit detections show cyclic DNA be it is existing,
But agarose gel electrophoresis is difficult to observe by band.See Fig. 9.The elimination ratio and cyclic annular library that Qubit detections are calculated
Accounting is shown in that Figure 10, cyclic DNA library accounting are 6.04% (LRL), and 9.27% (LRC) has obtained result and embodiment 2-
1Qubit result trend is consistent.
Before excision enzyme combination is eliminated, we add with cyclic plasmid DNA, as a display internal reference.40ng
Plasmid pGL4.23 be added in the libraries 40ng, excision enzyme combines LRL, LRC, eliminate linear, after magnetic beads for purifying, carries out
Agarose gel electrophoresis detects and Qubit detections.Agarose gel electrophoresis the result is shown in Figure 11.It can be seen that use plasmid in
After ginseng, it can be seen that the cyclic plasmid DNA bands left after processing, Qubit detections are calculated cyclic annular ratio and see Figure 12, such as scheme
Shown, cyclic DNA library accounting is 87.5% (LRLP1), 52.5% (LRCP1), is added with cyclic plasmid DNA, linearly disappears
Except rear cyclic annular ratio accounting increases.This can also be used with plasmid strategy in C- technologies.
Embodiment 3, the excision enzyme combination application that noise is eliminated in Hi-C technologies.
Hi-C is high-throughput chromatin conformation crawl technology, based on 3C technologies, can capture the mediation of albumen corpusculum
Genome whole interaction.Neighbouring connection is the key that one step of Hi-C technologies.Neighbouring connection is high noisy at present, which is mark
One of the main reason for quasi- effective interaction rates of Hi-C are low (see Fig. 1 and Fig. 2).1 excision enzyme of embodiment combines removing method in plasmid
It is verified in experiment.2 excision enzyme of embodiment combines removing method after C- technologies share core procedure-" neighbouring connection "
Noise reduction obtains compliance test result.The present embodiment is quasi- to confirm that excision enzyme combination eliminates noise method in one of specific C- technologies member-
The eradicating efficacy applied in Hi-C technologies：Improve effective interaction ratio.
1, the Lambda+RecJF combinations application effect that noise is eliminated in Hi-C technologies
First, formaldehyde crosslinking, cell cracking, HindIII digestions, DNA are carried out to C2C12 cells by Hi-C conventional steps
Terminal biotin marks polishing, neighbouring blunt ends to connect, and the combination of LRL excision enzymes is added according to 2 same system of embodiment later,
That is Lambda+RecJF.And it is added simultaneously with cyclic plasmid DNA.37 DEG C of digestion 1h eliminate linear voice.Have in increase system
Imitate the ratio of interaction ring.Purified library.Then, continue to operate according to standard Hi-C steps：Utilize sonicator Covaris
S220 interrupts library, utilizes MyOneTMStreptavidin T1 immunomagnetic beads capture the mutual of biotin labeling
Make DNA, building library kit using NGS carries out building library amplification, and machine sequencing kit on Illumina is utilized to carry out NovaSeq sequencings
Machine is sequenced on instrument.It is operated in strict accordance with kit specification.By software raw2clean (novogene) after lower machine, filter out
Sequence containing connector, the low sequence of base mass value read N sequences.Then, pass through HiCUP 0.5.9 (http://
Www.bioinformatics.babraham.ac.uk/projects/hiccup/) software carries out effective interaction data filtering,
It is removed including truncating, theoretical digestion reference gene group, compares and arrive reference gene group, filter the false positive interaction from experiment,
PCR is removed to repeat.It is final to obtain effective and invalid interaction.The LRL excision enzymes combination application that noise is eliminated in Hi-C technologies
Effect is shown in Figure 13.As shown, testing the Hi-C technology noises that can debase the standard by LRL excision enzyme combined treatments Hi-C, make
It obtains effectively interaction comparative example in LRL-Hi-C and reaches 86%, be twice of standard Hi-C.Noise ratio is reduced to by~60%~
14%.The present embodiment is to be based on standard Hi-C systems by the improvement of excision enzyme reducing noise for combined.Similarly, in situ Hi-C systems
System is added excision enzyme combination and may also used to noise reduction after in situ connections (connection in core) when reverse cross-link.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, it will be understood by those of ordinary skill in the art that：It still can be to aforementioned each implementation
Technical solution recorded in example is modified or equivalent replacement of some of the technical features；And these modification or
It replaces, the spirit and scope for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of combining the method for eliminating three-dimensional genomics technologies noise using excision enzyme, which is characterized in that in three-dimensional gene
Excision enzyme combination elimination linear DNA after connection is added when the committed step of omics technology is adjacent to connection, improves cyclic annular effective
Interaction ratio.
2. the method for utilizing excision enzyme combination to eliminate three-dimensional genomics technologies noise according to claim 1, feature exist
In the excision enzyme combination includes two excision enzymes Lambda and RecJF.
3. the method for utilizing excision enzyme combination to eliminate three-dimensional genomics technologies noise according to claim 2, feature exist
In, excision enzyme combination further include the Lambda Buffer for coordinating two excision enzymes Lambda and RecJF to use or
CutSmart Buffer。
4. the method for utilizing excision enzyme combination to eliminate three-dimensional genomics technologies noise according to claim 1, feature exist
In the three-dimensional genomics technologies is with the neighbouring three-dimensional genomics technologies for being connected as base growth and coming of 3C-.
5. the method for utilizing excision enzyme combination to eliminate three-dimensional genomics technologies noise according to claim 4, feature exist
In the three-dimensional genomics technologies include 3C, 4C, 5C, ChIA-PET and Hi-C technology.
6. as described in any one in claim 1-5 combined using excision enzyme eliminates answering for three-dimensional genomics technologies noise method
With, which is characterized in that using excision enzyme combination, the elimination of noise is answered in the neighbouring connection of three-dimensional genomics technologies committed step
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4108767A1 (en) * | 2021-06-22 | 2022-12-28 | The Procter & Gamble Company | Cleaning or treatment compositions containing nuclease enzymes |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8642295B2 (en) * | 2007-01-11 | 2014-02-04 | Erasmus University Medical Center | Circular chromosome conformation capture (4C) |
| CN104561362A (en) * | 2015-02-03 | 2015-04-29 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing library and construction method thereof |
| CN106566828A (en) * | 2016-11-11 | 2017-04-19 | 中国农业科学院农业基因组研究所 | Efficient whole-genome chromosome conformation capture technology (eHi-C) |
| CN107208160A (en) * | 2015-01-30 | 2017-09-26 | 哈佛学院院长及董事 | Without microscope imaging |
| CN107475394A (en) * | 2017-08-31 | 2017-12-15 | 中国农业科学院农业基因组研究所 | One kind is based on a small amount of cell full-length genome chromatin high-resolution conformation technology eHi C2.0 |
-
2018
- 2018-06-08 CN CN201810589904.0A patent/CN108804872A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8642295B2 (en) * | 2007-01-11 | 2014-02-04 | Erasmus University Medical Center | Circular chromosome conformation capture (4C) |
| CN107208160A (en) * | 2015-01-30 | 2017-09-26 | 哈佛学院院长及董事 | Without microscope imaging |
| CN104561362A (en) * | 2015-02-03 | 2015-04-29 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing library and construction method thereof |
| CN106566828A (en) * | 2016-11-11 | 2017-04-19 | 中国农业科学院农业基因组研究所 | Efficient whole-genome chromosome conformation capture technology (eHi-C) |
| CN107475394A (en) * | 2017-08-31 | 2017-12-15 | 中国农业科学院农业基因组研究所 | One kind is based on a small amount of cell full-length genome chromatin high-resolution conformation technology eHi C2.0 |
Non-Patent Citations (1)
| Title |
|---|
| 张玉波: "三维基因组学与精准生物学", 《中国生物化学与分子生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4108767A1 (en) * | 2021-06-22 | 2022-12-28 | The Procter & Gamble Company | Cleaning or treatment compositions containing nuclease enzymes |
| WO2022272255A1 (en) * | 2021-06-22 | 2022-12-29 | The Procter & Gamble Company | Cleaning or treatment compositions containing nuclease enzymes |
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