CN106754868A - A kind of method of the DNA fragmentation interacted in capture Matrix attachment region - Google Patents
A kind of method of the DNA fragmentation interacted in capture Matrix attachment region Download PDFInfo
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- CN106754868A CN106754868A CN201611071277.9A CN201611071277A CN106754868A CN 106754868 A CN106754868 A CN 106754868A CN 201611071277 A CN201611071277 A CN 201611071277A CN 106754868 A CN106754868 A CN 106754868A
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- restriction enzyme
- dna fragmentation
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- attachment region
- matrix attachment
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1051—Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
Abstract
The present invention relates to a kind of high flux chromosomal conformation catching method, it is characterised in that comprise the following steps：S1：Sample cell is taken, is crosslinked；S2：Crack the sample cell；S3：Inactivating endogenous enzyme；S4：After digestion with restriction enzyme genome, produces with 5 ' prominent cohesive ends after the digestion with restriction enzyme double-stranded DNA, and the cohesive end is blunted with the restriction enzyme site that does not produce the restriction enzyme is connected after another cohesive end filling-in；S5：The cohesive end that digestion with restriction enzyme in filling-in S4 is produced, and add mark；S6：Connection；S7：By chromatin fragments, capture zone has the fragment of the molecular labeling, builds library.Method using the DNA fragmentation interacted in capture Matrix attachment region of the invention can reduce reaction system, simplify experimental procedure, and reduce noise pollution.
Description
Technical field
The present invention relates to molecular biology experiment technical field, more specifically it relates in a kind of capture Matrix attachment region mutually
The method of the DNA fragmentation of effect.
Background technology
The capture of high flux chromosomal conformation (High-throughput chromosome conformation capture,
Hi-C extensive use is subject to the interaction information between) technology is because that can obtain full-length genome all sites, its basic thought is
Their interaction relationship is reacted according to chromatinic physical spatial location.
Current Hi-C experimental programs mainly have dilution Hi-C (DilutionHi-C), original position Hi-C (in situ Hi-
C Hi-C (in nucleus Hi-C)) and in core.Common Hi-C methods generally comprise following steps：Formaldehyde treated cell,
The chromatin and its neighbouring protein cross in cell, cell lysis is set with the endogenous enzyme of SDS killed cells, to be used in combination
Triton X-100 are quenched SDS；Then with digestion with restriction enzyme chromatin generation cohesive end, filling-in cohesive end, and
A kind of ribonucleoside triphosphote of biotin labeling is added in filling-in system, so that the end by filling-in to digestion adds biotin
Mark, with the enzyme in SDS deactivation systems, prevent the fragment that follow-up coupled reaction is obtained because restriction enzyme site recovers by restriction endonuclease again
Secondary cut-out；Then reaction is attached, makes, because being combined with protein and the connection of the end of DNA close to each other, to make after connection
It is crosslinked with Proteinase K solution；Purifying DNA, and the biotin of disconnected DNA ends is removed, make DNA fragmentation, then with life
Thing element-Streptavidin effect fragment of the capture with biotin labeling, adjunction head is built into library, for later stage sequencing.
But existing Hi-C schemes there is a problem of certain：First, dilution Hi-C a large amount of examinations because its big system need to be consumed
Agent (linked system is more than 6 times of Hi-C in situ), and typically terminate all in a system to coupled reaction from cell cracking
In carry out, period is not purified, and so causes system increasing, causes experiment process than relatively time-consuming, and causes experiment
Poor reproducibility, the data reliability for obtaining and repeatability be not high；Secondly, it is reduction noise, Hi-C inactivating endogenous enzyme in core
Method be incubated 60 minutes in 37 DEG C by using SDS and carry out, because process time is long can influence some cell types
Nucleus internal structure, causes the sample applicability of such scheme relatively low；Again, Hi-C needs first to use restriction endonuclease enzyme in core
Cut, after mark is added in filling-in, then coupled together interaction fragment with ligase, in order to prevent the restriction endonuclease in connection procedure
Digestion again, so often through addition lauryl sodium sulfate (sodium dodecyl sulfate, SDS) inactivation restriction endonuclease
And SDS is quenched to cause that subsequent reactions are smoothed out, while increasing with Triton X-100 (Triton X-100)
Reaction system, increased follow-up reagent consumption.
Accordingly, it would be desirable to a kind of method of the DNA fragmentation interacted in new capture Matrix attachment region, reduces reaction system,
Experimental procedure is simplified, and reduces noise pollution.
The content of the invention
To solve problem above, the invention provides a kind of high flux chromosomal conformation catching method, it includes following step
Suddenly：
S1：Sample cell is taken, is crosslinked；
S2：Crack the sample cell；
S3：Inactivating endogenous enzyme；
S4：Digestion with restriction enzyme genome, produces prominent with 5 ' after the digestion with restriction enzyme double-stranded DNA
Cohesive end, and after the cohesive end is blunted be connected after another cohesive end filling-in do not produce it is described it is restricted in
The restriction enzyme site of enzyme cutting；
S5：The cohesive end that digestion with restriction enzyme in filling-in S4 is produced, and add mark；
S6：Make because with protein binding and DNA fragmentation close to each other；
S7：By chromatin fragments, capture zone has the fragment of the molecular labeling, builds library.
Being reconnected due to have chosen special restriction enzyme, after its restriction enzyme site filling-in will not recover protoenzyme and cut position
Point, can be directly added into linked system and be attached, it is not necessary to which restriction endonuclease is again switched off connection in considering connection procedure below
The problem of fragment, so without adding SDS to inactivate restriction endonuclease again in system after the digestion filling-in.Experiment step is simplified
Suddenly, reaction system is reduced.
Further, also digested using pancreatin after the crosslinking, disperseed the sample cell.So that follow-up cracking
Middle cell is fully contacted with lysate.
Further, by adding final concentration of 0.5%SDS in S3,62 DEG C of incubations carry out inactivating endogenous enzyme in 10 minutes；S4
Restriction endonuclease is not inactivated between S5.General method is that, by adding 0.1-0.3%SDS, 65 DEG C are incubated 10 minutes come in inactivating
Exogenous enzyme；Then by adding about 1.48%SDS after digestion, 65 DEG C are incubated 30 minutes to inactivate restriction endonuclease.But so lead
Cause experimental result noise is very big, and the ratio of cis interaction is only 45% effect.By to SDS final concentrations and incubation temperature and when
Between reset, the present invention substantially increases signal to noise ratio.
Further, after using SDS inactivating endogenous enzymes, with the SDS in also addition TritonX.To prevent SDS treatment
DNA structure is damaged too long.
Further, after the sample cell is cracked, supernatant is abandoned in centrifugation, and precipitation is used for into subsequent operation, in inactivation
After exogenous enzyme, supernatant is abandoned in centrifugation, and precipitation is used for into subsequent operation.Reaction system can significantly be reduced by the operation.
Further, the restriction enzyme is the one kind in BamHI, HindIII, NcoI, ScaI, SpeI, XbaI
Or several combinations.
Further, the mark is biotin.
Further, one or more nucleotides point that the biotin-conjugated is added when filling-in to the cohesive end
On son.
Brief description of the drawings
Fig. 1 is the electrophoretogram of HindIII digestion postgenomes；
Fig. 2 is the electrophoretogram of NheI digestions inspection；
Fig. 3 is detect the 2100 glue figures for obtaining to library DNA.
Specific embodiment
Principle of the invention and feature are described below in conjunction with example, example is served only for explaining the present invention, and
It is non-for limiting the scope of the present invention.
Our Secondary Culture Human cell line A549 cells first, make its cell fusion degree reach about 80%.We need
It is crosslinked, cracked, digestion, connection, ultrasound, two generations is built a series of experiments step such as storehouse and biotin capture, to obtain height
The Hi-C libraries of quality.Specifically experimental procedure is：
1) cell culture of culture abandons nutrient solution, is washed one time with 5mL 1x PBS (phosphate buffer), and PBS is abandoned in suction；
2) 5mL 1x PBS are added, be subsequently adding the paraformaldehydes of 5mL 2% or formaldehyde (final concentration of 1%, also directly add
The paraformaldehyde or formaldehyde of the final concentration 1% that 10mL 1x PBS are prepared), it is incubated 10 minutes at room temperature, rock one within every two minutes
It is secondary, or be placed directly on shaking table and slowly rock, make intracellular DNA with its near protein cross；
3) 1.11mL 2.0M glycine (final concentration 0.2M, 1x PBS preparations) are added, is incubated at room temperature 5 minutes, suitably shaken
Shake, neutralize paraformaldehyde or formaldehyde；
4) inhale and abandon buffer solution, (period is spaced a few minutes and rocks one to add 37 DEG C of 0.25% pancreatin of 2mL to be incubated 10 minutes
It is secondary), cell culture medium of the two volumes containing 10% hyclone is added, scrape collection cell with cell and managed (after crosslinking in 2 15mL
Cell be difficult digestion and get off), slightly blow and beat cell dispersion, cross cell sieve (typically directly toppling over), 650g room temperatures are centrifuged 5
Minute, supernatant is abandoned, -80 DEG C of cells for freezing crosslinking are stand-by；
5) crosslinking cell for freezing is taken out in thawed on ice, and cell is resuspended in the cold lysate (Tris-HCl of 250 μ L
PH8.0 10mM, sodium chloride 10mM, NP40 0.2%, 1X protease inhibitors (article No. Roche-4693116001)) in,
4 DEG C of 2500g is centrifuged 5 minutes, and supernatant is abandoned in suction；Precipitation is resuspended in the 250 cold lysates of μ L (Tris-HCl pH8.0 10mM, chlorine
Change sodium 10mM, NP40 0.2%, 1X protease inhibitors (article No. Roche-4693116001)) in, it is incubated 15 minutes；2500g
4 DEG C are centrifuged 5 minutes, abandon supernatant；
6) will precipitate and be resuspended in 1 × NEBuffer 2 of the 50 μ L containing 0.5%SDS, careful piping and druming is careful not to produce gas
Bubble, 62 DEG C are incubated 10 minutes；65 μ L water and 25 μ L TritonX-100 are added, careful piping and druming is careful not to produce bubble, 37 DEG C
15 minutes (the μ L of total system 140 take 10 μ L for genomic integrity Quality Control) is incubated, 3000g 2 minutes abandons supernatant；
7) precipitation is resuspended in (μ L, the 10U/ μ L of water 80 μ L, 10 × NEBuffer2.1 10.0 in 100 μ L digestion systems
The μ L of HindIII 10.0), mix, 37 DEG C are overnight (the μ L of total system 100 take 10 μ l for the Quality Control of genome digestion effect)；
8) filling-in, to filling-in system is added in the system after digestion, one of which ribonucleoside triphosphote is by biotin labeling (water
The μ L of 11.8 0.6 μ L, 10mM dTTP of μ L, 10 × NEBuffer2 2.0 μ L, 10mM dATP, 0.6 μ L, 10mM dGTP 0.6,
The μ L of 1.2 μ L, 5U/ μ L Klenow of 5mM biotin-14-dCTP 3.2), leniently piping and druming is mixed up and down, is incubated in 37 DEG C of rotations
Educate 45 minutes；
9) connect, to adding the 292 μ L linked systems (μ of 172 μ L, 10%Triton X-100 of water 40 in the system after filling-in
The μ L of 40 μ L, 1U/ μ L T4DNA ligase of L, 10 × T4ligation buffer 40), overturn and mix, 16 DEG C are incubated 4 hours；
10) add 65 DEG C of 10 μ L 20mg/mL Proteinase Ks to be incubated 2 hours, be then loaded into 10 μ L 20mg/mL Proteinase Ks,
65 DEG C of overnight incubations (solution crosslinking)；
11) reaction mixture is cooled to room temperature, one times of chloroform separates and recovers supernatant, adds 1/10 volume sodium acetate and two
- 20 DEG C of times volume ethanol is precipitated 30 minutes, and 75% ethanol cleaning (precipitation of upspringing), dries up twice, and recovery product is dissolved in 50uL
EB, 30 minutes are incubated at 37 DEG C with all of RNA that degrades by adding 2uL 1mg/mL RNAse.NanoDrop determines concentration
(being stored in -20 DEG C)；
12) effect of digestion, connection is identified by running glue.DNA fragmentation, HindIII before A549 cell line genome digestions
Fragment after digestion, the electrophoresis result of DNA fragmentation after connection, as a result as shown in figure 1, digestion and the equal normal work of connection；
13) DNA that will be obtained carries out end Microbial anticorrosive additive treatment, and system is as follows：Water 84.83 μ L, DNA 2.5 μ g, 10
The μ L of 10.0 μ L, 10mM dGTP of × NEBuffer2.1,1.0 μ L, 3U/ μ L T4DNA Ploymerase 1.67, in 12 DEG C of incubations
2 hours, it is subsequently adding 2 μ L 0.5M EDTA terminating reactions；
14) 130 μ L are taken, using Covaris ultrasounds, DNA fragmentation is turned into 400bp or so, and (ultrasound procedures select 400bp journeys
Sequence)；
15) DNA is reclaimed in Ampure Beads purifying, and recovery product is dissolved in 30ul EB, and NanoDrop determines concentration,
Illumina kits build storehouse kit (NEBNext Ultra DNA Library Prep Kit for Illumina, NEB-
E7370S library standard step filling-in terminal bases dATP and joint) are built, Ampure Beads standard DNA purification recycling steps are used
DNA after recovery adjunction head, magnetic bead (MyOneTMStreptavidin C1, Thermo Fisher-650-
01), by specification standard step capture biotin labeling fragment, Illumina kits build storehouse (100 μ L, to 12 circulations), cut
Band (QIAquick Gel Extraction Kit, Qiagen-28706) between glue reclaim 400-600bp, with NheI pairs
Library carries out digestion, and digestion band substantially moves down (Fig. 2) compared to non-digestion band, illustrates to contain a number of newly formed in library
It is Chimeric fragment that NheI restriction enzyme sites, i.e. this Hi-C library are most of；
16) blunt end cloning kit (pEASY-Blunt Simple Cloning Kit, Quan Shi gold-CB111-01)
Library fragments are connected in T1 carriers, transformed competence colibacillus, and 16 monoclonals of picking are used to be sequenced, and sequencing result shows, library
The cis interactions of DNA, trans interaction, the ratio of unavailable fragment are 11:2:3, meet Quality Control of the cis interaction ratio more than 60%
It is required that；
17) detected using 2100 pairs of library fragments sizes, as shown in figure 3, the clip size in library compares concentration,
Suitable for carrying out high-flux sequence.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (8)
1. the method for the DNA fragmentation for being interacted in a kind of capture Matrix attachment region, it is characterised in that using restriction enzyme enzyme
Cut and reaction is directly attached after filling-in, comprise the following steps：
S1：Sample cell is taken, is crosslinked；
S2：Crack the sample cell；
S3：Inactivating endogenous enzyme；
S4：Digestion with restriction enzyme genome, produces with the viscous of 5 ' protrusions after the digestion with restriction enzyme double-stranded DNA
Property end, and do not produce the restriction enzyme with being connected after another cohesive end filling-in after the cohesive end is blunted
Restriction enzyme site；
S5：The cohesive end that digestion with restriction enzyme in filling-in S4 is produced, and add mark；
S6：Make because with protein binding and DNA fragmentation close to each other is connected；
S7：By chromatin fragments, capture zone has the fragment of the molecular labeling, builds library.
2. the method for the DNA fragmentation for being interacted in capture Matrix attachment region according to claim 1, it is characterised in that institute
State and also digested using pancreatin after being crosslinked, disperse the sample cell.
3. the method for the DNA fragmentation for being interacted in capture Matrix attachment region according to claim 1, it is characterised in that S3
In carry out inactivating endogenous enzyme by the following method：Final concentration of 0.5% SDS is added, is incubated 10 minutes in 62 DEG C.
4. the method for the DNA fragmentation for being interacted in capture Matrix attachment region according to claim 3, it is characterised in that make
After with SDS inactivating endogenous enzymes, with the SDS in also addition TritonX.
5. the method for the DNA fragmentation for being interacted in capture Matrix attachment region according to claim 1, it is characterised in that
After S2 cracks the sample cell, supernatant is abandoned in centrifugation, and precipitation is used for into subsequent operation, and after S3 inactivating endogenous enzymes, centrifugation is abandoned
Supernatant, subsequent operation is used for by precipitation.
6. the method for the DNA fragmentation for being interacted in capture Matrix attachment region according to claim 1, it is characterised in that institute
It is the one or more combination in BamHI, HindIII, NcoI, ScaI, SpeI, XbaI to state restriction enzyme.
7. the method for the DNA fragmentation for being interacted in the capture Matrix attachment region according to any one of claim 1-6, it is special
Levy and be, the mark is biotin.
8. the method for the DNA fragmentation for being interacted in capture Matrix attachment region according to claim 7, it is characterised in that institute
State on one or more nucleic acid molecule of the biotin-conjugated to the cohesive end addition when filling-in.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055491A (en) * | 2018-09-18 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of Hi-C high-flux sequence banking process suitable for plant |
| CN109056078A (en) * | 2018-09-18 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of Hi-C high-flux sequence banking process suitable for bacterium |
| WO2019134586A1 (en) * | 2018-01-05 | 2019-07-11 | 清华大学 | Method and system for identifying gene-regulatory chromatin interaction and application thereof |
| CN114891858A (en) * | 2022-07-13 | 2022-08-12 | 广州国家实验室 | Chromatin three-dimensional conformation capture method and application thereof |
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| CN105121661A (en) * | 2013-02-01 | 2015-12-02 | 加利福尼亚大学董事会 | Methods for genome assembly and haplotype phasing |
| CN105839196A (en) * | 2016-05-11 | 2016-08-10 | 北京百迈客生物科技有限公司 | Hi-C high-throughput sequencing and database building method for eukaryote DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105121661A (en) * | 2013-02-01 | 2015-12-02 | 加利福尼亚大学董事会 | Methods for genome assembly and haplotype phasing |
| CN105839196A (en) * | 2016-05-11 | 2016-08-10 | 北京百迈客生物科技有限公司 | Hi-C high-throughput sequencing and database building method for eukaryote DNA |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019134586A1 (en) * | 2018-01-05 | 2019-07-11 | 清华大学 | Method and system for identifying gene-regulatory chromatin interaction and application thereof |
| CN109055491A (en) * | 2018-09-18 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of Hi-C high-flux sequence banking process suitable for plant |
| CN109056078A (en) * | 2018-09-18 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of Hi-C high-flux sequence banking process suitable for bacterium |
| CN114891858A (en) * | 2022-07-13 | 2022-08-12 | 广州国家实验室 | Chromatin three-dimensional conformation capture method and application thereof |
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Application publication date: 20170531 |