CN107217309A - Build the method and its application in the DNA sequencing library of testing gene group - Google Patents
Build the method and its application in the DNA sequencing library of testing gene group Download PDFInfo
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Abstract
The present invention proposes a kind of method in the DNA sequencing library for building testing gene group, and this method includes：(1) digestion process is carried out to testing gene group using restriction enzyme, to obtain digestion process product；(2) the digestion process product is subjected to biotin labeling processing, to obtain biotin labeling processing product；(3) processing is attached to biotin labeling processing product using DNA ligase, to obtain connection product；(4) connection product is subjected to solution crosslinking Treatment；(5) solution crosslinking Treatment product is subjected to purification process；(6) purification process product is subjected to ultrasound and precipitation process, the precipitation process is by carrying out contacting progress ultrasonically treated product and streptavidin magnetic bead, to obtain the target DNA fragments for being combined with streptavidin magnetic bead；And (7), based on the target DNA fragments for being combined with streptavidin magnetic bead, storehouse is built in progress.
Description
Technical field
The present invention relates to biological technical field.In particular it relates to build the DNA sequencing library of testing gene group
Method and its application.More particularly it relates to build the method in the DNA sequencing library of testing gene group, determine base to be measured
Because of the method for the method and determination testing gene group three-D space structure of the DNA sequence dna information of group.
Background technology
Recently as the development of genomics, the Human Genome Project successfully depicts human genome DNA's sequence chart
Spectrum.Human genome encyclopedia plan correlative study, analysis is found that tens of thousands of genes, the different gene regulation of hundreds of thousands
Element.It has recently been demonstrated that the three-D space structure of genome has important shadow to functions such as expression, the regulation and control of genome
Ring.Therefore, different cell categories how to be obtained in the different cell cycles, in different developments and differential period and in normal cell
The three-dimensional structure information of genome becomes urgent problem to be solved during changing to disease cells.And chromatin conformation is caught
Obtain the development popularization of technology and two generation sequencing technologies so that the full-length genome chromatin conformation capture technique being sequenced based on two generations
Hi-C (whole genome chromosome conformation capture) becomes research chromosome three-dimensional structure
Important means.However, full-length genome chromatin conformation capture technique is extremely limited to cell number, if sufficient amount can not be obtained
Being used for comprising enough effective connection events build the DNA in storehouse, be that can not obtain effective and high-resolution enough gene
Group three-dimensional structure information.
So as to how set up and be difficult DNA sequencing library obtain, a small amount of quantity type cell and surveyed effective for DNA
Sequence and acquisition genome three-dimensional structure information high-resolution enough, are the problem of having to be solved.
The content of the invention
The application is that the discovery of problems with is made based on inventor：
Traditional Hi-C technology reaction systems are larger, centrifuge, wash, to change pipe step various, fragment is carried out before DNA cloning
Size selection can cause substantial amounts of DNA to lose.And DNA fragmentation size range of choice is narrower, interrupted pair considerably beyond ultrasonic wave
The enrichment degree of DNA fragmentation, further reduces the amount of DNA being sequenced for two generations.Some reason above, causes Hi-C skills
Art is generally used for studying the order of magnitude being readily available 107Common type cell above, in embryonic development early stage
This extremely difficult cell type for obtaining a large amount of numbers is helpless.Based on hair of the inventor to above conventional art problem
It is existing, inventor by reducing reaction system, change cell branch mode, changing scope and time that DNA fragmentation size is selected etc.,
Creatively propose improved complete genome DNA sequencing library construction method (in this application, referred to as sisHi-C
(small scale in situ Hi-C) technology), this method can be for the research cell number as little as precious cell of 10
Type, to study, the genome three-dimensional structure of early embryo development is set up and gene expression regulation brings hope.
In the first aspect of the present invention, the present invention proposes a kind of method in the DNA sequencing library for building testing gene group.
Embodiments in accordance with the present invention, methods described includes：(1) digestion process is carried out to testing gene group using restriction enzyme,
To obtain digestion process product；(2) the digestion process product is subjected to biotin labeling processing, to obtain biotin mark
Note processing product；(3) processing is attached to biotin labeling processing product using DNA ligase, to be connected
Product；(4) connection product is subjected to solution crosslinking Treatment；(5) solution crosslinking Treatment product is subjected to purification process；(6) by purifying
Manage product and carry out ultrasound and precipitation process, the precipitation process is by the way that ultrasonically treated product and streptavidin magnetic bead are carried out
What contact was carried out, to obtain the target DNA fragments for being combined with streptavidin magnetic bead；And (7) are based on being combined with chain enzyme parent
The target DNA fragments of biscuit porcelain pearl are closed, storehouse is built in progress.Utilize the DNA sequencing of structure testing gene group according to embodiments of the present invention
The method in library can be entered for building as little as 10 cells of cell number or genome amount as little as 50pg DNA sequencing library
And realize the capture for being difficult genome chromatin conformation obtain, a small amount of quantity type cell.
In the second aspect of the present invention, the present invention proposes a kind of sequencing library.Embodiments in accordance with the present invention, the survey
Preface storehouse is obtained by the method in the DNA sequencing library of foregoing structure testing gene group.Using according to the present invention
The sequencing library of embodiment is sequenced, and can obtain cell or genome the amount as little as 50pg gene of cell number as little as 10
Group sequence information and genome three-dimensional structure information high-resolution enough.
In the third aspect of the present invention, the present invention proposes a kind of method for the DNA sequence dna information for determining testing gene group.
Embodiments in accordance with the present invention, methods described includes：The DNA sequencing text of testing gene group is built according to foregoing method
Storehouse；The DNA sequencing library is sequenced, to obtain sequencing result；And based on the sequencing result, it is determined that described treat
The DNA sequence dna information of cls gene group.Utilize the side of the DNA sequence dna information of determination testing gene group according to embodiments of the present invention
Method, can obtain cell or genome the amount as little as 50pg Genomic sequence information of cell number as little as 10.
In the fourth aspect of the present invention, the present invention proposes a kind of side for being used to determine testing gene group three-D space structure
Method.Embodiments in accordance with the present invention, methods described includes：The DNA sequencing of testing gene group is built according to foregoing method
Library；The DNA sequencing library is sequenced, to obtain sequencing result；And based on the sequencing result, it is determined that described
The three-D space structure information of testing gene group.Utilize determination testing gene group three-D space structure according to embodiments of the present invention
Method, the cell of cell number as little as 10 or genome amount as little as 50pg genome three high-resolution enough can be obtained
Tie up structural information.
It should be noted that testing gene group proposed by the invention refers to the full-length genome or part base of cell or tissue
Because of group, and genome by chromatin or genome into.It will be understood to those skilled in the art that the source of genome not by
Especially limitation, can be obtained from any possible approach, can be directly obtained or from other laboratories by commercially available
Directly obtain, can also be what is directly extracted from cell or tissue sample.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
Fig. 1 is the flow chart of the method in the DNA sequencing library of structure testing gene group according to embodiments of the present invention；
Fig. 2 is the flow chart of the method in the DNA sequencing library of the structure testing gene group according to further embodiment of this invention；
Fig. 3 is the flow chart of the method in the DNA sequencing library of the structure testing gene group according to further embodiment of this invention；
Fig. 4 is the flow chart that TruSeq according to embodiments of the present invention builds storehouse；
Fig. 5 is the flow chart that TruSeq according to yet another embodiment of the invention builds storehouse；
Fig. 6 is the flow chart that TruSeq according to yet another embodiment of the invention builds storehouse；
Fig. 7 is the flow chart that TruSeq according to yet another embodiment of the invention builds storehouse；And
Fig. 8 is that the banking process that the checking embodiment of the present invention according to embodiments of the present invention is proposed has significant advantage
Result figure.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.
The method for building the DNA sequencing library of testing gene group
In the first aspect of the present invention, the present invention proposes a kind of method in the DNA sequencing library for building testing gene group.
Embodiments in accordance with the present invention, with reference to Fig. 1, this method includes：S100：Testing gene group is disappeared using restriction enzyme
Change is handled, to obtain digestion process product；S200：The digestion process product is subjected to biotin labeling processing, to obtain
Obtain biotin labeling processing product；S300：Processing is attached to biotin labeling processing product using DNA ligase,
To obtain connection product；S400：Connection product is subjected to solution crosslinking Treatment；S500：Solution crosslinking Treatment product is purified
Processing；S600：By purification process product carry out ultrasound and precipitation process, the precipitation process be by by ultrasonically treated product with
Streptavidin magnetic bead carries out contact progress, to obtain the target DNA fragments for being combined with streptavidin magnetic bead；And
S700：Based on the target DNA fragments for being combined with streptavidin magnetic bead, storehouse is built in progress.Utilize structure according to embodiments of the present invention
The method for building the DNA sequencing library of testing gene group can be low for building the cell or genome amount of cell number as little as 10
To 50pg DNA sequencing library, and then realization is difficult catching for genome chromatin conformation obtain, a small amount of quantity type cell
Obtain.
Embodiments in accordance with the present invention, with reference to Fig. 2, the testing gene group is obtained by cell lysis or tissue
, optionally, the cell is cell line or primary cell.Cell lysis or tissue and discharge the gene in cell or tissue
Group.
Embodiments in accordance with the present invention, with reference to Fig. 3, the cell or tissue first passes through formaldehyde crosslinking processing in advance.And then first
DNA- protein spatially close under nature and protein-protein are compound in aldehyde instantaneous set cell or tissue
Thing.
Embodiments in accordance with the present invention, the cell or genome carry out transfer processing by mouth suction pipe.Inventor has found, makes
Cell or tissue is accurately shifted with mouth suction pipe can be prevented effectively from the loss cell that centrifugal process replacing solution is brought.
Embodiments in accordance with the present invention, the restriction enzyme is MboI.Inventor has found that MboI recognition site is
GATC, base distribution is uniform, and when being cut on genome, obvious Preference is not had；While MboI recognition site
For four bases, compared to conventional hexabasic base restriction enzyme, its cutting frequency is higher, point of the data obtained in theory
Resolution is higher；Last MboI degree of commercialization is high, and use cost is low, can easily obtain, thus effectively controls whole structure
The cost in DNA sequencing library.
Embodiments in accordance with the present invention, the biotin labeling processing is carried out in the following way：By digestion process
Product and triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid thymidine deoxynucleotide,
The triphosphoric acid deoxycytidylic acid and archaeal dna polymerase large fragment of biotin labeling are contacted, and the contact is 37
Carried out 1.5 hours under conditions of DEG C.Through the above way can be by biotin highly efficient labeling to the end of MboI endonuclease bamhis.
Embodiments in accordance with the present invention, the connection is processed as T4 connections, and the solution crosslinking Treatment is by by the company
Thing of practicing midwifery contacts what is carried out with Proteinase K, SDS and sodium chloride.Being attached using T4 DNA ligases can be different by DNA
The chimeric molecule of nick end connection circlewise, and then by above-mentioned solution crosslinking Treatment, by protein in connection DNA point
Open and effectively discharge DNA.
Embodiments in accordance with the present invention, the purification process be by by it is described solution crosslinking Treatment product and precooling it is anhydrous
Ethanol carries out contact progress, and the contact is carried out 15 minutes under conditions of -80 DEG C.Utilize above-mentioned purification process side
Formula, DNA is effectively deposited in test tube bottom, and then realizes DNA purifying.
By the solution crosslinking Treatment product and hepatic glycogen and vinegar during embodiments in accordance with the present invention, the purification process
Sour sodium is contacted.Inventor has found that during DNA is purified, because cell number is few, amount of DNA is especially few, and adds liver
Glycogen can indicate the position that DNA is deposited in EP pipes with DNA co-precipitation, and then effectively prevent DNA losses in washing process.
Embodiments in accordance with the present invention, it in Peak Power is that 50, Duty Factor are 20, Cycles/ that the ultrasound, which is,
Burst be 200 under conditions of carry out 134s.Wherein, Peak Power represent highest incident power, are the winks acted on sample
When ultrasonic power；Duty Factor represent that service factor, i.e. ul-trasonic irradiation account for the percentage of total period in the time of sample
Number；Cycles/Burst represents the number of ul-trasonic irradiation transfer of ultrasonic energy in sample.Inventor has found, upper
State under ultrasound condition, be effectively reduced the DNA losses in DNA fragmentation size selection course.
Embodiments in accordance with the present invention, with reference to Fig. 4, storehouse is built in the storehouse of building in S700 for TruSeq, and S700 includes will knot
The target DNA fragments that closing has streptavidin magnetic bead carry out S710：End is repaired, S720：End adds Adenosine triphosphate purine deoxidation
Ribonucleotide and S730：Connect sequence measuring joints series processing.
Embodiments in accordance with the present invention, after the repair process of S710 ends, S720 ends add Adenosine triphosphate purine deoxidation core
Further comprise that tween washs the magnetic bead processing before ribotide processing；Preferably, S720 ends add Adenosine triphosphate purine
Further comprise that tween washs the magnetic bead after deoxyribonucleotide processing, before S730 connection sequence measuring joints series processings
Processing；Preferably, further comprise that tween washs the magnetic bead processing after S730 connections sequence measuring joints series processing.Each
After step reaction terminates, directly magnetic bead is washed twice with tween wash solution, you can simple and quick replacing solution, it is to avoid DNA
Loss in purge process.
Embodiments in accordance with the present invention, with reference to Fig. 5, further comprise carrying out connection sequence measuring joints series processing product
S740：The elutions of DNA first processing and S750：PCR is expanded.Handled by the first elution, can be by target DNA fragments from chain enzyme affinity
Biscuit porcelain pearl efficiently elutes, and is expanded by PCR, and the efficiently concentrating of target dna can be achieved.
Embodiments in accordance with the present invention, with reference to Fig. 6, further comprise S760：By pcr amplification product and AMPure XP magnetic
Pearl is combined processing, optionally, with reference to Fig. 7, further comprises S770：The PCR for being combined with AMPure XP magnetic beads is expanded into production
Thing carries out the elutions of DNA second processing.So as to obtain DNA fragmentation of the size in the range of 200 base-pairs to 1000 base-pairs.
The sequencing library that the above method according to embodiments of the present invention is obtained can be surveyed as sisHi-C libraries for two generations
Sequence.
The above method according to embodiments of the present invention uses the reaction system of very little, increases while cost is substantially reduced
The efficiency of digestion and coupled reaction；Cell cracking before, using mouth suction pipe accurately shift cell avoid centrifugal process change it is molten
The loss cell that liquid band is come；DNA is combined and TruSeq is carried out on streptavidin magnetic bead builds storehouse, washed using the magnetic bead of milder
Wash condition and control is built storehouse operation and carried out in same EP pipes, these measures successfully get around purifying recycling step, and effectively
Reduce the DNA losses during Jian Ku；Optimize the ultrasonic condition for interrupting DNA fragmentation, two-step method eluted dna, after PCR
DNA of the clip size in the range of 200bp to 1000bp is all remained, last available DNA is further increased
Amount.Whole method not only significantly reduces the consumption of initiator cell, and cost is also only 1/10th of conventional method, so that efficiently
Realize the capture of genome chromatin conformation.
It will be appreciated by persons skilled in the art that the structure testing gene group obtained based on method described herein
The unit in DNA sequencing library, unit, module also in the protection domain of the application, the unit, unit,
The advantage of module is similar with foregoing method, will not be described in detail herein.It will be appreciated to those of skill in the art that can adopt
With building block of any device for being adapted for aforesaid operations as known in the art as above-mentioned unit.Herein
Used term " connected " should broadly understood, and can be joined directly together, can also be indirectly connected to by intermediary, for
For one of ordinary skill in the art, the concrete meaning of above-mentioned term can be understood as the case may be.
Sequencing library
In the second aspect of the present invention, the present invention proposes a kind of sequencing library.Embodiments in accordance with the present invention, the survey
Preface storehouse is obtained by the method in the DNA sequencing library of foregoing structure testing gene group.Using according to the present invention
The sequencing library of embodiment is sequenced, and can obtain cell or genome the amount as little as 50pg gene of cell number as little as 10
Group sequence information and genome three-dimensional structure information high-resolution enough.
The method for determining the DNA sequence dna information of testing gene group
In the third aspect of the present invention, the present invention proposes a kind of method for the DNA sequence dna information for determining testing gene group.
Embodiments in accordance with the present invention, this method includes：The DNA sequencing library of testing gene group is built according to foregoing method；
The DNA sequencing library is sequenced, to obtain sequencing result；And based on the sequencing result, determine described to be measured
The DNA sequence dna information of genome.On build testing gene group DNA sequencing library method, before have been carried out in detail
Description, will not be repeated here.Embodiments in accordance with the present invention, the method and apparatus that sequencing library is sequenced are not limited especially
System, it is contemplated that the maturity of technology, embodiments in accordance with the present invention, can use second generation sequencing technologies, such as SOLEXA,
SOLID and 454 sequencing technologies.It is of course also possible to use developing or still undeveloped new sequencing technologies, such as single point
Sub- sequencing technologies, such as：The True Single Molecule DNA sequencing technologies of Helicos companies, Pacific
The single molecule, real-time (SMRT.TM.) technology of Biosciences companies, and Oxford
(Rusk, Nicole (2009-04-01) .Cheap such as nano-pore sequencing technology of Nanopore Technologies companies
Third-Generation Sequencing.Nature Methods 6(4)：244–245).
It is surprisingly found by the inventors that, utilize the DNA sequence dna information of determination testing gene group according to embodiments of the present invention
Method, can sensitive, accurately and efficiently determine the genome or minigene group of few cells (cell initial amount as little as 10)
The Genomic sequence information of (DNA initial amounts as little as 50pg).
The method for determining testing gene group three-D space structure
In the fourth aspect of the present invention, the present invention proposes a kind of side for being used to determine testing gene group three-D space structure
Method.Embodiments in accordance with the present invention, methods described includes：The DNA sequencing of testing gene group is built according to foregoing method
Library；The DNA sequencing library is sequenced, to obtain sequencing result；And based on the sequencing result, it is determined that described
The three-D space structure information of testing gene group.
According to a particular embodiment of the invention, methods described includes building testing gene group according to foregoing method
DNA sequencing library；Both-end sequencing is carried out for resulting library, is believed with the sequence for obtaining each DNA fragmentation two ends in library
Breath；The sequence information at two ends is compared on genome respectively, so as to obtain that linear range in genome is different every two
The intersegmental spatial proximity information of individual piece, and then combine the three-D space structure (Aidenet that mathematical method is inferred to genome
al.Comprehensive mapping of long-range interactions reveals folding principle
of the human genome.Science.2009)。
It is surprisingly found by the inventors that, utilize determination testing gene group chromatin target area according to embodiments of the present invention
The method of sequence information, can it is sensitive, accurately and efficiently determine few cells (cell initial amount as little as 10) genome or micro-
Measure the genome three-dimensional structure information high-resolution enough of genome (DNA initial amounts as little as 50pg).
Below with reference to specific embodiment, the present invention will be described, it is necessary to which explanation, these embodiments are only illustrative
, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, (write according to the technology described by document in the art or condition such as with reference to J. Pehanorm Brookers, Huang Peitang etc. is translated
's《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Unreceipted production firm person, is that be able to can for example be purchased from Illumina companies by the conventional products of acquisition purchased in market.
Embodiment 1 builds the DNA sequencing library of cellular genome
1.1 reagents prepare
Lysate (Lysis buffer)
10mM Tris-HCl., pH=7.4
·10mM NaCl
0.5%NP-40
·0.1mM EDTA
·1X Proteinase Inhibitor
2X biotins and streptavidin magnetic bead binding soln (2X Binding buffer)
10mM Tris-HCl., pH=8.0
·2M NaCl
·1mM EDTA
Tween wash solution (Tween wash buffer)
5mM Tris-HCl., pH=8.0
·1M NaCl
0.05%Tween
·0.5mM EDTA
1.2 formaldehyde crosslinking
The sample of collection is transferred in the PBS solution containing 1% formaldehyde of Fresh under stereoscope with mouth suction pipe,
Room temperature fixes 10 minutes, adds 2.5M glycine solutions to final concentration of 0.2M, is stored at room temperature 10 minutes.With mouth suction pipe by sample
It is transferred in PBS solution and washed once, is then transferred into PCR pipe.
1.3 lysate samples and digestion with restriction enzyme
50 microlitres of lysates are added into the PCR pipe containing sample, and pressure-vaccum is mixed repeatedly with low pipette tips of the absorption with filter core
It is even, stand 50 minutes on ice.Supernatant is removed in centrifugation.Add 10 microlitre 0.5% of SDS solution, 62 DEG C of metal baths 10 minutes.
25 microlitres of water and 10 microlitres of 10%Triton X-100 solution are added into sample.With pipettor, pressure-vaccum is mixed repeatedly
It is even, sample is placed in metal bath 37 DEG C and is incubated 15 minutes.Liquid is mixed to prevent follow-up SDS to restriction enzyme enzymatic activity
Influence.
Continue to add 5 microlitres of 10X NEB buffer 2 and 50U restriction enzyme MboI into sample, be placed in after mixing
Rotate on blending instrument, 37 DEG C of digestions 15 hours, chromatin is carried out fully to digest and separate.
1.4 end marks and connection
Second day, sample is removed from rotation blending instrument, be placed on 62 DEG C of metal baths and stand 20 minutes, to inactivate
MboI.On ice, 1mM triphosphoric acids adenyl-deoxyribonucleotide (dATP) is separately added into each sample, 1mM triphosphoric acid birds are fast
Purine deoxynucleotide (dGTP) and each 0.5 microlitre of 1mM triphosphoric acid thymidine deoxynucleotides (dTTP), and 3.75 microlitres of lifes
The 0.4mM triphosphoric acids deoxycytidylic acid (biotin-14-dCTP) of thing element mark.10U archaeal dna polymerases are added after mixing
Large fragment (Klenow), by sample be placed in rotation blending instrument on 37 DEG C handle 1.5 hours, by biotin labeling to MboI digestions
The end of fragment.
Solution containing sample is transferred to from PCR pipe in 1.5 milliliters of EP pipes of low absorption.And add 12 microlitres of 10X
NEB T4 DNA ligases reaction solution, 7 microlitres of 10%Triton X-100,1.2 microlitres 10 milligrams every microlitre of ox bloods are pure
Albumen, 1 microlitre of 400U/ul T4 DNA ligase and 39 microlitres of water.After mixing, sample is placed in constant temperature blending instrument (Thermo
Mixer on), 500 revs/min, 24 DEG C are handled 5.5 hours, are connected different nick ends circlewise using DNA ligase
Chimeric molecule.
1.5 solution crosslinkings and DNA purifying
After coupled reaction terminates, 100U Proteinase Ks and 12 microlitres of 10%SDS solution, 55 DEG C of gold are added into each sample
Category bath 30 minutes.Then 13 microlitres of 5M sodium chloride are added, are placed in after being mixed on vortex instrument in 65 DEG C of baking ovens, overnight solution crosslinking.
3rd day, sample is taken out from baking oven, after temperature is down to room temperature, 1 microlitre of glycogen is added into each sample
Former and 15 microlitres of 3M sodium acetates, are mixed on vortex instrument, are then added the absolute ethyl alcohol of 240 microlitres of precoolings, are turned upside down mixed
Even, -80 DEG C stand 15 minutes.High speed centrifugation, precipitation is washed with 75% absolute ethyl alcohol twice, with 50 microlitres of water after drying to be precipitated
Dissolving purifying obtained DNA, 37 DEG C of metal baths 15 minutes.During DNA is purified, because cell number is few, amount of DNA is special
It is few, add hepatic glycogen and be co-precipitated with DNA to indicate position that DNA is deposited in EP pipes, prevent that DNA loses in washing process.
1.6 ultrasounds interrupt DNA and biotin labeling precipitation
Sample DNA shearing is broken into 300-500 base-pairs (bp) size with Covaris M220 ultrasonic waves DNA broken instrument
Fragment, ultrasound condition is：Peak Power 50, Duty Factor 20, Cycles/Burst 200, time 134s.With
20 microlitres of water washing Covaris ultraphonic pipes are lost with reducing DNA.Inventor is had found, DNA can be reduced under above-mentioned ultrasound condition
DNA losses in clip size selection course.
Prepare Streptavidin MagneSphere (Dynabeads MyOne Streptavidin C1), each sample needs 100 are micro-
Gram magnetic bead.Magnetic bead is cleaned with tween wash solution twice, is then combined with 70 microlitres of 2X biotins and streptavidin magnetic bead molten
Magnetic bead is resuspended in liquid, and is added into each sample, is mixed with pipettor pressure-vaccum.Sample is placed on rotation blending instrument, room temperature
50 minutes, carry out biotin labeling precipitation to obtain markd target DNA fragment.
1.7 build storehouse and DNA fragmentation selection
After biotin labeling is precipitated, the target DNA fragments with biotin labeling are combined in Streptavidin MagneSphere
On.Because the DNA of biotin labeling is combined highly stable, these combining targets DNA magnetic bead with Streptavidin MagneSphere
Progress TruSeq can be used directly to and build storehouse.End reparation is carried out successively, add Adenosine triphosphate purine deoxidation core in DNA fragmentation end
Ribotide (dATP) and connection sequence measuring joints sequence.After every single step reaction terminates, directly magnetic is washed with tween wash solution
Pearl is twice, you can simple and quick replacing solution, it is to avoid losses of the DNA in purge process.
4th day, connect after sequence measuring joints sequence, sample be placed on magnetic frame, treats that magnetic bead is all adsorbed onto magnetic frame,
Solution becomes after clarification, abandons supernatant, magnetic bead is cleaned twice with tween wash solution, every time 2 points of the rocked at room temperature on rotation blending instrument
Clock.20 microlitres of water are added into sample, sample is placed on constant temperature blending instrument 66 DEG C, 1400 revs/min, 20 by pressure-vaccum after mixing
Minute, eluted dna is twice.Enter performing PCR amplification.After PCR terminates, 72 microlitre (1 is added into 150 microlitres of samples：0.48)
AMPure XP magnetic beads, after being mixed with pipettor pressure-vaccum, room temperature is combined 5 minutes on rotation blending instrument.Supernatant is transferred to new
In low absorption EP pipes, 78 microlitre (1 is added：1) AMPure XP magnetic beads, room temperature combines 5 points on rotation blending instrument after mixing
Clock, abandons supernatant.With 75% washes of absolute alcohol magnetic bead twice, after drying, 50 microlitres of water elutions are added into EP pipes, so as to obtain
DNA fragmentation of the size in the range of 200 base-pairs to 1000 base-pairs.The library obtained by above-mentioned steps is (in this application,
Above method acquisition library is referred to as sisHi-C libraries by inventor) it can be used to the sequencing of two generations.
Embodiment 2
In order to contrast the sisHi-C of the application and method (the Rao et al.A 3D map of the human of forefathers
genome at kilobase resolution reveals principles of chromatin
Looping.Cell.2014) the effect in a small amount of cell research, inventor accurately counts four groups of Development of Mouse Embryos using mouth suction pipe
Tire stem cell.Every group of cell number is 500.Then inventor is utilized respectively sisHi-C and the method for forefathers is each thin to two groups
Born of the same parents' (be respectively labeled as repeating experiment 1 and repeat experiment 2) carry out building storehouse.DNA concentration is measured before PCR, and in library sequencing
Ratio shared by the effective data of post analysis.Fig. 8 A results are shown, before PCR, use two groups of DNA concentrations of sisHi-C methods
Obviously higher than two groups of use forefathers' method；Fig. 8 B results show that sisHi-C significantly reduces DNA loss, in sequencing number
Seldom, ratio shared by effective data is apparently higher than before use for the ratio shared by identical sequence produced according to middle PCR
Two groups of the method for people.To sum up, compared with the method for forefathers, the sisHi-C of the application significantly reduces DNA in experimentation
In loss, be highly suitable for study very small amount cell genome three-dimensional structure.
In addition, term " first ", " second " are only used for describing purpose, and it is not intended that indicating or implying relative importance
Or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can express or
Implicitly include one or more this feature.In the description of the invention, " multiple " are meant that two or more,
Unless otherwise specifically defined.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the not be the same as Example or the feature of example and non-be the same as Example or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (12)
1. a kind of method in the DNA sequencing library for building testing gene group, it is characterised in that including：
(1) digestion process is carried out to testing gene group using restriction enzyme, to obtain digestion process product；
(2) the digestion process product is subjected to biotin labeling processing, to obtain biotin labeling processing product；
(3) processing is attached to biotin labeling processing product using DNA ligase, to obtain connection product；
(4) connection product is subjected to solution crosslinking Treatment；
(5) solution crosslinking Treatment product is subjected to purification process；
(6) purification process product is subjected to ultrasound and precipitation process, the precipitation process is by by ultrasonically treated product and chain
Enzyme avidin magnetic bead carries out contact progress, to obtain the target DNA fragments for being combined with streptavidin magnetic bead；And
(7) based on the target DNA fragments for being combined with streptavidin magnetic bead, storehouse is built in progress.
2. according to the method described in claim 1, it is characterised in that the testing gene group is by cell lysis or tissue
At least a portion of the genome of acquisition,
Optionally, the cell is cell line or primary cell.
3. method according to claim 2, it is characterised in that the cell or tissue first passes through formaldehyde crosslinking processing in advance,
Preferably, the cell or tissue carries out transfer processing by mouth suction pipe.
4. according to the method described in claim 1, it is characterised in that the restriction enzyme is MboI.
5. according to the method described in claim 1, it is characterised in that the biotin labeling processing is to carry out in the following way
's：
By digestion process product and triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid thymus gland
Pyrimidine deoxynucleotide, the triphosphoric acid deoxycytidylic acid of biotin labeling and archaeal dna polymerase large fragment are contacted,
The contact is carried out 1.5 hours under conditions of 37 DEG C.
6. according to the method described in claim 1, it is characterised in that the connection is processed as T4 connections, the solution crosslinking Treatment
Be by the way that the connection product is contacted into progress with Proteinase K, SDS and sodium chloride,
Optionally, the purification process is by the way that the absolute ethyl alcohol of the solution crosslinking Treatment product and precooling is carried out into contact progress
, the contact is carried out 15 minutes under conditions of -80 DEG C；
Preferably, the solution crosslinking Treatment product is contacted with hepatic glycogen and sodium acetate during the purification process.
7. according to the method described in claim 1, it is characterised in that it in Peak Power is 50, Duty that the ultrasound, which is,
Factor be 20, Cycles/Burst be 200 under conditions of carry out 134s.
8. according to the method described in claim 1, it is characterised in that storehouse is built in the storehouse of building for TruSeq, including will be combined with chain
The target DNA fragments of enzyme avidin magnetic bead carry out end reparation, end and add Adenosine triphosphate purine deoxyribonucleotide and connection
Sequence measuring joints series processing,
Preferably, after the repair process of end, end adds the bag that takes a step forward of Adenosine triphosphate purine deoxyribonucleotide processing
Include tween and wash the magnetic bead processing；
Preferably, end add Adenosine triphosphate purine deoxyribonucleotide handle after, connection sequence measuring joints series processing before
Further comprise that tween washs the magnetic bead processing；
Preferably, further comprise that tween washs the magnetic bead processing after connection sequence measuring joints series processing.
9. method according to claim 8, it is characterised in that further comprise that sequence measuring joints series processing product will be connected
The elutions of DNA first processing and PCR amplifications are carried out,
Optionally, further comprise pcr amplification product and AMPure XP magnetic beads being combined processing,
Optionally, further comprise the pcr amplification product for being combined with AMPure XP magnetic beads carrying out the elutions of DNA second processing.
10. a kind of sequencing library, it is characterised in that the sequencing library is by the structure described in any one of claim 1~9
What the method in the DNA sequencing library of testing gene group was obtained.
11. a kind of method for the DNA sequence dna information for determining testing gene group, it is characterised in that including：
Method according to any one of claim 1~9 builds the DNA sequencing library of testing gene group；
The DNA sequencing library is sequenced, to obtain sequencing result；And
Based on the sequencing result, the DNA sequence dna information of the testing gene group is determined.
12. a kind of method for determining testing gene group three-D space structure, it is characterised in that including：
Method according to any one of claim 1~9 builds the DNA sequencing library of testing gene group；
The DNA sequencing library is sequenced, to obtain sequencing result；And
Based on the sequencing result, the three-D space structure information of the testing gene group is determined.
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| PCT/CN2017/114475 WO2019006975A1 (en) | 2017-07-07 | 2017-12-04 | In situ whole genome chromatin conformation capture method for infinitesimal cells |
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| CN108265104A (en) * | 2018-01-02 | 2018-07-10 | 北京诺禾致源科技股份有限公司 | Chromosome configuration captures library and its construction method |
| WO2019006975A1 (en) * | 2017-07-07 | 2019-01-10 | 清华大学 | In situ whole genome chromatin conformation capture method for infinitesimal cells |
| CN109735900A (en) * | 2019-03-20 | 2019-05-10 | 嘉兴菲沙基因信息有限公司 | A kind of small fragment DNA library construction method suitable for Hi-C |
| CN109797436A (en) * | 2018-12-29 | 2019-05-24 | 阅尔基因技术（苏州）有限公司 | A kind of sequencing library building kit and its application method and application |
| CN110396534A (en) * | 2019-08-12 | 2019-11-01 | 华大生物科技（武汉）有限公司 | The construction method of gene library, determined nucleic acid sample gene mutation detection method and kit |
| CN111364105A (en) * | 2020-04-30 | 2020-07-03 | 华中农业大学 | Simple and effective construction method of plant long fragment in situ DLO Hi-C sequencing library |
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| CN108265104A (en) * | 2018-01-02 | 2018-07-10 | 北京诺禾致源科技股份有限公司 | Chromosome configuration captures library and its construction method |
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| CN109735900A (en) * | 2019-03-20 | 2019-05-10 | 嘉兴菲沙基因信息有限公司 | A kind of small fragment DNA library construction method suitable for Hi-C |
| CN110396534A (en) * | 2019-08-12 | 2019-11-01 | 华大生物科技（武汉）有限公司 | The construction method of gene library, determined nucleic acid sample gene mutation detection method and kit |
| CN111364105A (en) * | 2020-04-30 | 2020-07-03 | 华中农业大学 | Simple and effective construction method of plant long fragment in situ DLO Hi-C sequencing library |
| CN111364105B (en) * | 2020-04-30 | 2021-09-07 | 华中农业大学 | Simple and effective construction method of plant long fragment in situ DLO Hi-C sequencing library |
| CN112522251A (en) * | 2020-12-29 | 2021-03-19 | 上海派森诺生物科技股份有限公司 | Method for extracting Hi-C animal tissue |
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