CN105754995B - Construct the method and its application in the DNA sequencing library of testing gene group - Google Patents
Construct the method and its application in the DNA sequencing library of testing gene group Download PDFInfo
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Abstract
The invention proposes a kind of methods in DNA sequencing library for constructing testing gene group, this method comprises: (1) carries out the first digestion process to testing gene group using micrococcal nuclease, to obtain the first digestion process product；(2) the first digestion process product is subjected to co-immunoprecipitation processing, so that adaptive immune is co-precipitated processing product；(3) the second digestion process is carried out to co-immunoprecipitation processing product using Proteinase K, to obtain the second digestion process product；(4) using shrimp alkaline phosphotase to described second；Digestion process product directly carries out dephosphorylation process, to obtain dephosphorylation process product；(5) the dephosphorylation process product is directly subjected to denaturation treatment, to obtain the denaturation treatment product containing single strand dna；And (6) obtain sequencing library according to TELP method based on the denaturation treatment product containing single strand dna.
Description
Technical field
The present invention relates to field of biotechnology.In particular it relates to construct the DNA sequencing library of testing gene group
Method and its application.More particularly it relates to construct the method in the DNA sequencing library of testing gene group, building base to be measured
Because of the equipment in the DNA sequencing library of group, the method for the DNA sequence dna information for determining testing gene group, the DNA for determining testing gene group
The method of the system of sequence information and the sequence information for determining testing gene group chromatin target area.
Background technique
In recent years epigenetics field studies have shown that histone modification to maintain cell growth and development have to
Close important role.Therefore, variety classes cell how is obtained in the comprehensive histone modification of the system of different developmental phases
Information is at urgent problem to be solved.And two generation sequencing technologies is universal, so that the chromatin immune of sequencing is combined to be co-precipitated skill
Art becomes the important technical of researching DNA and histone interaction, for the comprehensive DNA histone of system for disclosing cell
Modification map provides strong technical support.However, chromatin immune chemical coprecipitation technique be extremely limited to cell number and
Various factors such as the quality of antibody are to be unable to get effective group if the DNA for being used to build library of sufficient amount can not be obtained
Protein modified information.Traditional chromatin immune chemical coprecipitation technique includes crosslinking, interrupts DNA, antibody incubation, solution crosslinking and wash
Several important steps such as de- DNA, however traditional chromatin immune chemical coprecipitation technique is usually used for research is easy to get, number
Magnitude is 106The cell of above common type is in this extremely unobtainable, a small amount of number of embryonic development early stage for studying
The cell of purpose type is helpless.
To how establish the DNA sequencing library of quantity type cell that be not easy to obtain, a small amount of and be surveyed effective for DNA
Sequence is problem to be solved.
Summary of the invention
The application is the discovery based on inventor to following problems and makes:
The process of crosslinking and DNA breakage in traditional chromatin immune chemical coprecipitation technique will cause a large amount of DNA loss,
And it is subsequent several times purify DNA process make again the recovery efficiency of DNA become influence acquisition amount of DNA it is crucial because
Element.Discovery based on inventor on acquisition amount of DNA factor is influenced in the above traditional dyeing matter Immunoprecipitation, inventor couple
Traditional Immunoprecipitation has carried out creative improvement, proposes a kind of new building based on chromatin immune co-precipitation
Library and sequencing technologies.The database technology and sequencing technologies can carry out building library to the DNA of primary cell or number down to 500 cell
With effective sequencing, hope is brought to study the finely regulating research of epigenetics of embryonic development early stage.
In the first aspect of the present invention, the invention proposes a kind of methods in DNA sequencing library for constructing testing gene group.
According to an embodiment of the invention, this method comprises: (1) using microballoon nuclease to testing gene group carry out the first digestion process,
To obtain the first digestion process product；(2) the first digestion process product is subjected to co-immunoprecipitation processing, to obtain
Co-immunoprecipitation handles product；(3) the second digestion process is carried out to co-immunoprecipitation processing product using Proteinase K, with
Just the second digestion process product is obtained；(4) dephosphorization is directly carried out to the second digestion process product using shrimp alkaline phosphotase
Acidification, to obtain dephosphorylation process product；(5) the dephosphorylation process product is directly subjected to denaturation treatment,
To obtain the denaturation treatment product containing single strand dna；And (6) based on the denaturation containing single strand dna at
Product is managed, obtains sequencing library according to TELP method.
Microballoon core is used using the method method in the DNA sequencing library of building testing gene group according to an embodiment of the present invention
Sour digestion is broken chromatin, and then avoids the local damage of DNA caused by ultrasonication, substantially increases Ag-Ab
Recognition efficiency；It is not required to disappear to second using the method in the DNA sequencing library of building testing gene group according to an embodiment of the present invention
Change processing product and carry out purification and recovery, directly carries out using shrimp alkaline phosphotase dephosphorylation process, and pass through shrimp alkalinity phosphorus
The dephosphorylation process product of sour enzyme does not need purification and recovery yet, and directly can be denaturalized and be obtained sequencing library, Jin Erxian
Write the amount for improving the DNA that can be used for building library.The method that the embodiment of the present invention is proposed can efficiently establish starting cell concentration
Down to 500 or so cells genome or starting amount of DNA down to 2.5 nanograms genome DNA sequencing library, according to this hair
Bright specific embodiment, it might even be possible to efficiently establish starting cell concentration down to 200 cells genome or DNA initial amount even
Down to the DNA lateral order library of the genome of 1 nanogram.The method that the embodiment of the present invention is proposed is especially suitable for primary cell or stripping
The building in the DNA sequencing library of the equal few cells genome from tissue, and then efficiently, delicately can be applied to high pass and measure
Sequence technology, based on the data analysis to sequencing result, it will be able to effectively obtain gene sequence information.
In the second aspect of the present invention, the invention proposes a kind of equipment in DNA sequencing library for constructing testing gene group.
According to an embodiment of the invention, the equipment includes: the first digestion process device, the first digestion process device is for utilizing
Microballoon nuclease carries out digestion process to testing gene group, to obtain the first digestion process product；Co-immunoprecipitation processing dress
It sets, the co-immunoprecipitation processing unit is connected with the first digestion process device, and is used for first digestion
It manages product and carries out co-immunoprecipitation processing, so that adaptive immune is co-precipitated processing product；Second digestion process device, described second
Digestion process device is connected with the co-immunoprecipitation processing unit, and for utilizing Proteinase K to the co-immunoprecipitation
It handles product and carries out the second digestion process, to obtain the second digestion process product；Dephosphorylation process device, it is described to remove phosphoric acid
Change processing unit to be connected with the second digestion process device, the dephosphorylation process device is used to utilize shrimp alkaline phosphotase
Dephosphorylation process is directly carried out to the second digestion process product, to obtain dephosphorylation process product；Denaturation treatment
Device, the degeneration processing device are connected with the dephosphorylation process device, and for producing the dephosphorylation process
Object directly carries out denaturation treatment, to obtain the denaturation treatment product containing single strand dna；And TELP device, it is described
TELP device is connected with the degeneration processing device, and the TELP device is based on the denaturation treatment containing single strand dna
Product obtains sequencing library according to TELP method, and optionally, the degeneration processing device is suitable for thermal denaturation and obtains described single-stranded
DNA。
It is surveyed using the DNA according to an embodiment of the present invention using according to an embodiment of the present invention for constructing testing gene group
The equipment in preface library can establish starting cell concentration and receive down to the genome of 500 or so cells or starting amount of DNA down to 2.5
Gram genome DNA sequencing library, according to a particular embodiment of the invention, it might even be possible to efficiently establish starting cell concentration down to
The genome or DNA initial amount of 200 cells are even as low as the DNA sequencing library of the genome of 1 nanogram.And the present invention is implemented
The equipment that example is proposed remains the specificity of single stranded DNA chain, and DNA loss is few, and gene information keeps complete, especially suitable for original
For the building in the DNA sequencing library of cell or the equal few cells genome of removing tissue.
In the third aspect of the present invention, the invention proposes a kind of methods of the DNA sequence dna information of determining testing gene group.
According to an embodiment of the invention, the described method includes: constructing the DNA sequencing text of testing gene group according to mentioned-above method
Library；The DNA sequencing library is sequenced, to obtain sequencing result；And be based on the sequencing result, determine it is described to
The DNA sequence dna information of cls gene group.
It, can be sensitive, accurate, high using the method for the DNA sequence dna information of testing gene group according to an embodiment of the present invention
Effect ground determines that few cells (cell initial amount down to 500 or so, even as low as 200) genome or minigene group (rise
Beginning amount of DNA is down to 2.5 nanograms, even as low as 1 nanogram) single strand dna sequence information.
In the fourth aspect of the present invention, the invention proposes a kind of systems of the DNA sequence dna information of determining testing gene group.
According to an embodiment of the invention, the system comprises: sequencing library constructs equipment, and the sequencing library building equipment is front institute
It states；Sequencing equipment, the sequencing equipment is connected with sequencing library building equipment, for use in the base to the genome
Because sequencing library is sequenced, the sequencing result of the genomic DNA is obtained；And analytical equipment, to the genomic DNA
Sequencing result is analyzed, to obtain the DNA sequence dna information.
It, can be sensitive, accurate, high using the system of the DNA sequence dna information of testing gene group according to an embodiment of the present invention
Effect ground determines few cells (cell initial amount down to 500 or so, even as low as 200) genome or minigene group
The sequence information of the single strand dna of (originating amount of DNA down to 2.5 nanograms, even as low as 1 nanogram).
In the fifth aspect of the invention, the invention proposes a kind of for determining testing gene group chromatin target area
The method of sequence information.According to an embodiment of the invention, the described method includes: constructing testing gene according to mentioned-above method
The target area sequencing library of group, wherein target area described in the histone antibodies specific recognition；To the testing gene
The target area sequencing library of group is sequenced, to obtain sequencing result；And be based on the sequencing result, determine it is described to
The sequence information of cls gene group chromatin target area.
Utilize the method for the sequence information of determining testing gene group chromatin target area according to an embodiment of the present invention, energy
Enough genomes that is sensitive, accurately and efficiently determining few cells (cell initial amount down to 500 or so, even as low as 200)
Or the sequence information of the chromatin target area of minigene group (originating amount of DNA down to 2.5 nanograms, even as low as 1 nanogram), from
And realize the detection of the chromatin target area to the genome of few cells.
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 at.It will be understood to those skilled in the art that the source of genome not by
Especially limitation, can obtain from any possible approach, can be and directly obtained by commercially available, is also possible to from other laboratories
It directly acquires, can also be and directly extracted from cell or tissue sample.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Fig. 1 is the flow chart of the method in the DNA sequencing library of building testing gene group according to an embodiment of the present invention；
Fig. 2 is the flow chart of the method in the DNA sequencing library of the building testing gene group of another embodiment according to the present invention；
Fig. 3 be it is according to an embodiment of the present invention using micrococcal nuclease to testing gene group carry out the first digestion process
Flow chart；
Fig. 4 is the structural schematic diagram of the equipment in the DNA sequencing library of building testing gene group according to an embodiment of the present invention；
Fig. 5 is that the structure of the equipment in the DNA sequencing library of building testing gene group according to yet another embodiment of the invention is shown
It is intended to；
Fig. 6 is the structural schematic diagram of the first digestion process device according to an embodiment of the present invention；
Fig. 7 is the structural schematic diagram of co-immunoprecipitation device according to an embodiment of the present invention；
Fig. 8 is the structural schematic diagram of the second digestion process device according to an embodiment of the present invention；
Fig. 9 is the structural schematic diagram of TELP device according to an embodiment of the present invention；
Figure 10 is the structural schematic diagram of TELP device according to yet another embodiment of the invention；
Figure 11 is the structural schematic diagram of connector connection unit according to an embodiment of the present invention；
Figure 12 is the structural representation of the system of the DNA sequence dna information of determining testing gene group according to an embodiment of the present invention
Figure；
Figure 13 is the method for the sequence information of determining testing gene group chromatin target area according to an embodiment of the present invention
Flow chart；
Figure 14 is the DNA gel electrophoretogram of dephosphorylation enzyme screening experiment according to an embodiment of the present invention；
Figure 15 is the DNA gel electrophoretogram of dephosphorylation enzyme screening experiment according to yet another embodiment of the invention；
Figure 16 is the result for the banking process validity that the verifying embodiment of the present invention according to an embodiment of the present invention is proposed
Figure；And
Figure 17 is the banking process validity that the verifying embodiment of the present invention according to yet another embodiment of the invention is proposed
Result figure.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
The method for constructing the DNA sequencing library of testing gene group
In the first aspect of the present invention, the invention proposes a kind of methods in DNA sequencing library for constructing testing gene group.
According to an embodiment of the invention, disappearing with reference to Fig. 1 this method comprises: (1) carries out first to testing gene group using microballoon nuclease
Change processing, to obtain the first digestion process product；(2) the first digestion process product is subjected to co-immunoprecipitation processing；So as to
Adaptive immune co-precipitation processing product；(3) the second digestion process is carried out to co-immunoprecipitation processing product using Proteinase K, with
Just the second digestion process product is obtained, wherein the corresponding histone that Proteinase K is used to digest in co-immunoprecipitation processing product is anti-
Body, so as to naked DNA, thus the second digestion process product herein is naked DNA；(4) using shrimp alkaline phosphotase to second
Digestion process product directly carries out dephosphorylation process, to obtain dephosphorylation process product；(5) dephosphorylation process is produced
Object directly carries out denaturation treatment, to obtain the denaturation treatment product containing single strand dna；And (6) are based on containing single-stranded
The denaturation treatment product of DNA molecular obtains sequencing library according to TELP method.Utilize building base to be measured according to an embodiment of the present invention
Because the method in the DNA sequencing library of group splits chromatin using the cutting of microballoon nuclease, and then avoid DNA caused by ultrasonication
Local damage, substantially increase the recognition efficiency of Ag-Ab；Utilize building testing gene group according to an embodiment of the present invention
The method in DNA sequencing library be not required to carry out purification and recovery to the second digestion process product, directly carry out using shrimp alkaline phosphatase
Enzyme dephosphorylation process, and purification and recovery is not needed yet by the dephosphorylation process product of shrimp alkaline phosphotase, and can be straight
It taps into row denaturation and obtains sequencing library, and then significantly improve the amount that can be used for building the DNA in library.The embodiment of the present invention is proposed
Method can efficiently establish starting cell concentration and received down to the genome of 500 or so cells or the initial amount of DNA down to 2.5
Gram genome DNA sequencing library, according to a particular embodiment of the invention, it might even be possible to efficiently establish starting cell concentration down to
The genome or DNA initial amount of 200 cells are even as low as the DNA lateral order library of the genome of 1 nanogram.Institute of the embodiment of the present invention
Structure of the method for proposition especially suitable for the DNA sequencing library of primary cell or the genome of the equal few cells of removing tissue
Build, so can efficiently, delicately be applied to high throughput sequencing technologies, based on to sequencing result data analysis, it will be able to have
Effect ground obtains gene sequence information.
According to an embodiment of the invention, the testing gene group is obtained by lytic cell or tissue with reference to Fig. 2
, wherein the cell is cell line or primary cell, and then discharges the genome in cell or tissue, in favor of micrococcus luteus
Nuclease directly digests chromatin.The method that the embodiment of the present invention is proposed is directly to the cell or tissue of native state
Cracking processing is carried out, and is to fix cell using formaldehyde crosslinking, and the problem of covering epitope information occur in the prior art,
The method that the embodiment of the present invention is proposed substantially increases the recognition efficiency of subsequent Ag-Ab.It is according to an embodiment of the present invention
Construct testing gene group DNA sequencing library method, be using micrococcal nuclease by chromatin be broken into mononucleosome or
The state of double-core corpusculum, i.e., the first above-mentioned digestion process product are the chromatin of fragmentation, and then effectively expose a group egg
White related antigen further effectively increases subsequent antigen-antibody reaction efficiency.
According to another specific embodiment, with reference to Fig. 3, inventor's discovery carries out testing gene group using micrococcal nuclease
First digestion process can further comprise: (1-1) makes the testing gene group and micrococcal nuclease working solution carry out pre-contact,
To obtain mixture after pre-contact.Product and micrococcal nuclease working solution carry out pre-contact after cracking processing, can make in advance
It obtains chromatin and adapts to digestion system, and then be conducive to the raising of digesting efficiency；(1-2) is using micrococcal nuclease to the pre-terminated
Mixture carries out the first digestion process after touch, to obtain the first digestion process product；(1-3) is terminated using micrococcal nuclease
Liquid terminates digestion process.By aforesaid operations, the chromatinic efficiency that micrococcal nuclease digests cell sample to be measured is significantly mentioned
Height, and then chromatin more can thoroughly be fractured into mononucleosome or double-core corpusculum.
According to another embodiment of the present invention, co-immunoprecipitation processing is realized by following operation: by described first
Digestion process product is incubated for histone antibodies, so that adaptive immune is co-precipitated processing product, optionally, is further wrapped
It includes: co-immunoprecipitation processing product being contacted with the magnetic bead for carrying protein A, optionally, further comprises: utilizing
RIPA buffer and lithium chloride buffer start the cleaning processing co-immunoprecipitation processing product.Histone antibodies specificity
It identifies the specific antigen site of histone on nucleosome, and then by the co-immunoprecipitation of antigen-antibody, chromatin is carried out
Enrichment can further be enriched with chromatin in addition, co-immunoprecipitation product is contacted with the magnetic bead for carrying protein A, improve dyeing
The purity of matter further cleans the co-immunoprecipitation product using RIPA buffer and lithium chloride buffer, can
Go the purity that enrichment purpose product (chromatin) is further increased unless specially combine.
Still another embodiment according to the present invention realizes second digestion process by following operation: utilizing elution
Buffer and Proteinase K carry out the second digestion process to co-immunoprecipitation processing product, to obtain the second digestion process
Product, optionally, second digestion process are carried out 1 hour under conditions of 55 degrees Celsius, optionally, further comprise:
Inactivation processing is carried out to the Proteinase K.According to an embodiment of the invention, above-mentioned elution buffer is for producing co-immunoprecipitation
Object is eluted from magnetic bead, and then under digestion of the Proteinase K to histone antibodies, the naked DNA without histone
Separate out, the optimum concentration reaction temperature of Proteinase K are 55 degrees Celsius, in 55 degrees Celsius of 1 hours of reaction, invention human hair
Existing, completely, shorter than 1 hour, digestion is insufficient, is longer than 1 hour for naked DNA release, and DNA can be made to generate non-specific drop
Solution.After protease K digesting processing 1, inactivation processing further is carried out to Proteinase K, according to specific example, inventor is lost using heat
Processing living is so that Proteinase K terminates reaction in due course.
According to a particular embodiment of the invention, the item that the dephosphorylation process of shrimp alkaline phosphotase is at 37 degrees Celsius is utilized
It is carried out 1 hour under part.Under conditions of 37 degrees Celsius, the enzyme activity highest of shrimp alkaline phosphotase is carried out at 1 hour dephosphorylation
Reason both can guarantee the abundant reaction of reaction substrate, ensure that not overreaction and nonspecific products be caused to increase.Further
Ground, inactivation processing can be carried out after dephosphorylation process to shrimp alkaline phosphotase, and inactivation processing is that 10 points are carried out under 65 degrees Celsius
Clock, inactivation processing can effectively terminate dephosphorylation reaction.
According to a particular embodiment of the invention, above-mentioned denaturation treatment can be used but be not limited to thermal denaturation processing.At denaturation
Reason be in order to which double-stranded DNA is changed into single stranded DNA, and then further according to TELP method obtain sequencing library, thermal denaturation processing tool
Have be denaturalized it is high-efficient, small on DNA damage, DNA mutation probability is low and will not introduce influence after continue library the factor advantage.
In addition, applicant elaborates TELP method in patent 201410466261.2.It is according to the present invention
Specific embodiment, TELP method are realized in the following way:
Firstly, the denaturation treatment product containing single strand dna is directly formed poly (C) in 3 ' endsnTail portion, so as to
Obtaining has Poly (C)nThe single strand dna of tail portion, wherein n represents the number of base C, and n is whole between 5~30
Number.According to specific example, the amount of the single strand dna is >=5pg.The initial amount that the present invention obtains sequencing library as a result, is shown
The initial amount for being less than other two generations sequencing libraries is write, is suitable for trace sample and constructs sequencing library, particularly suitable for precious rare sample
Product or clinical patient sample or primary cell construct sequencing library.Specific example according to the present invention, the single strand dna
Amount is 5pg~10ng.The high-efficient of sequencing library is constructed as a result, and accuracy is good.According to specific example, Poly (C)nThe n of tail portion
It can be the integer between 15~25.According to specific example, n can be 20.The effect in conjunction with extension primer is good as a result,.Root
According to specific embodiment, Poly (C)nThe mode that tail portion is added to 3 ' end of single strand dna is not particularly limited.According to specifically showing
Example, the poly (C)nTail portion, which can be, utilizes terminal enzyme (DNA) formation.Effect of the single stranded DNA in terminal enzyme (DNA) (TdT)
Under, 3 ' ends of DNA chain can connect several polycytidine deoxynucleotides Poly (C)n, reaction process are as follows: be pre-mixed
28 μ l DNA solutions, 1 μ l 10x EX buffer (Takara, supplied with RR006A), 1 μ l 1mM dCTP (NEB,
N0446S), high temperature is denaturalized DNA, obtains single strand dna.It is subsequently added into 1 μ l terminal enzyme (DNA) (TdT；NEB, M0315S),
And 35min is reacted under the conditions of 37 DEG C.After reaction, 75 DEG C are heated to be kept for 20 minutes, so that TdT is inactivated, obtains 3 ' ends
End connection oligomerization Poly (C)nSingle strand dna.
Then, using extension primer, there is Poly (C) based on describednThe single strand dna of tail portion obtains double-stranded DNA point
Son, wherein extension primer includes H (G) in its 3 ' endmUnit, H is base A, base T or base C, m are the number of bases G,
And m is the integer between 5~15.Extension primer can be at Poly (C) as a result,nSuitable position on tail portion originates annealed pairs.
According to embodiments of the present invention, H (G)mThe m of unit can be 9.H (G) as a result,mUnit and oligomerization Poly (C)nOriginate annealed pairs
Position be suitable for.According to specific example, the sequence of extension primer is not particularly limited, as long as can guarantee that DNA extends can be into
Row.Specific example according to the present invention, the extension primer can the nucleotide shown in SEQ ID NO:1 constitute.As a result,
Extension primer is easy to and poly (C)nTail portion pairing, extension are high-efficient.Wherein, the particular sequence of SEQ ID NO:1 is as follows:
GTGACTGGAGTTCAGACGTGTGCTGGGGGGGGGH (SEQ ID NO:1).
According to specific example, it can use KAPA 2G Robust HS and single strand dna extended, described in acquisition
Double chain DNA molecule.For example, the reaction system extended are as follows: the 3 ' ends that previous step obtains connect oligomerization Poly (C)nIt is single-stranded
In DNA molecular, 6.2 μ l water, 0.8 μ l KAPA 2G Robust HS (KAPA, KK5515), 12 μ l 5x KAPA buffers are added
2 μM of extension primers of A (KAPA, KK5515), 4.8 μ l 2.5mM dNTP (Takara, RR006A) and 6 μ l.According to specific reality
Apply example, the program of extension are as follows: (1) 95 DEG C of 3min；(2) 47 DEG C of 1min, 68 DEG C of 2min, 16 circulations；(3)72℃10min.
After reaction, it adds exonuclease I (Exo I) to react 1 hour in 37 DEG C, digests extra extension primer, obtain
Extension products.Wherein illustrating, the extension chain end obtained using extension, i.e. 3 ' ends of extended chain are base A, by
This, convenient for being connect with 5 ' by half connector of head end of base T.
According to specific embodiment, the extension primer can have selection markers, wherein the selection markers are formed in institute
State 5 ' ends of extension primer.Efficiently the double-stranded DNA after extension is screened by selection markers as a result, is purified, is obtained
Target gene.According to specific example, the selection markers can be biotin.As a result, using the DNA fragmentation for being connected with biotin
The purifying of method in conjunction with magnetic bead to the double stranded DNA product of extension, substantially reduces the loss of DNA in purification process.According to this
Invention specific example, process of the biotin in conjunction with magnetic bead are as follows: using 1x Binding&Wash (B&W) buffer (10mM in advance
Tris-HCl pH 8.0,0.5mM EDTA, 1M NaCl) cleaning magnetic streptavidin C1 magnetic bead
(Invitrogen, 650.01), the incubation after then being mixed with the product after extension on temperature control blending instrument, condition are as follows: 23 DEG C
1400rpm concussion (oscillation frequency: concussion 10s stops 10s) 30min.After reaction, 100 μ l of the magnetic bead of DNA are combined
1x B&W buffer is washed once, and 150 μ l EBT buffers (10mM Tris-HCl pH 8.0,0.02%Triton X-100) are washed
Three times, finally with 8.4 μ l elution buffer (EB；10mM Tris-HCl pH 8.0) it is resuspended, it is anti-for connection later
It answers.
Finally, in the double chain DNA molecule far from H (G)mOne end jointing of unit, and obtained connection is produced
Object is expanded, and to obtain amplified production, the amplified production constitutes the sequencing library.According to embodiments of the present invention, should
Step may further include: the single stranded nucleic acid molecule for being respectively provided with the nucleotide sequence as shown in SEQ ID NO:2-3 is carried out
Annealing, to obtain half connector, one end of half connector and the double chain DNA molecule is attached, to be connected
There is the double chain DNA molecule of half connector.It should be noted that if pearl is connected with one end of double-stranded DNA, then inhibition is generated,
Therefore half connector is connected with one end far from double-stranded DNA pearl connecting pin.Wherein, the 3 ' of half connector SEQ ID NO:2-3 are held all
There is phosphate group to modify to prevent it from connecting, it is specific as follows that half connector connects nucleotide sequence shown in primer:
Adp_A:GACGCTCTTCCGATCT (SEQ ID NO:2)；
Adp_B:GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT (SEQ ID NO:3)
According to specific example, the condition of reaction is connected are as follows: by 1 μ l quick ligase (NEB, M2200L), 10 μ l 2x are quick
Buffer is connected, the magnetic bead for being combined with extension products and 0.6 μ l 10mM connector that 8.4 μ l are resuspended are placed in centrifuge tube, fill
Divide and mixes.Centrifuge tube is placed on rotary incubator to prevent magnetic bead sedimentation, entire reaction carries out (about 15 overnight under the conditions of 4 DEG C
Hour), obtain the double chain DNA molecule of jointing.The background connected as a result, is small, and joint efficiency is high.According to specific implementation
Example, after the double chain DNA molecule jointing, before being expanded, to the double chain DNA molecule for being connected with connector into
Row purifying.According to specific embodiment, the mode of purifying is not particularly limited.Specific example according to the present invention, purifying can be with
It is to be carried out using the pearl of specific recognition biotin.According to another specific example, the pearl can be magnetic bead, wherein
Streptavidin is connected on the magnetic bead.As a result, using method of the magnetic bead in conjunction with the DNA fragmentation for being connected with biotin to prolonging
The purifying for the double stranded DNA product stretched, purification effect is good, and can substantially reduce the loss of DNA in purification process.According to specific
Embodiment can be eluted after purification using the 72 DEG C of heating of ultrapure distilled water, and the double chain DNA molecule for being connected with half connector exists
In eluent, the double chain DNA molecule for being connected with half connector of purifying is obtained.As a result, containing the double-stranded DNA point for being connected with half connector
The eluent of son can be directly used for the amplified production of next step, reduces intermediate operation step, DNA is avoided to lose.
After acquisition is connected with the double chain DNA molecule of half connector, it is expanded, to obtain amplified production, the expansion
Increase production object and constitutes the sequencing library.According to embodiments of the present invention, the mode of amplification is not particularly limited, and can according to need
PCR is taken turns using a wheel PCR or more.For example, this method uses two-wheeled PCR, first round PCR, using as shown in SEQ ID NO:4-5
Nucleotide as primer, the double chain DNA molecule for being connected with half connector is expanded, as a result, DNA molecular amplification effect
Rate is high；Second wheel PCR, according to embodiments of the present invention, second wheel PCR using the nucleotide as shown in SEQ ID NO:4-5 as
Primer.The amplification efficiency of DNA molecular is high as a result,.Wherein, the specific nucleotide sequence of amplimer is as follows:
Amplimer:
First round PCR:
MP24_G5:GTGACTGGAGTTCAGACGTGTGCTGGGGG (SEQ ID NO:4),
P1_FL:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC TTCCGATCT (SEQ
ID NO 5)；
Second wheel PCR:
P1_Sh:AATGATACGGCGACCACCGA (SEQ ID NO:6)
Remove the Index sequence of label: CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGG AGTTCAGACG
(SEQ ID NO:7)
According to embodiments of the present invention, in the amplification unit, the primer comprising sequence label can be used, to the connection
Product is expanded, and can survey multiple and different samples in a high-flux sequence as a result,.For example, in one end quilt of DNA library
It added diacritic index sequence label, which can be used for building for the standard library sample of Illumina high-flux sequence
It is vertical.
Term " sequence label primer " as used in the present invention refers to is embedded in sequence mark in PCR primer sequence
Sequence label primer, as a result, during carrying out amplification reaction using sequence label primer, can be introduced into purpose piece by label
Nucleic acid tag can be both introduced into 5 ' ends by one end of section, and label can also be introduced into 3 ' ends.For example, with reference to Fig. 6, when adopting
Use label PCR primer as upstream primer, i.e., when the 5 ' of target fragment hold calling sequence label, sequence label primer specificity
Ground identifies the sequence of 5 ' connectors, and then downstream primer can be using the sequence for specifically identifying 3 ' connectors.Pass through sequence label
Primer is connected with DNA molecular, can accurately characterize the sample source of DNA molecular.Above-mentioned nucleic acid tag is utilized as a result, it can be with
The DNA library for sequencing of a variety of DNA moleculars is constructed, simultaneously so as to by the way that the DNA library of different samples will be derived from
It is mixed, while being sequenced, classified based on sequence label to DNA sequence dna, obtain the sequence letter of a variety of DNA moleculars
Breath.So as to making full use of high-throughput sequencing technologies, such as using Solexa sequencing technologies, while to a variety of DNA moleculars
It is sequenced, to improve the efficiency and flux of DNA molecular sequencing.According to embodiments of the present invention, the particular sequence of sequence label
It is not particularly limited, can be designed according to their own needs, as long as can distinguishing sequence source.For example, using such as
The PCR primer that nucleotide shown in any one of SEQ ID NO:8~19 is constituted in table 1 is sequenced as a result, as label PCR primer
Accuracy and precision further increase.In the present specification, nucleic acid tag is named as IndexN respectively, wherein N=1-
Arbitrary integer in 12, sequence are as shown in table 1 below:
Table 1: the sequence of nucleic acid tag aligning primer Index1-12
Thus, it is possible to easily construct the gene sequencing library of a variety of samples simultaneously, and high pass can be effectively applied to
Microarray dataset is measured, so that the sequence information based on label can be distinguished accurately more after carrying out data analysis to sequencing result
The sequence information in the gene sequencing library of kind of sample can fully utilize high-flux sequence platform in turn, and save the time,
Reduce cost.
The equipment for constructing the DNA sequencing library of testing gene group
In the second aspect of the present invention, the invention proposes a kind of equipment in DNA sequencing library for constructing testing gene group.
With reference to Fig. 4, which includes: the first digestion process device 100, and the first digestion process device 100 is used to utilize microballoon core
Sour enzyme carries out digestion process to testing gene group, to obtain the first digestion process product；Co-immunoprecipitation processing unit 200,
The co-immunoprecipitation processing unit 200 is connected with the first digestion process device 100, and for first digestion
It handles product and carries out co-immunoprecipitation processing, so that adaptive immune is co-precipitated processing product；Second digestion process device 300, institute
It states the second digestion process device 300 to be connected with the co-immunoprecipitation processing unit 200, and for utilizing Proteinase K to institute
It states co-immunoprecipitation processing product and carries out the second digestion process, to obtain the second digestion process product；Dephosphorylation process dress
400 are set, the dephosphorylation process device 400 is connected with the second digestion process device 300, the dephosphorylation process dress
400 are set for directly carrying out dephosphorylation process to the second digestion process product using shrimp alkaline phosphotase, to obtain
Dephosphorylation process product；Degeneration processing device 500, the degeneration processing device 500 and the dephosphorylation process device 400
It is connected, and for the dephosphorylation process product directly to be carried out denaturation treatment, to obtain containing single strand dna
Denaturation treatment product；And TELP device 600, the TELP device 600 is connected with the degeneration processing device 500, described
TELP device 600 obtains sequencing library based on the denaturation treatment product containing single strand dna, according to TELP method, optional
Ground, the degeneration processing device 500 are suitable for thermal denaturation and obtain the single stranded DNA.Utilize utilization according to an embodiment of the present invention
It is low can to establish starting cell concentration for equipment according to an embodiment of the present invention for constructing the DNA sequencing library of testing gene group
To 500 or so cells genome or starting amount of DNA down to 2.5 nanograms genome DNA sequencing library, according to the present invention
Specific embodiment, it might even be possible to the genome or DNA initial amount for efficiently establishing starting cell concentration down to 200 cells are even low
To the DNA lateral order library of the genome of 1 nanogram.And the equipment that the embodiment of the present invention is proposed remains the special of single stranded DNA
Property, DNA loss is few, and gene information keeps complete, the gene of the equal few cells especially suitable for primary cell or removing tissue
The building in the DNA sequencing library of group.
With reference to Fig. 5, the equipment for constructing the DNA sequencing library of testing gene group can further comprise: cracker 700, institute
It states cracker 700 and is connected 100 with the first digestion process device, and the cracker 700 is used for cell or group
It knits and carries out cracking processing, to obtain testing gene group.
According to still another embodiment, with reference to Fig. 6, the first digestion process device 100 further comprises: pre-contact list
Member 110, the pre-contact unit 110 is connected with the cracker 700, and the pre-contact unit 110 be used for described in
Cls gene group is contacted with micrococcal nuclease working solution, to obtain mixture after pre-contact, genome and microballoon sclerotium
Sour enzyme working solution carries out pre-contact in pre-contact unit 110, chromatin can be made to adapt to digestion system in advance, and then be conducive to enzyme
Cut the raising of efficiency；First digestion process unit 120, the first digestion process unit 120 and 110 phase of pre-contact unit
Even, the first digestion process unit 120 is used for using the micrococcal nuclease to described in mixture progress after the contact
First digestion process, to obtain the first digestion process product；And digestion unit 130 is terminated, the termination digestion is single
Member 130 is connected with the first digestion process unit 120, and the termination digestion unit 130 is used for whole using micrococcal nuclease
Only liquid terminates digestion process.The dyeing of micrococcal nuclease digestion testing gene group in above-mentioned first digestion process device 100
The efficiency of matter significantly improves, and chromatin more can thoroughly be fractured into mononucleosome or double-core corpusculum, after further greatly improving
The joint efficiency of continuous Ag-Ab.
According to specific embodiment, with reference to Fig. 7, the co-immunoprecipitation device 200 includes: histone antibodies hatch unit
210, the histone antibodies hatch unit 210 is connected with the first digestion process device 100, and the histone antibodies are incubated for
Unit 210 is for the first digestion process product to be incubated for histone antibodies, so as to adaptive immune co-precipitation processing
Product, optionally, the co-immunoprecipitation device 200 include further comprising: protein A magnetic bead osculating element 220, described
Protein A magnetic bead osculating element 220 is connected with the histone antibodies hatch unit 210, and the pr-otein A magnetic
Pearl osculating element 220 is contacted for co-immunoprecipitation processing product with the magnetic bead for carrying protein A, optionally, described
Co-immunoprecipitation device 200 further comprises: cleaning unit 230, the cleaning unit 230, the cleaning unit 230 with it is described
Protein A magnetic bead osculating element 220 is connected, and for described immune total to what is contacted with the magnetic bead for carrying protein A
Precipitation process product starts the cleaning processing.First digestion process product is passed sequentially through in above-mentioned histone antibodies hatch unit 210
With the effect in protein A magnetic bead osculating element 220, chromatin is enriched with, and the purity of purpose product (chromatin) obtains
Improve, further under the action of cleaning unit 230, can remove it is non-specially combine, further increase enrichment purpose product (dyeing
Matter) purity.
According to still another embodiment, with reference to Fig. 8, the second digestion process device 300 further comprises: the second digestion
Processing unit 310, the second digestion process unit 310 are connected with the co-immunoprecipitation processing unit 200, and described second disappears
Change processing unit 310 to be used to carry out the second digestion to co-immunoprecipitation processing product using elution buffer and Proteinase K
Processing, to obtain the second digestion process product, optionally, the second slaking apparatus 300 further comprises: Proteinase K inactivation is single
Member 320, the Proteinase K inactivation unit 320 are connected with the second digestion process unit 310, and the Proteinase K inactivates unit
320 for carrying out inactivation processing to the Proteinase K.It is washed according to an embodiment of the invention, the second digestion process unit 310 utilizes
De- buffer elutes co-immunoprecipitation product from magnetic bead, and then in Proteinase K to the digestion of histone antibodies
Under, when by the naked DNA separate out for being free of histone.After protease K digesting processing, digestion product travels further into protease
K inactivation unit 320 carries out inactivation processing to Proteinase K wherein, and according to specific example, Proteinase K inactivates unit 320 and uses
Heat inactivation processing is so that Proteinase K terminates reaction in due course.
According to a particular embodiment of the invention, with reference to Fig. 9, the TELP device 600 includes: tail portion connection unit 610, institute
It states tail portion connection unit 610 to be connected with the degeneration processing device 400, and is used for the change containing single strand dna
Property processing product directly carries out 3 ' end modified processing, so as to 3 ' ends of the single strand dna formation poly (C) n tail
Portion, to obtain the single strand dna with the tail portion Poly (C) n, wherein n represents the number of base C, and n be 5~30 it
Between integer；Extension apparatus 620, the extension apparatus 620 are connected with the tail portion connection unit 610, and for being based on institute
The single strand dna with the tail portion Poly (C) is stated, obtains double chain DNA molecule, wherein extension primer includes H in its 3 ' end
(G) m unit, H is base A, base T or base C, m are the number of bases G, and m is the integer between 5~15；Connector connection
Unit 630, the connector connection unit 630 are connected with the extension apparatus 620, and for remote in the double chain DNA molecule
One end jointing from H (G) m unit；And amplification unit 640, the amplification unit 640 and the connector connection unit
630 are connected, and for expanding to the connection product of the connector connection unit 630, described to obtain amplified production
Amplified production constitutes the sequencing library and is optionally provided with terminal enzyme (DNA) in the tail portion connection unit 610, optional
Ground is provided with KAPA 2G Robust HS in the extension apparatus 620, to obtain the double chain DNA molecule, optionally, institute
It states extension primer nucleotide shown in SEQ ID NO:1 to constitute, optionally, what is be arranged in the extension apparatus 620 described prolongs
Extend object, has selection markers, wherein the selection markers are formed in 5 ' ends of the extension primer, optionally, the sieve
Choosing is labeled as biotin.Using TELP device according to an embodiment of the present invention, the sequencing based on single stranded DNA of acquisition can be passed through
Library, and the specificity of DNA chain is remained, sample loss is less, and gene information keeps more complete.
Optionally, with reference to Figure 10, the TELP device 600 further comprises: purification unit 650, the purification unit pair
The double chain DNA molecule for being connected with connector is purified, wherein the purifying is the pearl using specific recognition biotin
It carries out, optionally, the pearl is magnetic bead, wherein is connected with Streptavidin on the magnetic bead.Optionally, the purifying
Unit 650 further comprises: elution module, the elution module utilize ultrapure 72 degrees Centigrade of distilled water, will be with pearl knot
The double chain DNA molecule for being connected with connector closed is separated.Elution as a result, containing the double chain DNA molecule for being connected with half connector
Liquid can be directly added into amplification unit 640, be expanded, and reduce intermediate operation step, DNA is avoided to lose.
According to a particular embodiment of the invention, with reference to Figure 11, the connector connection unit 630 further comprises: half connector
Module 631 is formed, the single stranded nucleic acid molecule of the nucleotide sequence as shown in SEQ ID NO:2-3 respectively is annealed, to obtain
Obtain half connector；And link block 632, the link block is for connecting half connector with the double chain DNA molecule
It connects, to obtain the double chain DNA molecule for being connected with half connector, optionally, is provided with quick connection in the link block 632
Enzyme optionally, is made to obtain the double chain DNA molecule for being connected with half connector using the nucleotide as shown in SEQ ID NO:4-7
For primer, the double chain DNA molecule for being connected with half connector is expanded, optionally, in the amplification unit 640, is used
Primer comprising sequence label expands the connection product, and optionally, the primer containing sequence label is one group of mark
Sign one kind of aligning primer, wherein a group of labels aligning primer nucleotide shown in SEQ ID NO:8-19 is constituted.
About these units, unit, module implement processing the advantages of, before detailed description has been carried out,
This is no longer described in detail.It will be appreciated to those of skill in the art that can using it is as known in the art it is any be adapted for it is above-mentioned
Building block of the device of operation as above-mentioned each unit.Term " connected " used in herein should broadly understood,
It can be directly connected, can also indirectly connected through an intermediary, for the ordinary skill in the art, Ke Yigen
The concrete meaning of above-mentioned term is understood according to concrete condition.
The method for determining the DNA sequence dna information of testing gene group
In the third aspect of the present invention, the invention proposes a kind of methods of the DNA sequence dna information of determining testing gene group.
According to an embodiment of the invention, this method packet includes: the DNA sequencing text for constructing testing gene group according to mentioned-above method
Library；The DNA sequencing library is sequenced, to obtain sequencing result；And be based on the sequencing result, determine it is described to
The DNA sequence dna information of cls gene group.About building testing gene group DNA sequencing library, before detailed description has been carried out,
Details are not described herein.According to an embodiment of the invention, the method and apparatus that sequencing library is sequenced are not particularly limited, examine
Consider the maturity of technology, according to an embodiment of the 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 novel sequencing technologies, such as single-molecule sequencing
Technology, such as: the True Single Molecule DNA sequencing technology of Helicos company, Pacific
The single molecule, real-time (SMRT.TM.) technology and Oxford of Biosciences company
(Rusk, Nicole (2009-04-01) .Cheap such as nano-pore sequencing technology of Nanopore Technologies company
Third-Generation Sequencing.Nature Methods 6(4):244–245)。
It is surprisingly found by the inventors that utilizing the DNA sequence dna information of determining testing gene group according to an embodiment of the present invention
Method, can it is sensitive, accurately and efficiently determine few cells (cell initial amount down to 500 or so, even as low as 200)
Genome or minigene group (DNA initial amount down to 2.5 nanograms, even as low as 1 nanogram) single strand dna sequence letter
Breath.
The system for determining the DNA sequence dna information of testing gene group
In the fourth aspect of the present invention, the invention proposes a kind of system of the DNA sequence dna information of determining testing gene group,
It is characterized in that, the system includes: sequencing library building equipment 1000 with reference to Figure 12, the sequencing library constructs equipment 1000
It is described above；Sequencing equipment 2000, the sequencing equipment 2000 are connected with sequencing library building equipment 1000, with
Just for the gene sequencing library of the genome to be sequenced, the sequencing result of the DNA of the genome is obtained；And point
Desorption device 3000 analyzes the sequencing result of the DNA of the genome, to obtain the DNA sequence dna information.This ability
Field technique personnel are, it is understood that can be using any equipment for being adapted for aforesaid operations as known in the art as upper
State the building block of each unit.Term " connected " used in herein should broadly understood, and can be directly connected,
Can indirectly connected through an intermediary, for the ordinary skill in the art, can understand as the case may be on
State the concrete meaning of term.
Inventors have found that the system of the DNA sequence dna information using determining testing gene group according to an embodiment of the present invention, energy
Enough genomes that is sensitive, accurately and efficiently determining few cells (cell initial amount down to 500 or so, even as low as 200)
Or the sequence information of the single strand dna of minigene group (DNA initial amount down to 2.5 nanograms, even as low as 1 nanogram).
The method for determining the sequence information of testing gene group chromatin target area
In the fifth aspect of the invention, the invention proposes a kind of for determining testing gene group chromatin target area
The method of sequence information.According to an embodiment of the invention, with reference to Figure 13, this method comprises: being constructed according to mentioned-above method
The target area sequencing library of testing gene group, wherein target area described in the histone antibodies specific recognition；To described
The target area sequencing library of testing gene group is sequenced, to obtain sequencing result；And it is based on the sequencing result, really
The sequence information of the fixed testing gene group chromatin target area.
It is surprisingly found by the inventors that utilizing determining testing gene group chromatin target area according to an embodiment of the present invention
The method of sequence information sensitive, accurately and efficiently can determine few cells (cell initial amount down to 500 or so, even
Down to 200) the chromatin target of genome or minigene group (DNA initial amount down to 2.5 nanograms, even as low as 1 nanogram)
The sequence information in region, to realize the detection of the chromatin target area to the genome of few cells.
Below with reference to specific embodiment, the present invention will be described, it should be noted that 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.Particular technique or item are not specified in embodiment
Part, described technology or conditions (such as with reference to the work such as J. Pehanorm Brooker, translate by Huang Peitang etc. according to the literature in the art
" Molecular Cloning:A Laboratory guide ", the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Production firm person is not specified, being can be with conventional products that are commercially available, such as can purchase from Illumina company.
The DNA sequencing library of the building cellular genome of embodiment 1
1.1 reagents prepare
Lysate (Lysis buffer)
0.5%NP-40
0.5%Tween
0.1%SDS
MNase working solution (MNase working buffer)
·100mM Tris-HCl ph8.0
·2mM CaCl2
MNase dilution (MNase Dilute buffer)
·50mM Tris‐HCl,pH 8.0
·1mM CaCl2
0.2%Triton X-100
10x MNase terminate liquid (10xMNase Stop buffer)
·110mM Tris‐HCl pH 8.0
·55mM EDTA
2x RIPA buffer 2xRIPA buffer (on ice)
·280mM NaCl
1%Triton X-100
0.1%SDS
0.2%Na-Deoxycholate
·5mM EGTA
RIPA buffer RIPA buffer
·10mM Tris pH 8.0
·1mM EDTA
·140mM NaCl
1%Triton X-100
0.1%SDS
0.1%Na-Deoxycholate
Lithium chloride buffer (LiCl wash buffer)
·250mM LiCl
·10mM Tris pH 8.0
·1mM EDTA
0.5%NP-40 (now known as Igepal CA-630)
0.5%Na-deoxycholate
1.2 lytic cell samples
Supernatant is removed in the cell centrifugation of collection to save on ice to be placed on, configures fresh lysate, protease is added
Inhibitor (PI).19 microlitres of lysates are added in each sample, with blowing and beating repeatedly for several times under the pipette tips of low adsorption, until cell
Cracking completely avoids blowout bubble during paying attention to.After cell cracks completely, 19 microlitres of micrococcus luteus nucleic acid are added into sample
Enzyme (MNase) working solution, mixes well.
Fresh MNase dilution is configured, MNase is diluted to suitable concentration.So that chromatin is digested fracture and arrives
The state of mononucleosome.MNase is a kind of very sensitive enzyme, and the activity of nuclease can be slightly changed with the difference of batch,
Before the MNase every time using new lot, need to detect best MNase diluted concentration again.Into sample, 2 microlitres of addition is dilute
MNase after releasing, is put into 37 DEG C water baths and stands 5 minutes after mixing well, 5 microlitres of 10xMNase are added later and terminate
Liquid is mixed well to prevent undue interruption.
Into sample be added 50 microlitres of 2xRIPA buffers, supplement protease inhibitors, after mixing well in 4 degrees Celsius from
High speed centrifugation 15 minutes in scheming.It takes supernatant solution into 150 microlitres of new pipes, is placed in and saves on ice.
1.3 antibody incubation
40 microlitres of RIPA buffers are added into sample, the corresponding histone modification antibody of 1-2 microgram is added after mixing.On
Lower reverse soft mixing, is incubated overnight in 4 degrees Celsius of test tube suspension equipment.
1.4DNA elution
Second day, prepare the magnetic bead for having proteinA.Each co-immunoprecipitation sample (IP sample) needs 10 micrograms
Magnetic bead.Twice with RIPA buffer solution for cleaning magnetic bead, it is then resuspended with RIPA buffer.10 microgram magnetic are added into each sample
Pearl, mixing of turning upside down.It is put back on test tube suspension instrument, 4 degrees Celsius combine 2 hours.
Sample is placed on magnetic frame, after magnetic bead is all adsorbed onto the change clarification of magnetic frame solution, sops up supernatant.With
RIPA buffer solution for cleaning magnetic bead four times is suspended in 4 degrees Celsius of test tubes doubts upper rotation five minutes every time.Finally use lithium chloride buffer
Cleaning is primary.
By magnetic bead of short duration centrifugation rapidly, it is placed in the surplus liquid for removing on magnetic frame and attaching on magnetic bead.Into each sample
27 microlitres of water, one microlitre of 10xExTaq buffer, one microlitre of Proteinase K is added.It is mixed in vortex instrument and is placed on 55 degrees Celsius
A hour on shaking table.Invisible spectro liquid is drawn onto a new centrifuge tube, heats 40 minutes, makes under 72 degrees celsius
Proteinase K loses activity completely.
1.5 dephosphorylation process
Because of the particularity of MNase nuclease itself, what DNA was generated after being cut by it is 5 ' terminal hydroxyls, 3 ' end phosphorus
Acid groups.Library is built for downstream, needs to carry out it reparation of 3 ends.1 microlitre is added into the DNA sample after elution
Shrimp alkaline phosphotase (rSAP) reacts a hour under 37 degrees celsius, guarantees that 3 ' ends become hydroxyl by dephosphorylation.With
After be warming up to 65 degrees Celsius, continuing heating inactivates enzyme in 10 minutes.
1.6 denaturation treatments and TELP build library
The double-stranded DNA obtained after above-mentioned steps obtains single stranded DNA, list obtained after being directly over thermal denaturation processing
Chain DNA is directly used in TELP and builds library.
The screening of 2 dephosphorylation process enzyme of embodiment
Repair result to obtain best dephosphorylation end and guarantee the yield of final DNA, inventor from
T4PNK, T4DNA polymerase, Klenow tri- combination or T4PNK or rSAP in screen most suitable dephosphorylation process enzyme,
Specific experiment process is as described below:
The mouse embryo stem cell being grown in six porocyte culture plates is taken out from incubator, is used after sopping up culture medium
1ml PBS is softly cleaned one time, washes off remaining culture medium.The pancreatin that 0.5ml concentration is 0.05% is added later, disappears at 37 °
Change three minutes, terminates digestion reaction with the fresh culture of 1ml.To make cell go completely into single suspension cell state, 1ml is used
Big pipette tips blow and beat repeatedly, until the cell mass being visible by naked eyes.Later with the revolving speed low-speed centrifugal of 2000rmp 5 minutes.It inhales
Fall and cell is resuspended with PBS after supernatant, carries out cell count.Seven groups of 10k cells are taken to manage to 250ul low adsorption EP after calculating concentration
In, supernatant is carefully removed after being centrifuged again, ready cell is put and is kept on ice.
New lysate is configured, the concentration that 50x protease inhibitors (PI) arrives 1x is added.It is micro- that 19 are added into every group of sample
Lysate is risen, is blown and beaten repeatedly up and down with the pipette tips of low adsorption for several times, until cell cracks completely, avoids puffing away during paying attention to
Bubble.After cell cracks completely, 19 microlitres of MNase working solutions are added into every group of sample, mix well.
Fresh MNase dilution is configured, MNase is diluted to suitable concentration (0.01U), so that chromatin is digested
It is broken the state of mononucleosome.The MNase after 2 microlitres of dilutions is added into every group of sample, it is Celsius to be put into 37 after mixing well
It spends water-bath and stands 5 minutes, 5 microlitres of 10xMNase terminate liquids are added later, mix well to prevent undue interruption.
50 microlitres of 2xRIPA buffers are added into every group of sample, supplement protease inhibitors, it is Celsius in 4 after mixing well
High speed centrifugation 15 minutes are spent in centrifuge.It takes supernatant solution into 150 microlitres of new pipes, is placed in and saves on ice.
40 microlitres of RIPA buffers are added into every group of sample, it is anti-that 1.5 microgram H3K4me3 histone modifications are added after mixing
Body.It turns upside down soft mixing, is incubated overnight in 4 degrees Celsius of test tube suspension equipment.
Second day, prepare the magnetic bead (total 700ug) for having proteinA.100ug magnetic bead is added into every group of sample.With
Twice, then magnetic bead 10ul RIPA buffer is resuspended for 500ulRIPA buffer solution for cleaning magnetic bead.It is added into each sample
10ul magnetic bead, mixing of turning upside down.It is put back on test tube suspension instrument, 4 degrees Celsius combine 2 hours.
Sample is placed on magnetic frame, after magnetic bead is all adsorbed onto the change clarification of magnetic frame solution, sops up supernatant.With
150ulRIPA buffer solution for cleaning magnetic bead four times is suspended in 4 degrees Celsius of test tubes doubts upper rotation five minutes every time.Finally use 150ul chlorine
It is primary to change lithium buffer solution for cleaning.
By magnetic bead of short duration centrifugation rapidly, it is placed in the surplus liquid for removing on magnetic frame and attaching on magnetic bead.Into each sample
It is added 20 microlitres of Tris-EDTA buffers (10mM Tris, 1mM EDTA), one microlitre of Proteinase K carries out elution of reactive.It is vortexed
It is mixed on instrument and is placed on a hour on 55 degrees Celsius of shaking table.Invisible spectro liquid is drawn onto a new centrifuge tube, 72
It is heated 40 minutes under degrees celsius, Proteinase K is made to lose activity completely.
Match the reaction system (unit: ul) of set terminal reparation according to experiment condition shown in table 2,
Table 2:
After reacting a hour under 23 degrees celsius, A-F group is purified using Ampure beads, and G group uses
The method for crossing column purification.After obtaining DNA after purification, carries out TELP and build library, experimental result is as shown in figure 15.
Figure 14 shows, the purpose band that stripe size is intended in the range of 200bp-500bp, wherein the band of D group
Brightness highest shows the DNA output highest finally obtained, illustrates that exclusive use PNK enzyme is sufficient for dephosphorylized end and repairs
It is multiple.Within the scope of the considerations of can be included in optimization experiment condition in next step.
During next optimum experimental, it is contemplated that when further decreasing cell quantity, any purification process all can
Final DNA output is seriously affected, this is based on, attempts to remove the purification step after end is repaired.It is true and reliable in order to obtain
Experimental result, the initiator cell amount of experiment are reduced to 500 and compare experiment.When DAN is eluted, it is micro- that 27 are added into each sample
Rise water, 1 microlitre of elution buffer 10xExTaq, 1 microlitre of Proteinase K.It mixes and is placed on 55 degrees Celsius of shaking table in vortex instrument
One hour.Invisible spectro liquid is drawn onto a new centrifuge tube, is heated 40 minutes under 72 degrees celsius, makes protease
K loses activity completely.
When carrying out end reparation, it is directly added into 1 microlitre of rSAP enzyme into one group of sample, is directly added into another set
One microlitre of T4Polynucleotide Kinase (T4PNK) enzyme.Reaction system is as shown in table 3:
Table 3:
| rSAP | PNK | |
| 10xEx Taq | 1 | 1 |
| Proteinase K | 1 | 1 |
| ddH2O | 27 | 27 |
| T4 polynueleotide kinase (T4PNK) | 0 | 1 |
| rSAP | 1 | 0 |
A hour is reacted under 37 degrees celsius, heating makes enzyme lose activity later, without any purification step, directly
It meets TELP and builds library, experimental result is as shown in figure 15.
Figure 15 is the results show that the purpose band that stripe size is intended in the range of 200bp-500bp, wherein rSAP group
The amount of purpose band to be significantly more than PNK experimental group, the macroscopic amount of DNA on glue, the survey in enough downstreams can be obtained
Sequence experiment, illustrates to test feasible, selects end repair enzyme of the rSAP as subsequent experimental.
Embodiment 3
In order to verify the validity of the proposed banking process of the application, inventor takes most stringent measuring means: logical
It crosses and is based on being immunized altogether with a large amount of (1x10e7) the mouse embryo stem cell H3K4me3 for being treated as gold standard in ENCODE database
The result of the DNA sequencing technology (ChIP-sequence) of precipitating compares, and sees correlation between the two.
Specifically, on the one hand inventor compares the obtained spectrum data of inventor and ENCODE spectrum data, and will
It is loaded into UCSC browser, and then by the peak Distribution of observation H3K4me3, in conjunction with the position of itself and gene promoter
Relationship, judges whether banking process proposed by the invention succeeds.
As a result as shown in figure 16, can be seen from result (genomic locations: chr6:51,271,690-52,394,589)
Out, the ChIP- of the result (ENCODE) that is either obtained from a cells up to a million or inventor down to 200 cells
Sequence as a result, the gene in transcriptionally active promoter region, have the distribution of H3K4me3.Its signal-to-noise ratio also makes us full
Meaning, intuitively sees, the similitude of five tracks is high, and then effectively demonstrates the effective of banking process proposed by the invention
Property.
Still further aspect, inventor have also carried out more accurate big data analysis, inventor by whole gene group according to
Position is divided into the bin of continuous 2000bp, calculates the numerical value of its FPKM in each position, by comparing different cell concentrations with
FPKM value of the ENCODE data at same position, and then calculate its correlation.
As a result as shown in figure 17, can see from Figure 17 result, 5000 cell number data and ENCODE data
Correlation is up to 0.8, and cell quantity least (200) also has 0.75.Correlation is reduced with the reduction of cell quantity,
It is reasonable.Because being the comparison of full-length genome, there is such correlation to be enough to illustrate the reality even if in hundreds of order of magnitude cells
Under the conditions of testing, inventor still is able to obtain believable ChIP-sequence result.
In addition, term " first ", " second " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance
Or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or
Implicitly include one or more of the features.In the description of the present invention, the meaning of " plurality " is 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 specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (11)
1. a kind of method in the DNA sequencing library for constructing testing gene group characterized by comprising
(1) the first digestion process is carried out to testing gene group using micrococcal nuclease, to obtain the first digestion process product；
(2) the first digestion process product is subjected to co-immunoprecipitation processing, so that adaptive immune is co-precipitated processing product, institute
Stating co-immunoprecipitation processing is by carrying out the first digestion process product and histone antibodies to be incubated for realization；
(3) the second digestion process is carried out to co-immunoprecipitation processing product using Proteinase K, to obtain at the second digestion
Product is managed, second digestion process is by handling product to the co-immunoprecipitation using elution buffer and Proteinase K
Digestion process realization is carried out, inactivation treatment further is carried out to the Proteinase K after second digestion process；
(4) dephosphorylation process is directly carried out to the second digestion process product using shrimp alkaline phosphotase, to be gone
Phosphatizing treatment product, the dephosphorylation process are carried out 1 hour under conditions of 37 degrees Celsius；
(5) the dephosphorylation process product is directly subjected to thermal denaturation processing, to obtain the denaturation containing single strand dna
Handle product；And
(6) the denaturation treatment product based on described containing single strand dna obtains sequencing library according to TELP method.
2. the method according to claim 1, wherein further including: to described after the dephosphorylation process
Shrimp alkaline phosphotase carries out inactivation processing, and the inactivation processing is carried out 10 minutes under 65 degrees Celsius.
3. the method according to claim 1, wherein the TELP method is realized in the following way:
The denaturation treatment product containing single strand dna is directly carried out 3 ' end modified processing by (6-1), so as to described
3 ' ends of single strand dna form the tail portion poly (C) n, so that the single strand dna with the tail portion Poly (C) n is obtained,
In, n represents the number of base C, and n is the arbitrary integer between 5~30；
(6-2) utilizes extension primer, based on the single strand dna with the tail portion Poly (C) n, obtains double chain DNA molecule,
Wherein, the extension primer includes H (G) m unit in 3 ' ends, and H is base A, base T or base C, m are the number of bases G,
And m is the integer between 5~15；And
(6-3) in one end jointing of the double chain DNA molecule far from H (G) m unit, and to obtained connection product into
Row amplification, to obtain amplified production, the amplified production constitutes the DNA sequencing library.
4. the method according to claim 1, wherein the testing gene group is by lytic cell or tissue
The full-length genome or portion gene group of acquisition.
5. according to the method described in claim 4, it is characterized in that, step (1) further comprises:
(1-1) makes the testing gene group and micrococcal nuclease working solution carry out pre-contact, to mix after obtaining pre-contact
Object；
(1-2) carries out first digestion process to mixture after the contact using the micrococcal nuclease, to obtain
The first digestion process product；And
(1-3) terminates digestion process using micrococcal nuclease terminate liquid.
6. the method according to claim 1, wherein further comprising: the co-immunoprecipitation is handled product
It is contacted with the magnetic bead for carrying protein A.
7. the method according to claim 1, wherein further comprising: slow using RIPA buffer and lithium chloride
Fliud flushing starts the cleaning processing co-immunoprecipitation processing product.
8. the method according to claim 1, wherein second digestion process is under conditions of 55 degrees Celsius
It carries out 1 hour.
9. according to the method described in claim 3, it is characterized in that, step (6-3) further comprises:
(6-3-1) anneals the single stranded nucleic acid molecule of the nucleotide sequence as shown in SEQ ID NO:2-3 respectively, to obtain
Half connector；
One end of half connector and the double chain DNA molecule is attached by (6-3-2), is connected with half connector to obtain
Double chain DNA molecule；
(6-3-3) using the nucleotide as shown in SEQ ID NO:4-7 as primer, to the double-strand for being connected with half connector
DNA molecular is expanded.
10. a kind of method of the DNA sequence dna information of determining testing gene group characterized by comprising
The DNA sequencing library of described in any item method building testing gene groups according to claim 1~9；
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.
11. a kind of method for determining the sequence information of testing gene group chromatin target area characterized by comprising
The chromatin target area sequencing library of described in any item method building testing gene groups according to claim 1~9,
In, target area described in the histone antibodies specific recognition；
The target area sequencing library of the testing gene group is sequenced, to obtain sequencing result；And
Based on the sequencing result, the sequence information of the testing gene group chromatin target area is determined.
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| CN106191037B (en) * | 2016-08-08 | 2018-10-30 | 中国科学院北京基因组研究所 | A kind of construction method of multiple sequencing library |
| CN106192022B (en) * | 2016-08-08 | 2018-07-03 | 中国科学院北京基因组研究所 | The construction method of the multiple sequencing libraries of 16SrRNA |
| CN107217309A (en) * | 2017-07-07 | 2017-09-29 | 清华大学 | Build the method and its application in the DNA sequencing library of testing gene group |
| CN109517889B (en) | 2017-09-18 | 2022-04-05 | 苏州吉赛基因测序科技有限公司 | Method for analyzing oligonucleotide sequence impurities based on high-throughput sequencing and application |
| CN108181461A (en) * | 2017-12-29 | 2018-06-19 | 上海嘉因生物科技有限公司 | Applied to histone modification chromosome co-immunoprecipitation optimization method in tissue samples |
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