CN105008536A - Isolated oligonucleotide and use thereof - Google Patents
Isolated oligonucleotide and use thereof Download PDFInfo
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- CN105008536A CN105008536A CN201480011125.2A CN201480011125A CN105008536A CN 105008536 A CN105008536 A CN 105008536A CN 201480011125 A CN201480011125 A CN 201480011125A CN 105008536 A CN105008536 A CN 105008536A
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Abstract
Disclosed in the present invention are an isolated oligonucleotide and use thereof. The isolated oligonucleotide comprises: a first nucleic acid molecule, the first nucleic acid molecule encoding a double-base recognition module, wherein the double-base recognition module consists of a first single-base recognition module and a second single-base identification module in series, and the first single-base recognition module and the second recognition module both contain repeat variable di-residue amino acids; a first amplification sequence, wherein the first amplification sequence is located at the 5' side of the first nucleic acid molecule, and the first amplification sequence comprises a lis-type endoenzyme recognition sequence; and a second amplification sequence, wherein the second amplification sequence is located at the 3' side of the first nucleic acid molecule, and the second amplification sequence comprises a lis-type endoenzyme recognition sequence. The oligonucleotide can quickly obtain various combinations of RVD, thereby being capable of identifying any target nucleic acid sequence.
Description
Oligonucleotides of separation and application thereof
Technical field
The present invention relates to biological technical field.Specifically, the present invention relates to oligonucleotides of separation and application thereof.More specifically, the present invention relates to a kind of oligonucleotides of separation, a kind of oligonucleotide library, a kind of method of nucleic acid of construction expression TALE repetitive sequences and a kind of method of change cellular genome.Background technology
TALEN (Transcription activator-like effectors nucleases) directed gene modification technique is made up of two parts, a part of TALEN directional cuttings target sequence formation DNA double chain otch（DSB), another part Doner carriers provide the sequence for needing to edit, and complete to orient editing in genome.TALEN is made up of more than 12 series protein modules and Fokl restriction endonucleases, and each module includes 34 amino acid, and the 12nd and 13 amino acids residues are the critical sites of base identification, referred to as bis-amino acid residue（RVD).Target-gene sequence can be just recognized by changing RVD sequence of modules, TALEN is attached at target sequence under cutting DNA double-strand formation DSB, the mechanism of nonhomologous end repair in the cell, frameshift mutation occurs at DBS, terminator codon is formed.Due to target gene premature transcription termination, complete target gene and knock out purpose.TALEN technologies have knockout efficiency high, the features such as selectivity is strong.TALEN can select target spot in genome any part, accomplish gene knockout, change target gene sequence, so TALEN technologies are research gene function prefered methods.
However, due to there is the repeatability of sequence in its recognition sequence in Talen carriers so that the directly synthesis to RVD module groups is extremely difficult.Thus, the ways and means that TALE repetitive sequences are built at present still has much room for improvement.The content of the invention
It is contemplated that at least solving one of above-mentioned technical problem to a certain extent or providing at a kind of useful business selection.Therefore, it is an object of the present invention to propose a kind of oligonucleotides that can be effective for building TALE repetitive sequences and application thereof.
In the first aspect of the present invention, the present invention proposes a kind of oligonucleotides of separation.Embodiments in accordance with the present invention, the oligonucleotides of the separation includes：First nucleic acid molecules, the first nucleic acid molecule encoding double alkali yl identification module, the double alkali yl identification module is made up of the first single base identification module and the second single base identification module connected, and the first single base identification module and the second single base identification module are comprising the variable bis-amino acid residue of repetition；First extension increasing sequence, first extension increasing sequence is located at the 5' sides of first nucleic acid molecules, and first extension increasing sequence includes lis type endonuclease recognition sequences；And second extension increasing sequence, second extension increasing sequence is located at the 3' sides of first nucleic acid molecules, and the second nucleotide sequence includes lis type endonuclease recognition sequences.Due to including the nucleotide sequence of two single base identification modules of coding in the oligonucleotide molecules, thus, parent material is used as by the use of this kind of oligonucleotide molecules for encoding different RVD, it can be cut by using restriction enzyme, cohesive end is formed in the both sides of oligonucleotide molecules, can be directly attached, so that, the RVD of various various combinations can be quickly obtained, so as to recognize any target nucleic acid sequence.Digestion connection can be greatly reduced
Number of times, and it is surprisingly found by the inventors that, can realize that the mismatch rate of modular unit connection is substantially reduced, typically, be combined for longer RVD, digestion connection number of times is more, correct connection is more difficult.
Embodiments in accordance with the present invention, the oligonucleotides can also have following additional technical feature：
In one embodiment of the invention, the first single base identification module and the second single base identification module are respectively provided with following amino acid sequences：
LTPDQVVAIAS*RVD*GGKQALETVQRLLPVLCQDHG, wherein the * RVD* of the 12nd and the 13rd represent RVD sequences, that is, repeats variable bis-amino acid, and it determines different identification bases.Wherein, the corresponding RVD of base A are NI;The corresponding RVD of base C are HD;The corresponding RVD of base T are NG;And the corresponding RVD of bases G is N.Thus, the first single base identification module and the second single base identification module are respectively provided with selected from one of following amino acid sequence：
Recognize base A: LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG;
Recognize base C: LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG;
Recognize base T:LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG and
Recognize bases G: LTPDQWAIASNNGGKQALETVQRLLPVLCQDHG.
It is surprisingly found by the inventors that, can be effectively in the function including playing specific recognition base in the various kinds of cell including zooblast and plant cell by using above-mentioned single base identification module single base identification module.
And it is surprisingly found by the inventors that, compared with the TALEN of wild type, optimized by shortening the length of RVD both sides C-terminal and N-terminal, and to partial amino-acid, recognition efficiencies of the TALEN to target nucleotide obtained by can further improving.
In one embodiment of the invention, the RVD in the first single base identification module and the second single base identification module meets one of following condition：The RVD of the first single base identification module is NI, and the RVD of the second single base identification module is I;The RVD of the first single base identification module is NI, and the RVD of the second single base identification module is NG;The RVD of the first single base identification module is NI, and the RVD of the second single base identification module is HD;The RVD of the first single base identification module is NI, and the RVD of the second single base identification module is NN;The RVD of the first single base identification module is NG, and the RVD of the second single base identification module is NI;The RVD of the first single base identification module is NG, and the RVD of the second single base identification module is NG;The RVD of the first single base identification module is NG, and the RVD of the second single base identification module is HD;The RVD of the first single base identification module is NG, and the RVD of the second single base identification module is N;The RVD of the first single base identification module is HD, and the RVD of the second single base identification module is NI;The RVD of the first single base identification module is HD, and the RVD of the second single base identification module is NG;The RVD of the first single base identification module is HD, and the RVD of the second single base identification module is HD;The RVD of the first single base identification module is HD, and the RVD of the second single base identification module is N;The RVD of the first single base identification module is NN, and the RVD of the second single base identification module is NI;The RVD of the first single base identification module is NN, and the RVD of the second single base identification module is NG;The RVD of the first single base identification module is N, and the RVD of the second single base identification module is HD;Or the RVD of the first single base identification module is NN, the RVD of the second single base identification module is NN.Thus, the oligonucleotides, which can be encoded, can recognize AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CG, CC, GA, GT, GC, GG one kind.It is thus possible to by by all above-mentioned possible first bases
The combination of identification module and the second base identification module, build respectively after oligonucleotide molecules, the oligonucleotide molecules storehouse that can be used for building any RVD combinations can be effectively obtained, so as to, only need to corresponding oligonucleotide molecules being attached, you can combine to obtain the desired RVD that can recognize predetermined target nucleotide sequences.
According to one embodiment of present invention, the lis types restriction endonuclease is at least one selected from Bsal, Bbsl and BsmBI.Wherein, preferably described lis types restriction endonuclease is Bsal, and the lis types endonuclease recognition sequence is GGTCTCNNNN, wherein, ^^ is, T, G or C.Thus, it is possible to further improve by inscribe cleavage, and cleaved products are attached, obtain the efficiency of different RVD combinations.
According to one embodiment of present invention, first extension increasing sequence further includes Xbal restriction enzyme sites, and the first extension increasing sequence can have formula (M)1().2()TCTAGA(;M;)2.8GGTCTC(H; 8.25, wherein M is A, T, C or G;H is A, T, C or G, and consistent with the coded sequence of the first single base identification module.Optionally, second extension increasing sequence further includes Xhol restriction enzyme sites, and the second extension increasing sequence has formula (M') io-2oCTCGAG (M') 2-8GGTCTC (H') i8-25, wherein M' are A, T, C or G;11' is A, T, C or G, and is matched with the coded sequence of the second single base identification module.M and M' sequence is variable, in one embodiment of the invention, first nucleic acid molecules or separate room base identification module coded sequence are connected to carrier as amplification template, M and M' sequence can be respectively set to consistent with the 5' ends of carrier and with carrier 3 ' terminal sequences and match.Thus, it is possible to corresponding oligonucleotides easily be preserved or expand, consequently facilitating subsequent builds talen efficiency.Selection markers thing, such as drug resistance gene, so as to easily be expanded or screening vector or plasmid can be included in embodiments in accordance with the present invention, carrier or plasmid.
According to one embodiment of present invention, first extension increasing sequence and the second extension increasing sequence meet following condition：First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 1, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 10；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 2, second extension increasing sequence has such as SEQ ID N0:Nucleotide sequence shown in 11；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 3, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 12；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 4, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 13；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 5, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 14；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 6, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 15；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 7, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 16；First extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 8, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 17；Or first extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 9, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 18.
On SEQ ID NO:1-18 sequence is summarized as follows table 1,
0Ϊ869Ϊ/ 0Ζ OAV
It is surprisingly found by the inventors that, by using the combination of above-mentioned first extension increasing sequence and the second extension increasing sequence, the efficiency expanded to oligonucleotide molecules can be effectively improved, and inventor has found, based on above-mentioned first extension increasing sequence and the second extension increasing sequence, by for each double alkali yl identification module, above-mentioned first extension increasing sequence and the second extension increasing sequence is respectively adopted, each double alkali yl identification module can be directed to, build the parent material for connection, so as to when being attached larger RVD combinations, only need to select the first extension increasing sequence and the second extension increasing sequence of each oligonucleotide molecules, it can just be connected by a digestion, complete the structure combined to final RVD, the displacement and amplification of intermediate carrier need not be carried out, only need to converted after once connecting, in theory, only needing to 6 hours can be obtained by correct plasmid, again by the way that the experiment in downstream can be carried out after once converting amplification identification.Whole experiment process is very simple, has really tested procedure, experiment is had controllability.
In the second aspect of the present invention, the present invention proposes a kind of oligonucleotide library, it is characterised in that including：The oligonucleotides of multiple separation, wherein, the oligonucleotides of the separation is foregoing oligonucleotides.Thus, the oligonucleotide library, can be efficiently used for building different RVD combinations.Embodiments in accordance with the present invention, different oligonucleotides is separately positioned in different containers.Thus, it is possible to easily carry out the structure of RVD combinations.In addition, embodiments in accordance with the present invention, a large amount of oligonucleotides types are pre-set in the oligonucleotide library, first, for the various combinations of the first base identification module and the second base identification module, respective oligonucleotide molecules are built respectively, then, for each double alkali yl identification module, above-mentioned first extension increasing sequence and the second extension increasing sequence is respectively adopted, respective oligonucleotide molecules are built.So as to, it is only necessary to corresponding oligonucleotide molecules are attached, you can combined with obtaining the desired RVD that can recognize predetermined target nucleotide sequences.And, when being attached RVD combinations, only need to select the first extension increasing sequence and the second extension increasing sequence of each oligonucleotide molecules, it can just be connected by a digestion, complete the structure combined to final RVD, it is not necessary to carry out the displacement and amplification of intermediate carrier, only need to converted after once connecting, in theory, it is only necessary to which 6 hours can be obtained by correct plasmid, then by the way that the experiment in downstream can be carried out after once converting amplification identification.Whole experiment process is very simple, has really tested procedure, experiment is had controllability.In addition, PCR amplifications need not be used to obtain each Dan Shuan unit module in an experiment, but directly each Dan Shuan unit module is building up on plasmid, substantial amounts of fragment library is obtained by the amplification of plasmid, reduce due to the mutation that PCR is introduced, and make experiment controllability more preferable.Directly connect to obtain final carrier by the digestion of plasmid and plasmid, and can be screened using different resistances and obtain correct carrier.
According to the third aspect of the invention we, the present invention proposes a kind of method of the nucleic acid of construction expression TALE repetitive sequences.This method includes：The first oligonucleotides and the second oligonucleotides are provided, first oligonucleotides and the second oligonucleotides are foregoing oligonucleotides；First oligonucleotides and the second oligonucleotides are cut using lis types restriction endonuclease, to obtain the first oligonucleotides cleaved products and the second oligonucleotides cleaved products, wherein, the first oligonucleotides cleaved products are formed with cohesive end in the first extension increasing sequence and the second extension increasing sequence, and the second oligonucleotides cleaved products are formed with cohesive end in the first extension increasing sequence and the second extension increasing sequence, and the cohesive end that the cohesive end that the second extension increasing sequence of the first oligonucleotides cleaved products is formed is formed with the first extension increasing sequence of the second oligonucleotides cleaved products is matched；And be attached the first oligonucleotides cleaved products with the second oligonucleotides cleaved products, to obtain the oligonucleotides of expression TALE repetitive sequences.Can be effectively by the way that different oligonucleotides be attached, so as to obtain different RVD combinations using this method.In actual mechanical process, it is only necessary to which the first extension increasing sequence and the second extension increasing sequence of oligonucleotide molecules both sides are selected realize, big RVD combinations are obtained by once connecting.It should be noted that
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, " first " is defined, one or more this feature can be expressed or be implicitly included to the feature of " second ".In the description of the invention, " multiple " are meant that two or more, unless otherwise specifically defined.Thus, embodiments in accordance with the present invention, can complete any number of RVD combinations disposably in a digestion linked system, by a digestion coupled reaction.The displacement and amplification of intermediate carrier need not be carried out, it is only necessary to converted after once connecting, only needing to 6 hours theoretically can be obtained by correct plasmid, then the experiment in downstream can be carried out after identifying by once converting amplification.Whole experiment process is very simple, has really tested procedure, experiment is had controllability.And according to current conventional method, only building process, it is necessary to expend the time of 2 ~ 5 days.In addition, PCR amplifications need not be used to obtain each Dan Shuan unit module in an experiment, but directly each Dan Shuan unit module is building up on plasmid, substantial amounts of fragment library is obtained by the amplification of plasmid, reduce due to the mutation that PCR is introduced, and make experiment controllability more preferable.Directly connect to obtain final carrier by the digestion of plasmid and plasmid, and can be screened using different resistances and obtain correct carrier.
Embodiments in accordance with the present invention, the above method can further have following additional technical feature：
In one embodiment of the invention, the RVD sequences of first oligonucleotides and second oligonucleotide molecules are determined based on predetermined target nucleic acid sequence.It is preferred that, based on following relationship, determine the RVD sequences of first oligonucleotides and second oligonucleotide molecules：
The corresponding RVD of base A are I;
The corresponding RVD of base C are HD;
The corresponding RVD of base T are NG;And
The corresponding RVD of bases G is NN.Thus, it is possible to effectively improve the efficiency of the designed predetermined target nucleotide sequences of RVD combination identifications.
In addition, embodiments in accordance with the present invention, at least one of the first oligonucleotides and the second oligonucleotides include a variety of different oligonucleotides, wherein, a cohesive end of every kind of oligonucleotides is at most matched with a cohesive end of other oligonucleotides.For example, for multiple oligonucleotides, the forward primer cohesive end F of each oligonucleotidesnSimply with the reverse primer cohesive end R of another oligonucleotidesn+1It is connected, and can not be connected with the cohesive end on other primers, thus, it is possible to is disposably attached multiple oligonucleotides, so as to improve structure efficiency.
In the fourth aspect of the present invention, the present invention proposes a kind of method for changing cellular genome, it is characterised in that including:For target nucleic acid sequence predetermined in cellular genome, TALE RVD sequences are determined；According to foregoing method, the oligonucleotides of construction expression TALE repetitive sequences, the TALE repetitive sequences being capable of target nucleic acid sequence described in specific recognition；And the oligonucleotides of TALE repetitive sequences and the nucleic acid introducing cell of coding TALE DNA modification enzymes will be expressed.Thus, embodiments in accordance with the present invention, can effectively be directed to predetermined target nucleic acid sequence, specific RVD combinations be built, thus, it is possible to effectively improve the efficiency for changing cellular genome.Embodiments in accordance with the present invention, the target nucleic acid sequence length is 4 ~ 20nt, preferably 12 ~ 20nt.Embodiments in accordance with the present invention, TALE DNA modifications enzyme is Fok I.It is preferred that, the nucleic acid of the oligonucleotides and coding TALE DNA modification enzymes of the expression TALE repetitive sequences is fabricated on the same vector.Thus, it is possible to effectively improve the efficiency for changing cellular genome.
In the fifth aspect of the present invention, the present invention proposes a kind of polypeptide of separation, it is characterised in that the polypeptide is by preceding
What the oligonucleotides described in face was encoded.Thus, resulting polypeptide being capable of specific recognition base sequence.
In the fifth aspect of the present invention, the present invention proposes a kind of carrier.Embodiments in accordance with the present invention, the carrier includes the nucleic acid molecules for encoding foregoing oligonucleotides.Wherein, preferably described carrier is plasmid.Thus, it is possible to corresponding oligonucleotides easily be preserved or expand, consequently facilitating subsequent builds talen efficiency.Selection markers thing, such as drug resistance gene, so as to easily be expanded or screening vector or plasmid can be included in embodiments in accordance with the present invention, carrier or plasmid.In addition, embodiments in accordance with the present invention, the selection markers thing of carrier comprising oligonucleotides, the selection markers thing of expression vector of such as drug resistance gene from finally building can be different, so as to more easily realize that a coupled reaction can just filter out required identification module combination.
In the sixth aspect of the present invention, the present invention proposes a kind of plasmid library.Embodiments in accordance with the present invention, the plasmid library includes：Multiple plasmids, wherein, the plasmid is foregoing carrier.It is preferred that, different plasmids is separately positioned in different containers.
In the seventh aspect of the present invention, the present invention proposes a kind of cell.Embodiments in accordance with the present invention, the cell is to be changed genome by the method for foregoing change genome and obtain.Thus, resulting cell is compared with initial cell, and its genome carries mutation, the silence of such as gene expression.
In the eighth aspect of the present invention, the present invention proposes a kind of method modified gene.Embodiments in accordance with the present invention, this method includes：Based on the target sequence of the gene, corresponding at least two plasmid is selected from foregoing plasmid library；At least two plasmid is cut using lis types restriction endonuclease, to obtain a variety of plasmid cleavage products, wherein, the two ends of the plasmid cleavage product are respectively formed with cohesive end, and a cohesive end of every kind of plasmid is at most matched with a cohesive end of other plasmids；The multiple plasmid cleavage product is attached, to obtain the oligonucleotides of expression TALE repetitive sequences, the target sequence of gene is stated in the TALE repetitive sequences identification；The target sequence for stating gene is recognized based on the TALE repetitive sequences, by TALE DNA modification enzymes, the gene is changed.Thus, it is possible to effectively modify predetermined gene, such as gene silencing reaches the purpose of gene knockout.In embodiments in accordance with the present invention, multiple plasmids, a cohesive end of each plasmid is at most matched with a cohesive end of other plasmids.For example, for multiple plasmids, the forward primer cohesive end F of each plasmidnSimply with the reverse primer cohesive end R of another plasmidn+1It is connected, and can not be connected with the cohesive end on other primers, thus, it is possible to is disposably attached multiple plasmids, so as to improve structure efficiency.
Embodiments in accordance with the present invention, this method can also have following additional technical feature：
In one embodiment of the invention, based on following relationship, corresponding plasmid is determined：
The corresponding RVD of base A are I;
The corresponding RVD of base C are HD;
The corresponding RVD of base T are NG;And
The corresponding RVD of bases G is N.
In one embodiment of the invention, the gene is at least one of Ρ Ρ Α Λ γ, the target sequence TGACACAGAGATGCCATT and GAATCAGCTCTGTGG.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become apparent from the description below, or be recognized by the practice of the present invention.Embodiment
Below by specific embodiment, the present invention will be described.It should be noted that, what the embodiments described below were merely exemplary, limitation is not made to the scope of the present invention, and the reagent used in the following embodiments is commercially available, in addition, unless otherwise instructed, the method for step is not yet explicitly illustrated in embodiment below, is that conventional method well known by persons skilled in the art is carried out.Embodiment 1 builds oligonucleotide library
1st, identification module is determined
Inventor selection can distinguish AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC,
CG, GA, GT, GC, GG (16 double alkali yls）16 identification modules NI-NI, NI-NG, NI-HD, NI- it is suitable, NG-NK NG-NG NG-HD, NG- is suitable, HD-NK HD-NG, HD-HD, HD- are beautiful, cis- NI, NN-NG, NN-HD, NN-NN sequence is linked in sequence on carrier p-FUS-B2 respectively, and the template expanded is used as after identification is correct.
The base sequence of individual module is as follows：
NI(A)
CTGACCCCGG ACCAAGTGGT GGCTATCGCC AGCAACATTG GCGGCAAGCA AGCGCTCGAA HD(C)
CTGACTCCGG ACCAAGTGGT GGCTATCGCC AGCCACGATG GCGGCAAGCA AGCGCTCGAA
NG(T)
CTGACCCCGG ACCAAGTGGT GGCTATCGCC AGCAACGGTG GCGGCAAGCA
AGCGCTCGAA beautiful (G)
CTGACCCCGG ACCAAGTGGT GGCTATCGCC AGCAACAATG GCGGCAAGCA AGCGCTCGAA wherein p-FUS-B2 come from purchase from addgene, a carrier is used as by the use of p-FUS-B2, two single modules are connected together, the basic amplification template that inventor uses is constituted.It is stand-by after sequencing is correct.
2nd, identification module is expanded
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In superincumbent 18 primers of 5'-ctagacgtctcgatagcctcgagTGGTTggtctcTcAGTCCATGGTCCTGGCA C, all with Bsal restriction endonuclease recognition sequences GGTCTCN'NNNN.This is in type lis enzymes, and same restriction endonuclease recognition sequence can produce multiple viscosity identification ends, and 4 can be produced in theory4Individual viscosity identification end.Because the amino acid of ending and the beginning of each module is Gly and Leu, thus in view of degenerate codon（4 kinds of Gly codons, 6 kinds of Leu codons）Limitation, can produce 24 kinds of joints using a type lis enzyme.We choose 18 kinds therein and devise primer, except F 1 is with foretelling at R9 sunset, FnCan be with Rn+1Cohesive end be connected, and can not be connected with the cohesive end on other primers.It should be noted that in superincumbent amplimer, introducing Xhol and Xbal I restriction enzyme sites respectively at the two ends of amplified production respectively：C'TCGAG and T'CTAGA.
The program of PCR amplifications is as follows：
PCR reaction systems include：
Single double-template plasmid (50ng/ul) lul
Phusion High-Fidelity PCR Master Mix 25ul
Positive anti-primer（lOuM) 5ul
Distilled water 19ul
Cumulative volume 50ul
Reaction condition：
35 circulations
After PCR PCR primer recovery purifying is carried out for single module product；Due to there is the interference of single module, it is necessary to carry out rubber tapping recovery in the PCR primer of Dual module, the product that selection size is about 280bp carries out recovery purifying.
3rd, the plasmid library of identification module is built
Due to there is Xhol and Xbal restriction enzyme site on PNN4- plasmids, so inventor have selected the PNN4 plasmids that resistance is tetracyclin resistance and be connected as the background plasmid and single Dual module in plasmid storehouse, it is all step connection to be realized with more efficient because PNN4 resistance is different with the kalamycin resistance of final carrier.Wherein PNN4 is purchased from Addgene.
By based on PNN-4 module carriers plasmid and above resulting identification module amplified production by reclaiming purpose fragment after Xhol and Xbal digestions, digestion products are attached respectively, so as to obtaining this plasmid library to each identification module, 180 kinds of plasmids altogether.Sequence number according to primer is respectively designated as aN, and (wherein a represents 1-9 primer codes, the base of N representation modules identification, can be tetra- kinds of single base A, T, C, G, or 16 kinds of double alkali yls AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, GG)
Wherein endonuclease reaction condition is as follows：
Digestion system：
Xbal, lO U /μΙ 0.75ul
Xhol lO U /μΙ 0.75ul
NEB buffer 4 5ul
PNN-4 or single Dual module 500ng
Distilled water supplies volume to 50ul
Cumulative volume 50ul
Reaction condition： 37°C 4h
Wherein coupled reaction condition is as follows：
Linked system：
T4 liagase 0.4ul
T4 buffer lul
The PNN-4 20ng that digestion is obtained after reclaiming
Obtained single Dual module 200ng is reclaimed after digestion
Distilled water supplies volume to lOul
Cumulative volume lOul reaction conditions： 16°C 2h.After connection product transformed competence colibacillus bacterium TransTl, plasmid is extracted, plasmid is identified by double digestion（Xhol and Xbal) plasmid of fragment of size that obtains corresponding single Dual module send Sanger to be sequenced, and confirms sequence.
The plasmid for finally giving all single Dual modules being connected on PNN-4 is stand-by.
4th, the plasmid of the repeat unit of construction expression half
In Talen albumen, last half of repeat unit module actually includes half of RVD sequence and the also C-terminal of the talen albumen of part, in the present embodiment, this 2 parts are merged into a modular unit, it is attached simultaneously with other modules, connection flow can so be simplified, final carrier is reduced to a kind of final carrier without selecting different correspondences to recognize ATCG according to different end recognition sequence ATCG every time.
Because last module is half of unit, and the sequence of part N-terminal is contained, so inventor obtains corresponding sequence using the method for synthesis and PCR.C terminal seq of the present invention to synthesize enters performing PCR amplification as module using positive fragment primer and half-R.
The PCR amplification method is as follows：
PCR amplification system：
The C terminal seq 10ng of synthesis
Phusion High-Fidelity PCR Master Mix 25ul
half-R ( lOuM) 2ul
Distilled water supplies 50ul
Cumulative volume 50ul
Reaction condition is as follows：
98 °C 30s
98 °C 10s
60 °C of 20s J, 20 circulations
72 °C 20s
72 °C 5min
After first step PCR amplifications, 10ul is taken out from PCR system, lOul positive fragment primer is added, is heated to be placed in the annealing of room temperature natural cooling after 95 ° of 5min after mixing.Lul 1 Ox Klenow fragment Buffer and lul klenow fragment, 37 °C of processing 30min is added after annealing.
The product of 3ul above-mentioned steps is taken out, as module, enters performing PCR using amplimer F/R and reacts, condition is as follows：
PCR amplification system：
The product 3ul that Klenow ferment treatments are crossed
Phusion High-Fidelity PCR Master Mix 25ul
Amplimer F R (lOuM) 2ul
Distilled water supplies 50ul
Cumulative volume 50ul
Reaction condition is as follows：
98 °C 30s
98 °C 10s
60 °C of 20s, 35 circulations
72 °C 20s
72 °C 5min
PCR primer is connected to P-easy carriers up after reclaiming the fragment that size is 200bp or so length, rubber tapping recovery purifying.Reaction condition is as follows：
P-easy blunt vector lul
PCR primer 05.ul-4ul (20ng)
Picking monoclonal carries out Sanger sequencings with M13F after direct transformed competence colibacillus cell after room temperature connection 15min after mixing.Preserve correct plasmid stand-by.
Enter performing PCR amplification using amplimer F/R, it is available relative to recognition sequence last half and partial C-terminal modular unit, by the modular unit, with other single Dual modules, the same and PNN-4 is attached, method is the same with other single Dual modules, obtains that correct plasmid is sequenced.Because half is on the 10th of connection, 10A, 10T, 10C, 10G are respectively designated as according to the base of its identification.
Its full length sequence is as follows：
HD:
gcgcTCTAGACCTTAAACCGGCCAACATACCggtctcCactgaccCCGGACCAAGTGG
αι Das ) IOODDIIIDDDDIIIDIOIDIOVDO VODIDODOD , s ^-K^^
:MM
ON
( ΐ £ : ON ai 03S ) VVV03I3030VV30VV3003§§IIV3VV3§¾33§3^ §0I
:IM
(οε :OMaiDas) VVVODIDODOVVDOVVDOO^SSIVODVD^^^^S^^ ^OI
OH
(6 : OM ai Das ) § ¾§¾O¾§¾¾¾§§§§¾¾¾§§3 o
(82 OMaiDas) DDIIIDDDDIIIDIOIDIOVDOVODIDODOD :¾-/#|& f Ji
:OM(H0aS) 33VIV3VV330033VVVII33VOVI3I3§30 :J-/#|& f Ji
:Native ^ sfei hey Ife liJf ^ si
ςζ :OM CH 03S) §0§0OVO3I3¾0¾§¾0¾§¾¾¾§§§§¾¾¾§§00¾§¾§0§0¾¾00¾§W§°§00§ oi §11§ο§§οοΐ¾§ 裏οο§¾§ §¾οο裏1§11¾ο§¾¾¾§ο §ο§¾¾ο§¾¾ο§§ο§§ ) )3Υγο§¾οο§οΐ¾ § )
ON
:OM n Das) §o§oovo3i3¾0¾§¾0¾§¾¾¾§§§§¾¾¾§§00¾§¾§0§0¾¾00¾§w§°§00§
§11§ο§§οοΐ¾§ 裏οο§¾§ §¾οο裏1§11¾ο§¾¾¾§ο §ο§¾¾ο§¾¾ο§§ο§§ γ3Υγο§¾οο§οΐ¾ § ) ς
:IM
:OM n Das) §o§oovo3i3¾0¾§¾0¾§¾¾¾§§§§¾¾¾§§00¾§¾§0§0¾¾00¾§w§°§00§
§11§ο§§οοΐ¾§ 裏οο§¾§ §¾οο裏1§11¾ο§¾¾¾§ο §ο§¾¾ο§¾¾ο§§ο§§ γ )3Υ3θ§¾οο§οΐ¾ § ) £0tSL0/n0ZSLJ/∑Jd 0Ϊ869Ϊ/ 0Ζ OAV
Embodiment 2 is directed to target sequence, builds the selection of targeting vector talen target sites
The identification of talen target sites is since 5' to 3', and the previous position recognized is T.Online tool htt s can be utilized：The selection in/bogl ab .pip.iastate. edii/node/add/taien fi talen sites.Build N=A in 20bp target, G, T or C), middle 18bp is that we select the TALEN carrier target sequences to be built as needed.
1st, target sequence is selected
In this embodiment, inventor have selected gene PPARY2 (NCBI Shelf numbers ACCESSION: NG_011749 REGION:5001..151507), the position of the target sequence of the invention is located on 2 exons of the genes of Ρ Ρ Α Ι γ 2, and sequence is TGACACAGAGATGCCATTctggcccaccaacttcgGAATCAGCTCTGTGGA.Thus, the Talen of structure left arm recognizes 17 base sequence TGACACAGAGATGCCATT, right arm recognizes 15 base sequence GAATCAGCTCTGTGG, it is middle at intervals of 17 bases, advise the talen for including restriction enzyme site in selection interval in all designs in the present invention, be so conducive to follow-up target practice efficiency to verify.The foundation of talen designs is that the 5' ends of the gene order in target practice site are started with T, i.e., sequence is started with T, and is ended up with A.This T is extremely important for the efficiency for improving talen.
2nd, RVD modules are determined
Based on above identified target sequence, determine that the sequence of RVD modules is as follows：
Talen left arm RVD sequences：
Talen right arm RVD sequences：
HD HD NI HD NI are along NI NH HD NG along NI NG NG HD
After the RVD of target practice Talen identifications for determining Ρ Ρ Α Ι γ 2, we select plasmid from plasmid storehouse：1TG, 2AC, 3AC, 4AG, 5AG, 6AT, 7GC, 8CA, 9T, 10T have 10 plasmids altogether and the final vector plasmid of the present invention carries out digestion connection, finally give the correct Talen left arms of sequencing.Select plasmid：1CC, 2AC, 3AG, 4AG, 5CT, 6G, 7A, 8T, 9T, 10C-totally 10 plasmids and progress digestion connection of the final vector plasmid of the present invention, finally give the correct Talen right arms of sequencing.
3rd, module is connected
The plasmid concentration in each plasmid storehouse is unanimously diluted to 100ng/ul, because restriction enzyme site is Esp3I on carrier, therefore first carried out final carrier after digestion, the carrier that clip size is 4K is reclaimed in rubber tapping：
Digestion component volume is as follows：
Esp3I (BsmBI), lO U /μΙ 0.75ul
T4 buffer lul
Distilled water 14.85ul
Final carrier lmg
Cumulative volume 20ul
Reaction condition is rrc 4h.
The reaction system that digestion connection obtains final talen left and right arms is as follows:
Component volume
Carrier after digestion recovery purifying（100 ι^μΐ—1) lul
10 module plasmid (100 ng μ factories1) each 0.5ul
Bsal 0.75ul
BSA(IOX) lul
ATP(lOmM) lul
T7 ligase 0.25ul
Distilled water supplies volume to 20ul cumulative volumes 20ul
Reaction condition：
37°C 5min
20 °C lOmin J 35 cycles
Reaction converts connection product after terminating, and resistance is Ka, filters out stand-by after clone carries out Sanger correctly.
4th, verify
By sequencing, the assembling and comparison of above-mentioned Ρ Ρ Α Ι γ 2 Talen left arm RVD sequences：The RVD sequences of Talen left arm are：Corresponding RVD amino acid sequence is:
ETVQRLLPVLCQDHG (SEQIDNO: 37)
TVQRLLPVLCQDHG (SEQIDNO: 38)
TVQRLLPVLCQDHG (SEQIDNO: 39) TVQRLLPVLCQDHG (SEQIDNO: 40)
TVQRLLPVLCQDHG (SEQIDNO: 41)
TVQRLLPVLCQDHG (SEQIDNO: 42)
ETVQRLLPVLCQDHG (SEQIDNO: 43)
TVQRLLPVLCQDHG ( SEQ ID NO: 44)
LTPDQVVAIAS
E ( SEQ ID NO: 45 )
RVD is made up of altogether 598 amino acid, including 34* 17+20=598 ^
Sanger sequencing primer sequence be：
TAL-F1 5'-ttggcgtcggcaaacagtgg ( SEQ ID NO: 35 )
TAL-R2 5'-ggcgacgaggtggtcgttgg ( SEQ ID NO: 36)
Because whole Talen RVDs identification length is about 2k or so, for the correctness of the sequence that ensures each RVD, whole sequence RVDs points of three parts are carried out Sanger sequencings by us, the principle of sequence alignment is to ensure that three sections of sequences can be with overlapping, since the 1st amino acids LTPE, QAHG to last half ends, it is ensured that the complete comparison of each amino acid.
The carrier matched by detection sequence can be used to the detection of the target practice efficiency in downstream.
Targeting vector constructed in preceding embodiment 2 is transfected 293T cells by the gene knockout of embodiment 3：
Cell transfecting
Cell is adjusted before transfection to preferable state（Cellular morphology is clear, full bright）, one day before formal transfection, by passage in 6 orifice plates, cell density in transfection is reached that 80-90% is advisable.It is that negative control, a hole are positive control that a hole is reserved in addition to experimental group.
This experiment is to carry out cell transfecting using X-tremeGENE HP DNA Transfection Reagent（By taking 6 lonely L plates as an example）, in short, its step is as follows：
Prepare transfection composite：A) 200 μ serum free mediums or Opti-MEM I culture mediums.B) 2 μ are added§DNA (experimental groups：Each 1 μ of Talen plasmids§ ;Positive controls：The μ of EGFP-N1 plasmids 2§；Negative control group： none) .C) of short duration soft whirlpool（No more than 10s).D) the X-tremeGENE HP DNA DNA Transfection Reagent of 6 μ 1 are added.E) of short duration soft whirlpool.F) 15-30 minutes (15 to 25 °C) are incubated at room temperature.
Transfection composite is uniformly added dropwise in ready cell in advance, cross method gently shakes culture dish.Cell is placed in 37 °C, 5%C02Cultivated in incubator.
24h after transfection, by observing positive controls green fluorescence ratio, to speculate cell transfecting efficiency（Chinese hamster ovary celI is normally more than 80%）.Nutrient solution is discarded, the complete medium more renewed.
Transfect after 24h, culture medium can be replaced by the Selective agar medium containing puromycin, culture changes common complete medium into after about 7 days, now negative control group cell death ray, and experimental group still has survivaling cell（Antibiotic concentration will shift to an earlier date preliminary experiment measure with the medicine sieve time）
During medicine is sieved, it may appear that the phenomenon in culture medium is suspended in after a large amount of cell deaths, liquid can be changed with every 1 ~ 2 day, and remain that puromycin concentration is constant.
Change after common complete medium, treat experimental group cell length to about more than 80%, you can to extract genomic DNA progress
Subsequently identification discards culture medium, and PBS is used one time in each hole respectively, adds the digestion of 0.25% pancreatin, terminates and digests into addition complete medium after circle after cell dissociation, and supernatant is abandoned after collecting cell suspension, lOOOrpm centrifugations 5min.
Utilize blood tissues cellular genome extracts kit（TIANGEN cell genomic dna) is carried.
Using genomic DNA as template, enter performing PCR using the primer designed in advance according to target site, whether agarose electrophoresis testing goal clip size is correct, and PCR primer sample presentation is sequenced.
If there is set peak in target site in sequencing result, illustrate that target site is knocked out, Talen effects.Correct PCR primer will be sequenced and carries out a large amount of digestions, gene is not knocked out to remove, digestion products carry out digestion again after TA clones, bacterium colony PCR after reclaiming, to verifying that correct clone carries out sample presentation sequencing, the correctness of sequencing result confirmatory experiment.
The preparation of monoclonal cell
The gelatin for adding 0.1% on 10cm culture dishes in advance is put into 37 °C of incubator bed boards, there is the cell of knockout to be digested to cell suspension using pancreatin Preliminary Identification, counted by cell counting count board, cell suspension is added in culture dish with each 2000 cells of ware, 37 °C, 5%C02 incubators culture one week, forms the sharp-edged population of cells of island, as cell monoclonal.
The detection of monoclonal cell
The single cell clone that will have been grown after Method of Limited Dilution, suction out to be transferred in 96 orifice plates under the microscope using liquid-transfering gun and cultivate, it is passaged to after cell density reaches 90% in 48 orifice plates, and isolate a small amount of cell using fl40 phire animal tissue direct per kit enter performing PCR amplification, shown by agarose gel electrophoresis, wherein 93,96 samples are having band at 530bp.75 °C of digestions are carried out to PCR primer by Phol restriction enzymes again, electrophoresis showed go out that former 530bp bands are not cut open for the positive colony that filters out, its PCR result is sent into sequencing.Then sequencing result is analyzed, Phol sites（GGCC it is the single cell system after TALEN knockouts) to have the corresponding clone cell of knockout.Preliminary sequencing results display sequencing sample is 23, wherein being 6 without cell is knocked out, knocks out but has heteroproteose cell to pollute 2, single knockout 13（9 sequencing results are not obvious）, it is double to knock out 2（27,64) .
Further 27 are connected with No. 64 PCR primers with PMD18-T, T cloning and sequencings are carried out.Analysis result is as follows：
Thus, demonstrate and tested more than, successfully obtain the cell clone that many plants of pure and mild ppair2 are knocked out, and there are 2 plants to be knocked out for pure and mild pair.
In the description of this specification, the description of reference term " one embodiment ", " some embodiments ", " example ", " specific example " or " some examples " etc. means to combine specific features, structure, material or the feature that the embodiment or example describe and is contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referring to the schematic representation of above-mentioned term.Moreover, specific features, structure, material or the feature of description can in an appropriate manner be combined in any embodiment or example.
Although embodiments of the invention have been shown and described above, it is appreciated that, above-described embodiment is exemplary, it is not considered as limiting the invention, one of ordinary skill in the art can be changed to above-described embodiment, changes, replace and modification within the scope of the invention in the case where not departing from the principle and objective of the present invention.
Claims (1)
- Claims1st, a kind of oligonucleotides of separation, it is characterised in that including：First nucleic acid molecules, the first nucleic acid molecule encoding double alkali yl identification module, the double alkali yl identification module is made up of the first single base identification module and the second single base identification module connected, and the first single base identification module and the second single base identification module are comprising the variable bis-amino acid residue of repetition；First extension increasing sequence, first extension increasing sequence is located at the 5' sides of first nucleic acid molecules, and first extension increasing sequence includes lis type endonuclease recognition sequences；AndSecond extension increasing sequence, second extension increasing sequence is located at the 3' sides of first nucleic acid molecules, and the second nucleotide sequence includes lis type endonuclease recognition sequences.2nd, oligonucleotides according to claim 1, it is characterised in that the first single base identification module and the second single base identification module are respectively provided with amino acid sequence：LTPDQVVAIAS*RVD*GGKQALETVQRLLPVLCQDHG,Wherein, * RVD* represent to repeat variable bis-amino acid residue.3rd, oligonucleotides according to claim 2, it is characterised in that the RVD in the first single base identification module and the second single base identification module meets one of following condition：The RVD of the first single base identification module is that the RVD of the second " single base identification module described in L is NI;The RVD of the-- single base identification module is that the RVD of the second " single base identification module described in L is that the RVD of the-- single base identification module described in NG is that the RVD of the second " single base identification module described in L is that the RVD of the-- single base identification module described in HD is that the RVD of the second " single base identification module described in L is that the RVD of the-- single base identification module described in NN is NG, described：The RVD of two single base identification modules is that the RVD of the-- single base identification module described in NI is NG, the RVD of the second single base identification module is that the RVD of the-- single base identification module described in NG is NG, the RVD of the second single base identification module is that the RVD of the-- single base identification module described in HD is NG, the RVD of the second single base identification module is that the RVD of the-- single base identification module described in NN is HD, and the RVD of the second single base identification module is NI;The RVD of the-- single base identification module is HD, the RVD of the second single base identification module is that the RVD of the-- single base identification module described in NG is HD, the RVD of the second single base identification module is that the RVD of the-- single base identification module described in HD is HD, the RVD of the second single base identification module is that the RVD of the-- single base identification module described in NN is N, and the RVD of the second single base identification module is NI;The RVD of the-- single base identification module is N, and the RVD of the second single base identification module is NG;The RVD of the-- single base identification module is N, and the RVD of the second single base identification module is HD;Or the RVD of the first single base identification module is N, the RVD of the second single base identification module is NN.4th, oligonucleotides according to claim 1, it is characterised in that the lis types restriction endonuclease is at least one selected from Bsal, Bbsl and BsmBI.5th, the oligonucleotides according to claim 1, it is characterised in that the lis types endonuclease recognition sequence is GGTCTCNNNN, wherein, N is A, T, G or C.6th, oligonucleotides according to claim 1, it is characterised in that first extension increasing sequence further includes Xbal restriction enzyme sites, the first extension increasing sequence has formula CM;>1Q.2QTCTAGA(M;)2.8GGTCTC(;H; 8.25, wherein M is A, T, C or G;！！For, T, C or G, it is and consistent with the coded sequence of the first single base identification module.7th, oligonucleotides according to claim 1, it is characterised in that second extension increasing sequence further includes Xhol restriction enzyme sites, the second extension increasing sequence has formula (M>.2QCTCGAG(M';)2.8GGTCTC(;H'; 8.25, wherein M' is A, T, C or G;11 are, T, C or G, and are matched with the coded sequence of the second single base identification module.8th, oligonucleotides according to claim 1, it is characterised in that first extension increasing sequence and the second extension increasing sequence meet following condition：First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 1, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 10；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 2, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 11；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 3, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 12；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 4, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 13；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 5, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 14；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 6, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 15；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 7, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 16；First extension increasing sequence has such as SEQ ID NO：Nucleotide sequence shown in 8, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 17；OrFirst extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 9, second extension increasing sequence has such as SEQ ID NO:Nucleotide sequence shown in 18.9th, a kind of oligonucleotide library, it is characterised in that including：The oligonucleotides of multiple separation, wherein, the oligonucleotides of the separation is the oligonucleotides described in any one of claim 1 ~ 8.10th, oligonucleotide library according to claim 9, it is characterised in that different oligonucleotides is separately positioned in different containers.11st, a kind of method of the nucleic acid of construction expression TALE repetitive sequences, it is characterised in that including：The first oligonucleotides and the second oligonucleotides are provided, first oligonucleotides and the second oligonucleotides are the oligonucleotides described in any one of claim 1 ~ 8；First oligonucleotides and the second oligonucleotides are cut using lis types restriction endonuclease, to obtain the first few nucleosides Sour cleaved products and the second oligonucleotides cleaved products, wherein, the first oligonucleotides cleaved products are formed with cohesive end in the first extension increasing sequence and the second extension increasing sequence, and the second oligonucleotides cleaved products are formed with cohesive end in the first extension increasing sequence and the second extension increasing sequence, and the cohesive end that is formed of the second extension increasing sequence of the first oligonucleotides cleaved products is matched with the cohesive end that the first extension increasing sequence of the second oligonucleotides cleaved products is formed；AndThe first oligonucleotides cleaved products are attached with the second oligonucleotides cleaved products, to obtain the oligonucleotides of expression TALE repetitive sequences.12nd, method according to claim 11, it is characterised in that based on following relationship, determine the RVD sequences of first oligonucleotides and second oligonucleotide molecules：The corresponding RVD of base A are I;The corresponding RVD of base C are HD;The corresponding RVD of base T are NG;AndThe corresponding RVD of bases G is N.13rd, method according to claim 11, it is characterized in that, at least one of first oligonucleotides and the second oligonucleotides include a variety of different oligonucleotides, wherein, a cohesive end of every kind of oligonucleotides is at most matched with a cohesive end of other oligonucleotides.14th, a kind of method for changing cellular genome, it is characterised in that including：For target nucleic acid sequence predetermined in cellular genome, TALE RVD sequences are determined；Method according to any one of claim 11 ~ 13, the oligonucleotides of construction expression TALE repetitive sequences, the TALE repetitive sequences being capable of target nucleic acid sequence described in specific recognition；AndThe nucleic acid that the oligonucleotides and coding TALE DNA modification enzymes of TALE repetitive sequences will be expressed introduces the cell.15th, method according to claim 14, it is characterised in that the target nucleic acid sequence length is 4 ~ 20nt, preferably 12-20nt.16th, method according to claim 14, it is characterised in that TALE DNA modifications enzyme is Fok I.17th, method according to claim 14, it is characterised in that the nucleic acid of the oligonucleotides of the expression TALE repetitive sequences and coding TALE DNA modification enzymes is fabricated on the same vector.18th, a kind of polypeptide of separation, it is characterised in that the polypeptide is encoded as the oligonucleotides described in any one of claim 1 ~ 8.19th, a kind of carrier, it is characterised in that include the nucleic acid molecules of the oligonucleotides described in coding any one of claim 1 ~ 8.20th, carrier according to claim 19, it is characterised in that the carrier is plasmid.21st, a kind of plasmid library, it is characterised in that including：Multiple plasmids, wherein, the plasmid is the carrier described in claim 19 or 20.22nd, plasmid library according to claim 21, it is characterised in that different plasmids is separately positioned in different containers.23rd, a kind of cell, it is characterised in that the cell is to be changed genome by the method described in any one of claim 14 17 and obtain. 24th, a kind of method modified gene, it is characterised in that including：Based on the target sequence of the gene, corresponding at least two plasmid is selected from the plasmid library described in claim 21 or 22；At least two plasmid is cut using lis types restriction endonuclease, to obtain a variety of plasmid cleavage products, wherein, the two ends of the plasmid cleavage product are respectively formed with cohesive end, and a cohesive end of every kind of plasmid is at most matched with a cohesive end of other plasmids；The multiple plasmid cleavage product is attached, to obtain the oligonucleotides of expression TALE repetitive sequences, the target sequence of gene is stated in the TALE repetitive sequences identification；The target sequence for stating gene is recognized based on the TALE repetitive sequences, by TALE DNA modification enzymes, the gene is changed.25th, method according to claim 24, it is characterised in that based on following relationship, determines corresponding plasmid:The corresponding RVD of base A are I;The corresponding RVD of base C are HD;The corresponding RVD of base T are NG;AndThe corresponding RVD of bases G is N.26th, the method according to claim 24, it is characterised in that the gene is Ρ Ρ Α Ι γ, at least one of described target sequence TGACACAGAGATGCCATT standing grain mouthful GAATCAGCTCTGTGG.
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| CN201480011125.2A CN105008536A (en) | 2013-04-16 | 2014-04-15 | Isolated oligonucleotide and use thereof |
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