CN105524919A - Ligation-independent cloning (LIC) joint sequence and application thereof - Google Patents
Ligation-independent cloning (LIC) joint sequence and application thereof Download PDFInfo
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- CN105524919A CN105524919A CN201511027677.5A CN201511027677A CN105524919A CN 105524919 A CN105524919 A CN 105524919A CN 201511027677 A CN201511027677 A CN 201511027677A CN 105524919 A CN105524919 A CN 105524919A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
Abstract
The present invention relates to the field of genetic engineering, and in particular relates to a ligation-independent cloning (LIC) joint sequence and application thereof. The ligation-independent cloning (LIC) joint sequence comprises an upstream sequence of gg gAg Cgg AAg Cgg, which is a nucleotide sequence shown as SEQ ID NO: 7; and a downstream sequence of gC Cag AgC CAC Cgc, which is a nucleotide sequence shown as SEQ ID NO: 8. The ligation-independent cloning (LIC) joint sequence is connected with a plasmid vector to form a new vector for achievement of ligation-independent cloning of target fragments.
Description
Technical field
The present invention relates to genetically engineered field, particularly a pair for connect non-dependent clone joint sequence and application.
Background technology
Enzyme is cut, DNA ligase connects the restriction that the method building plasmid vector is subject to the sequence, arrangement mode, direction etc. of restriction enzyme site to adopt restriction enzyme to carry out.
The clone technology (LIC) connecting non-dependent utilizes the exo-acting of archaeal dna polymerase, the longer sticky end of DNA double chain 5 ' is produced at object carrier and Insert Fragment, the cyclic DNA with nicking is formed by base pair complementarity, after transformation of E. coli, in bacterial cell, repair the plasmid DNA become without nicking, thus complete the clone of object fragment.LIC technology can overcome enzyme and cut the restriction connecting clone, and object fragment is not by the impact of carrier restriction enzyme site, and cloning efficiency is high, is applicable to high-throughout clone.
Connect the design that one of key of non-dependent clone is joint sequence, because plasmid vector is used for the expression of target protein, therefore joint sequence should be avoided introducing rare amino acid residue, charge residue residue etc., to ensure that protein properties noticeable change does not occur as far as possible.
Summary of the invention
The invention provides a pair for connecting the joint sequence of non-dependent clone, after it being connected to the multiple clone site position of plasmid vector, the clone of the connection non-dependent of exogenous genetic fragment can be carried out, for connecting the structure of the carrier of non-dependent clone.
The technical solution adopted for the present invention to solve the technical problems is:
For connecting a non-dependent clone's joint sequence, this joint sequence comprises
Upstream sequence: the nucleotide sequence shown in gggAgCggAAgCgg, SEQIDNO:7; With
Downstream sequence: the nucleotide sequence shown in gCCagAgCCACCgc, SEQIDNO:8.Of the present invention for connecting non-dependent clone (ligationindependentcloing, LIC) sequence, the new carrier formed after this joint sequence is connected into plasmid vector, the connection non-dependent clone of object fragment can be realized.
Described joint sequence, connecting the application in non-dependent clone, comprises the steps:
The acquisition synthetic joint sequence according to claim 1 of a, sequence;
B, utilize Protocols in Molecular Biology joint sequence to be connected to the multiple clone site position of plasmid vector after, the new carrier of formation, carries out connecting the clone of non-dependent to exogenous sequences.
As preferably, utilize Protocols in Molecular Biology, the primer of synthesis amplification Gateway box, wherein upstream and downstream joint sequence of the present invention lays respectively at 5 ' end of upstream and downstream primer, by pcr amplification Gateway box, cut to connect by enzyme and be cloned into the multiple clone site of object carrier, form the clone that new carrier may be used for as connecting non-dependent, wherein the upstream and downstream position of the multiple clone site of this carrier is respectively upstream and downstream joint sequence of the present invention.When carrying out connection non-dependent clone, upstream and downstream joint sequence of the present invention should design the upstream and downstream primer 5 ' end with goal gene respectively, obtains by pcr amplification the goal gene fragment that flanking sequence is upstream and downstream joint.By T4DNA polysaccharase process carrier and intermediary's fragment, obtain the outstanding carrier of 5 ' end and intermediate segment, end is wherein given prominence to as joint sequence.By by carrier outstanding for 5 ' end and fragment mixing, form base pair complementarity, transform as after intestinal bacteria, repair in intestinal bacteria and form complete plasmid vector, complete the clone to object fragment.
Two sections of nucleotide sequences of the present invention, after it being connected to the multiple clone site position of plasmid vector, can carry out the clone of the connection non-dependent of exogenous genetic fragment, for connecting the structure of the carrier of non-dependent clone.Sequence of the present invention can be used in being suitable for LIC plasmid vector joint.
The present invention utilizes molecular biology experiment technology that sequence of the present invention is linked the multiple clone site of plasmid vector, forms the plasmid vector for connecting non-dependent clone, may be used for the connection non-dependent clone of goal gene.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation make the present invention and/or change all will fall into scope.
In the present invention, if not refer in particular to, all parts, per-cent are weight unit, and the equipment adopted and raw material etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.
Embodiment provided by the present invention all conveniently experiment condition, wherein adopted primer sequence is as table 1.
Table 1 connects non-dependent cloning vector and builds primer
Embodiment 1: connect the design of non-dependent cloning adapters sequence, synthesis, amplification and mensuration
1. connect design, the synthesis of non-dependent cloning adapters sequence
Primer pair Licl1-FP (+) respectively containing the nucleotide sequence shown in joint sequence SEQIDNO:7 and SEQIDNO:8, Licl1-RP (-) are synthesized and purifying by nucleic acid Synesis Company
2.Gateway box increases
With primer pair Licl1-FP (+), Licl1-RP (-) amplification Gateway box sequence, PCR amplification system is: 38 μ L water, 5 μ L10 × PCRBufferforPfu, 4 μ L2.5mMdNTPs, each 0.5 μ L of upstream and downstream primer (10 μMs), 1 μ LGateway box plasmid template, 1 μ LPfu polysaccharase.Total reaction system is 50 μ L.The reaction conditions of each fragment is: 94 DEG C of 5min; 94 DEG C of 45s, 52 DEG C of 45s, 72 DEG C of 4min, 35 circulations; 72 DEG C of 10min.The Gateway box containing joint sequence border is obtained through order-checking splicing after the PCR primer purifying of amplification.
3. connect the structure of non-dependent cloning vector
In step 2, PCR primer is after Qiagen gel purification kit (German Qiagen company) purifying, DNA ligase is utilized to carry out the clone of PCR fragment, adopt 10 μ L system reactions, condition is as follows: 10 × ligasebuffer1ul, NdeI and XhaoI double digestion linearizing cloning vector pET3250ng, Gateway box reclaims fragment 50ng, T4-ligase1ul (Dalian is precious biological), finally uses ddH
2o adjusts system to 10 μ L, mixes rear 4 DEG C of reaction 16h.After completion of the reaction, get in the centrifuge tube that 5ul reaction solution joins containing 100ul intestinal bacteria Db3.1 competent cell, mix gently after placing 30m on ice, 42 DEG C of heat shock 90s, place 2-3 minute on ice immediately, add LB liquid nutrient medium 900ul in centrifuge tube.Be put in by centrifuge tube on 37 DEG C of shaking tables after vibrations cultivation 1.5hr, collected by centrifugation nutrient solution is coated on the solid LB media containing that penicillin of 50mg/L card, 10mg/L paraxin, 37 DEG C of incubated overnight.Bacterial plaque is selected to deliver to the order-checking of Hangzhou Qing Ke company.Clone designation containing correct insertion sequence is pET32a-LIC (SeqIDNo.5).
Embodiment 2: the connection non-dependent clone of foreign gene
1. the amplification of foreign gene
With primer pair Licl1-NbPin1FP (+), Licl1-NbPin1RP (-) amplification foreign gene NbPin1 sequence, PCR amplification system is: 38 μ L water, 5 μ L10 × PCRBufferforPfu, 4 μ L2.5mMdNTPs, each 0.5 μ L of upstream and downstream primer (10 μMs), 1 μ LNbpin1 template, 1 μ LPfu polysaccharase.Total reaction system is 50 μ L.The reaction conditions of each fragment is: 94 DEG C of 5min; 94 DEG C of 45s, 52 DEG C of 45s, 72 DEG C of 4min, 35 circulations; 72 DEG C of 10min.The Nbpin1 gene fragment containing joint sequence is obtained through order-checking splicing after the PCR primer purifying of amplification.
2. exogenous sequences is cloned into pET32-LIC
After PCR primer adopts PEG deposition and purification in step 1, carry out the clone connecting non-dependent.
1) PCR primer PEG deposition and purification
Equal-volume adds ddH
2o, 30%PEG-8000/30mMMgCl
2, vortex mixes.Centrifugal (14,000rpm, 20min), now PCR primer is deposited in bottom centrifuge tube; Remove liquid, add 1mL75% ethanol rinse DNA and precipitate, centrifugal (14,000rpm, 20min); Remove liquid, dry (37 DEG C, 5min); Add 1/10PCR reaction volume ddH
2o, dissolves PCR primer, electrophoresis detection purification effect.
2) PCR primer end-o f-pipe-control
Reaction system is prepared in ice bath, step 1) middle fragment concentrations 100ng/ μ L, dATP concentration is 5mM; Add 4 μ LDNA polysaccharases, temperature bath (37 DEG C, 20min) processes PCR primer, produces 5 '-end strand; Temperature bath (75 DEG C, 20min), deactivation T4DNA polysaccharase.
3) LIC vehicle treated:
Alkalinity extraction plasmid pET28-LIC; Adopt quick restriction enzyme SacI enzyme to cut, plasmid concentration 5 μ g/100 μ L, and remove RNA; Moisturizing to 400 μ L, chloroform: primary isoamyl alcohol (24:1) extracting 2 times; Alcohol settling, rinsing; Dry (37 DEG C, 5min), dissolve also electrophoresis detection enzyme and cut effect; Reaction system is prepared in ice bath, linearization plasmid concentration 5 μ g/100 μ L, and dTTP concentration is 5mM; Add 4DNA polysaccharase, temperature bath (37 DEG C, 20min), process linearization plasmid, produces 5 '-end strand; Temperature bath (75 DEG C, 20min), deactivation T4DNA polysaccharase.
4) clone of non-dependent is connected:
The step 2 of equal-volume mixing T4DNA polysaccharase process) intermediate segment and step 3) carrier; Temperature bath (70 DEG C, 5min), temperature bath (22 DEG C, 30min) forms end annealing; After completion of the reaction, transformation of E. coli DH5 α.Coat on the solid LB media containing 50mg/L penbritin, 37 DEG C of incubated overnight.Select bacterial plaque to deliver to the order-checking of Hangzhou Qing Ke company, the clone designation after testing containing correct insertion sequence is pET32a-Nbpin1 (SeqIDNo.6).
To sum up, utilizing sequence of the present invention, the carrier for connecting non-dependent clone can being built, thus carry out the connection non-dependent clone of foreign gene easily.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
Claims (3)
1. a pair for connect non-dependent clone joint sequence, it is characterized in that: this joint sequence comprises upstream sequence: the nucleotide sequence shown in gggAgCggAAgCgg, SEQIDNO:7; And downstream sequence: the nucleotide sequence shown in gCCagAgCCACCgc, SEQIDNO:8.
2. joint sequence according to claim 1 is connecting the application in non-dependent clone, it is characterized in that:
The acquisition synthetic joint sequence according to claim 1 of a, sequence;
B, utilize Protocols in Molecular Biology joint sequence to be connected to the multiple clone site position of plasmid vector after, the new carrier of formation, carries out connecting the clone of non-dependent to exogenous sequences.
3. application according to claim 2, it is characterized in that b step specifically: the primer of synthesis amplification Gateway box, wherein upstream and downstream joint sequence lays respectively at 5 ' end of upstream and downstream primer, by pcr amplification Gateway box, the multiple clone site connecting and be cloned into object carrier is cut by enzyme, form new carrier as the clone connecting non-dependent, wherein the upstream and downstream position of the multiple clone site of this carrier is respectively described upstream and downstream joint sequence;
When carrying out connection non-dependent clone, described upstream and downstream joint sequence designs the upstream and downstream primer 5 ' end with goal gene respectively, obtains by pcr amplification the goal gene fragment that flanking sequence is upstream and downstream joint;
By T4DNA polysaccharase process carrier and intermediary's fragment, obtain the outstanding carrier of 5 ' end and intermediate segment, end is wherein given prominence to as joint sequence; By by carrier outstanding for 5 ' end and fragment mixing, form base pair complementarity, transform, form complete plasmid vector, complete the clone to object fragment.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201511027677.5A CN105524919A (en) | 2015-12-31 | 2015-12-31 | Ligation-independent cloning (LIC) joint sequence and application thereof |
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| CN201511027677.5A CN105524919A (en) | 2015-12-31 | 2015-12-31 | Ligation-independent cloning (LIC) joint sequence and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497925A (en) * | 2016-06-22 | 2017-03-15 | 浙江省农业科学院 | A kind of connection non-dependent cloning adapters sequence and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001077344A1 (en) * | 2000-04-11 | 2001-10-18 | Cellzome Gmbh | Vectors |
| CN102604982A (en) * | 2012-03-16 | 2012-07-25 | 杭州师范大学 | Traceless cloning and reorganizing method by means of activity of exonuclease |
| CN104212827A (en) * | 2014-02-18 | 2014-12-17 | 四川大学 | Quick molecular cloning method independent of bioengineering enzyme |
-
2015
- 2015-12-31 CN CN201511027677.5A patent/CN105524919A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001077344A1 (en) * | 2000-04-11 | 2001-10-18 | Cellzome Gmbh | Vectors |
| CN102604982A (en) * | 2012-03-16 | 2012-07-25 | 杭州师范大学 | Traceless cloning and reorganizing method by means of activity of exonuclease |
| CN104212827A (en) * | 2014-02-18 | 2014-12-17 | 四川大学 | Quick molecular cloning method independent of bioengineering enzyme |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497925A (en) * | 2016-06-22 | 2017-03-15 | 浙江省农业科学院 | A kind of connection non-dependent cloning adapters sequence and application |
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Application publication date: 20160427 |