CN104212827B - It is independent of the rapid molecular cloning process of bioengineered enzyme - Google Patents
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Abstract
The invention belongs to biology field, and in particular to a kind of rapid molecular cloning process for being independent of bioengineered enzyme.The rapid molecular cloning process for being independent of bioengineered enzyme is comprised the following steps：A, design of primers and synthesis；Two pairs of primers are separately designed and synthesize, first pair is purpose gene forward primer IF and genes of interest reverse primer IR, and second pair is carrier forward primer VF and carrier reverse primer VR；B, enter performing PCR, linear genes of interest and linear purpose carrier DNA are obtained respectively；C, by twice PCR product mix place, make linear genes of interest and the linear spontaneous connection of purpose carrier DNA circlewise, obtain the carrier containing genes of interest.The present invention is independent of the rapid molecular cloning process of bioengineered enzyme compared with traditional molecule clone technology, with high speed, efficient, low cost, the low advantage of random error.Have broad application prospects.
Description
Technical field
The invention belongs to biology field, and in particular to a kind of rapid molecular cloning process for being independent of bioengineered enzyme.
Background technology
After the completion of mankind's genome sequencing collection of illustrative plates, the whole genome sequence collection of illustrative plates of multiple species is also successively performed.
The primary structural information of gene order, is that mankind's exploration individual gene and GAP-associated protein GAP structure, function provide Information base.By
This, the mankind enter the post-genomic study epoch.However, in face of the gene sequence information of magnanimity, conventional molecular clone technology effect
Rate is low, and cost consumption is too high, and random error is larger, can not meet scientific research demand, the bottleneck limitation of molecule clone technology
Comprehensive deep analysis and research to each species gene.With more several species genome sequencing work completion, with
And the discovery of more new species, post-genomic study is in the urgent need to there is a kind of brand-new high flux molecule clone technology.
On the basis of traditional molecular cloning method, many new gene clone methods are occurred in that, including making
With new technologies such as homologous recombination sequence, specific connector, quick ligases.But the foundation of all these new methods, is required for
Use corresponding bioengineered enzyme, such as all kinds of recombinases, T4 ligases, restriction enzyme.These bioengineered enzymes make
With the complexity of experiment flow being increased, while also increasing experimental cost.Answered to solve Molecular Cloning: A Laboratory flow
It is miscellaneous, costly, the bottleneck problem such as quality control difficulty is big, there is a need in the art for having new breakthrough in the methodology of molecular cloning.
The content of the invention
The technical problem to be solved in the present invention is that this area molecular cloning need to cause experiment to be flowed using various bioengineered enzymes
Journey is complicated, costly problem.
The technical scheme that the present invention solves above-mentioned technical problem is to provide a kind of rapid molecular for being independent of bioengineered enzyme
Cloning process.
The rapid molecular cloning process for being independent of bioengineered enzyme is comprised the following steps：
A, design of primers and synthesis；Separately design and synthesize two pairs of primers, first pair be purpose gene forward primer IF and
Genes of interest reverse primer IR, second pair is carrier forward primer VF and carrier reverse primer VR；
B, enter performing PCR, linear genes of interest and linear purpose carrier DNA are obtained respectively；
C, twice PCR product is mixed and is placed, make linear genes of interest and linear purpose carrier DNA spontaneous connection cyclization
Shape, obtains the carrier containing genes of interest.
Further, the above method also includes step d：With step c products therefrom transform bacterias.
Further, the above method also includes step e：Select monoclonal bacterium colony and extract plasmid, enter performing PCR checking and survey
Sequence identification, obtains the recombinant vector containing genes of interest.
Wherein, in genes of interest forward primer IF described in above method step a and genes of interest reverse primer IR, purpose base
Because forward primer IF contains Overlap I1 primers and I2 primer two parts, sequence and the purpose of Overlap I1 primer portions are carried
The sequence of the 12-21 base at 5 ' ends of body insertion position is identical, and the sequence of I2 primer portions is individual with target gene 5 ' end 10-30
The sequence of base is identical；IR equally contains two parts, respectively Overlap I3 primers and I4 primers, Overlap I3 primers
Sequence and 12-21 base sequence reverse complemental of 3 ' terminal sequence of carrier insertion position, 10-30 alkali of I4 and the end of genes of interest 3 '
The sequence reverse complemental of base, wherein I1 is equal with I3 base quantity, base quantity 12-21.
Preferably, the sequence of the I2 primer portions of genes of interest forward primer IF described in above method step a and purpose base
Because the sequence of preceding 20 bases at 5 ' ends is identical；Last 20 alkali that genes of interest reverse primer IR I4 hold with genes of interest 3 '
The sequence reverse complemental of base.
Wherein, carrier forward primer VF described in above method step a and carrier reverse primer VR are with the 5 ' of carrier to 3 '
Direction is reference, and VF is identical with the 24-30 base sequence that carrier is inserted into the end of position 3 ', and VR is inserted into the end of position 5 ' with carrier
24-30 base sequence reverse complemental.
The method of two pairs of design of primers of the invention can be found in schematic diagram 10.
Wherein, the bacterium in the above method is Escherichia coli.Preferably DMT competence Escherichia coli.
Wherein, the mixing described in above method step C is placed in is carried out at room temperature, and the time is 30~90 minutes.Enter one
Step, 30~60 minutes time that described mixing is placed.
Wherein, the carrier described in the above method is plasmid.
The present invention is independent of the rapid molecular cloning process of bioengineered enzyme compared with traditional molecule clone technology, with height
Speed, efficient, low cost, the low advantage of random error.There is more obvious advantage compared with current existing rapid clon method, that is, exist
In whole experiment flow, without using any restriction enzyme, the engineering enzyme such as ligase, restructuring enzyme system, it is only necessary to be polymerized using DNA
Enzyme.Particularly valuable is the technology connector method for designing unique, is not limited by restriction enzyme site in commercial vector, can be by
Gene is inserted into any position of plasmid, can greatly expand the species of the carrier that molecular cloning is used, and can carry out any transformation to carrier
In addition the process of the inventive method is extremely easy, the work that one those of skill in the art of past are needed into three to four days
Make, shorten in 2 days and complete, the working time shortens 30%~50%.Experimental cost expends to be reduced up to 50%, success efficiency
Reach 90%.Have broad application prospects.
Brief description of the drawings
Fig. 1, reaction sequence number A：EcoI-C domain, twice PCR obtains linear genes of interest and linear purpose carrier.
The monoclonal bacterium colony that carrier conversion Escherichia coli DMT obtained by Fig. 2, picking reaction sequence number A is obtained extracts plasmid,
PCR is identified；Plasimid1 is plasmid extraction result, and PCR of plasimid1 are the plasmid PCR the result.
Fig. 3, reaction sequence number B：Pc109, twice PCR is obtained respectively linear genes of interest and linear purpose carrier
Carrier conversion monoclonal bacterium colony obtained by Fig. 4, picking reaction sequence number B extracts plasmid, PCR identifications；Plasimid1 is
Plasmid extraction result, PCR of plasimid1 are the plasmid PCR the result.
Fig. 5, reaction sequence number C：BCRP1, genes of interest and linear purpose carrier that twice PCR is obtained respectively.
Connector is the Dan Ke that the carrier conversion Escherichia coli DMT obtained by 18bp and 21bp is obtained in Fig. 6, picking reaction sequence number C
Grand bacterium colony extracts plasmid, PCR identifications, testing when BCRP118 and BCRP121PCR the results are respectively connector for 18bp and 21bp
Card result, it can be seen that, up to 75%, positive colony rate is up to 100% when connector is 21bp for positive colony rate when connector is 18bp.
Connector is the monoclonal bacterium that the carrier conversion Escherichia coli DMT obtained by 12bp is obtained in Fig. 7, picking reaction sequence number C
Fall and extract plasmid, PCR identifications, plasimid1 is plasmid extraction result, PCR of plasimid1 are the plasmid PCR the result.
Connector is the monoclonal that the carrier conversion Escherichia coli DMT obtained by 15bp is obtained in Fig. 8, picking reaction sequence number C
Bacterium colony extracts plasmid, PCR identifications, and plasimid1 is plasmid extraction result, and PCR of plasimid1 are the plasmid PCR the result.
The comparative experiments result of Fig. 9, DMT competent cell and DH5 α competent cells, DH5 α competent cells must be with
DpnI is used cooperatively.
Figure 10, design of primers principle schematic.
Figure 11, incubation catenation principle schematic diagram.
Specific embodiment
The inventive method can specifically be realized in the following manner：
A, design of primers and synthesis；Separately design and synthesize two pairs of primers, first pair be purpose gene forward primer IF and
Genes of interest reverse primer IR, second pair is carrier forward primer VF and carrier reverse primer VR；
B, enter performing PCR, linear genes of interest and linear purpose carrier DNA are obtained respectively；
C, twice PCR product is mixed and is placed, make linear genes of interest and linear purpose carrier DNA spontaneous connection cyclization
Shape, obtains the carrier containing genes of interest.
Further, according to the requirement of cloning work, the inventive method can include step d or e.d：With step c gained
Product transform bacteria.e：Select monoclonal bacterium colony and extract plasmid, enter performing PCR checking and sequencing identification, obtain containing genes of interest
Recombinant vector.
The inventive method it is critical that step a.The method and operation principle of two pairs of design of primers of the invention can
Referring to Figure 10 and Figure 11.
Specifically, in genes of interest forward primer IF described in step a and genes of interest reverse primer IR, genes of interest is just
Contain Overlap I1 primers and I2 primer two parts to primer I F, sequence and the purpose carrier of Overlap I1 primer portions are inserted
The sequence for entering the 12-21 base at 5 ' ends of position is identical, sequence and 10-30, the target gene 5 ' end base of I2 primer portions
Sequence it is identical；IR equally contains two parts, respectively Overlap I3 primers and I4 primers, the sequence of Overlap I3 primers
Row and 12-21 base sequence reverse complemental of 3 ' terminal sequence of carrier insertion position, I4 and 10-30, the end of genes of interest 3 ' base
Sequence reverse complemental, wherein I1 are equal with I3 base quantity, base quantity 12-21.
Preferably, the sequence of the I2 primer portions of genes of interest forward primer IF described in above method step a and purpose base
Because the sequence of preceding 20 bases at 5 ' ends is identical；Last 20 alkali that genes of interest reverse primer IR I4 hold with genes of interest 3 '
The sequence reverse complemental of base.
Specifically, carrier forward primer VF described in above method step a and carrier reverse primer VR are with the 5 ' of carrier
It is reference to 3 ' directions, VF is identical with the 24-30 base sequence that carrier is inserted into the end of position 3 ', and VR is inserted into position with carrier
5 ' the 24-30 base sequence reverse complementals in end.
Wherein, above method step b is to carry out twice PCR respectively.It is respectively by genes of interest forward primer IF and purpose
Gene reverse primer IR enters performing PCR amplification and obtains linear genes of interest with the template of genes of interest；Carrier forward primer VF and
Carrier reverse primer VR and the support template of linearisation enter the purpose carrier DNA that performing PCR amplification is linearized.Obviously, linearly
The support template and carrier forward primer VF and carrier reverse primer VR of change match.
Wherein, the bacterium in the above method is Escherichia coli.Preferably DMT competence Escherichia coli.
Wherein, the mixing described in above method step C is placed in is carried out at room temperature, and the time is 30~90 minutes.Enter one
Step, 30~60 minutes time that described mixing is placed.
Wherein, the carrier described in the above method is plasmid.
Enter performing PCR using primer pair of the present invention to react, Overlap I1 and Overlap I3 respectively can be with purposes
Carrier forms spontaneous connection, is assembled into ring-type.Avoiding problems the use of ligase in conventional cloning methods.Overlap
Design of primers is produced in being reacted by PCR, and this also avoids the use of restriction enzyme in conventional cloning methods, while
Avoid the recovery of linear carrier in the gel after endonuclease reaction.So, also will not restriction enzyme site in commercial vector
Limitation, can be inserted into any position of plasmid by gene.
In order to verify that the inventive method is applied to the various length genes of interest different with base composition situation, and it is applicable
Different type carrier, has carried out a series of checking test.
Embodiment one carries out molecular cloning using the inventive method
The primer sequence used in the present embodiment is shown in Table 1, and each genes of interest information is shown in Table 2.The carrier for using has matter respectively
Grain carrier：PET28a, pTEV.Various reagents and carrier are conventional commercial.HS DNA Polymerase and
DpnI restriction enzymes are all from precious bioengineering (Dalian) Co., Ltd, and DMT competent cells are Transgen DMT
Chemically Competent Cell。
The various primer sequences that the embodiment one of table 1. is used
The information of the genes of interest that the embodiment one of table 2. is used
Specifically process is：
1st, according to the sequence of disclosed carrier to be used, and genes of interest fragment to be amplified in table 2, by the present invention
Various primer I F, IR and VF, VR needed for method is designed and synthesis obtains the inventive method, are specifically shown in Table 1.
The design principle of primer is referring to Figure 10
2nd, each experiment carries out twice PCR respectively using corresponding primer pair IF, IR and VF, VR described in table 2, obtains
Linear genes of interest and linear purpose vector DNA fragment.
Each twice PCR reaction system of experiment is respectively by as follows into being grouped into：1) IF and IR (or VF and VR), 2) contain
The DNA profiling of purposeful gene (or purpose carrier), 3) dNTP, 4) high-fidelity DNA polymerase PrimeSTAR, 5)
.PrimeSTAR matching used PCR buffer solutions, 6) ultra-pure waters.
Reaction condition is：98 DEG C of predegeneration 3min；98 DEG C of denaturation 15s, annealing temperature (Tm-5) DEG C, the time continues 30s, prolongs
It is 72 DEG C to stretch temperature, and the duration is set by 1000bp/min；Period is 18.
3. twice PCR product mixing is incubated：
Reaction system：Genes of interest 40ul~50ul+ carriers linear DNA 40ul~50ul, 37 DEG C of temperature, time：1h.
4th, conventional method draws the Escherichia coli DMT of 2-3ul product transformed competence colibacillus.
5. conventional method picking monoclonal bacterium colony extracts plasmid, enters performing PCR checking and sequencing identification, obtains conversion successful
Escherichia coli DMT.
(1) genetic fragment of different length is cloned using the inventive method
Experimentation：
1. performing PCR reaction is entered by the design of table 3 using primer provided in table 1
The reaction condition of the embodiment of table 3 ()
2. each product of PCR is incubated by the mixing distinguished of table 4.
The mixing incubation reaction design of table 4
| Mixing incubation reaction sequence number | Reactant (corresponding PCR reacts the product of sequence number) |
| A | 1+2 |
| B | 1+3 |
| C | 1+4 |
Reaction system：Genes of interest 40ul~50ul+ carriers linear DNA 40ul~50ul, 37 DEG C of temperature, time：1h.
3. conventional method draws the Escherichia coli DMT of 2-3ul product transformed competence colibacillus.
4. conventional method is pressed reaction sequence picking monoclonal bacterium colony and extracts plasmid, enters performing PCR checking and sequencing identification, is obtained
Convert successful Escherichia coli DMT.
5. experimental result：
Reaction sequence number A：Genes of interest is EcoI-C domain, as a result referring to Fig. 1, Fig. 2.Reaction sequence number B：Genes of interest
It is Pc109, as a result referring to Fig. 3, Fig. 4.Reaction sequence number C：Genes of interest is BCRP1, as a result referring to Fig. 5, Fig. 6, Fig. 7, Fig. 8.
What incubation reaction sequence number A, B, C embodied is that different length fragment is attached reaction, and the experiment purpose is to verify
Whether different length genes of interest has influenceed on joint efficiency, can be drawn from experimental result, different length genes of interest pair
The efficiency of rapid clon method presented herein does not simultaneously exist significant difference, there is efficiency higher, and satisfaction is applied to not
With the requirement of the structure of the recombinant vector of the genes of interest of length.
(2)：The comparative experiments of DMT competent cells and DH5 α competent cells
1st, enter performing PCR by the condition that table 5 is designed using primer provided in table 1 to react, PCR results are referring to Fig. 9.
The reaction condition of the embodiment of table 5 (two)
Each PCR primer carries out mixing incubation reaction by the design of table 6.
The mixing incubation reaction design of table 6
| Mixing incubation reaction sequence number | Reactant (corresponding PCR reacts the product of sequence number) |
| D | 5+6 |
| E | 5+6+DpnI+DpnI buffer solutions |
Reaction system：Genes of interest 40ul~50ul+ carriers linear DNA 40ul~50ul, 37 DEG C of temperature, time：1h.
2. Escherichia coli of the 2~3ul of incubation reaction product by reaction sequence number D transformed competence colibacillus are drawn with conventional method respectively
DMT, by reaction sequence number E transformed competence colibacillus cell DH5 α.
3. conventional method extracts plasmid by reaction sequence number D, E picking monoclonal bacterium colony, enters performing PCR checking and sequencing identification,
The successful Escherichia coli DMT and DH5 α of conversion is obtained, as a result referring to Fig. 9.
4. experimental result：The template plasmid that band methylates, reaction system group are directly removed using DMT competence Escherichia coli
Into being twice PCR product mixture (genes of interest 40ul~50ul+ carriers linear DNA 40ul~50ul), reaction condition is 37
DEG C incubate 1h；DMT competent cells do not influence cloning efficiency.In addition to this experiment, above and below clone uses DMT competence
Cell is carried out.
(3) cloned with the primer connector of different length
1. enter performing PCR by the design of table 7 using primer provided in table 1 to react.
The reaction condition of the embodiment of table 7 (three)
2. each PCR primer carries out mixing incubation reaction respectively by the design of table 8.
The mixing incubation reaction design of table 8
| Mixing incubation reaction sequence number | Reactant (corresponding PCR reacts the product of sequence number) |
| F | 7+8 |
| G | 7+9 |
| H | 7+10 |
| I | 7+11 |
Reaction system：Genes of interest 40ul~50ul+ carriers linear DNA 40ul~50ul, 37 DEG C of temperature, time：1h.
3. conventional method draws the Escherichia coli DMT of reaction sequence each 2~3ul product transformed competence colibacillus of F, G, H, I.
4. conventional method is pressed reaction sequence picking monoclonal bacterium colony and extracts plasmid, enters performing PCR checking and sequencing identification, is obtained
Convert successful Escherichia coli DMT.
5. experimental result
Experiment conclusion：When Overlap is 12bp, the company of BCRP1 and pET28a is carried out using the quick connection cloning process
Connect, successful connection, as a result referring to Fig. 7.
Experiment conclusion：When Overlap is 15bp, the connection of BCRP1 and pET28a is carried out using the inventive method, connected into
Work(, as a result referring to Fig. 6.
When Overlap is 18bp or 21bp, the connection of BCRP1 and pET28a is carried out using the inventive method, connected into
Work(, as a result referring to Fig. 8.
To sum up, Overlap successes of achievable coupled reaction between 12-21bp are carried out.
Generally speaking, the present embodiment demonstrates wherein the inventive method and can be successively inserted into the entrance of different size DNA fragmentation
Different carriers；The primer connector for demonstrating the different length being related in the scope of the invention can also be cloned successfully；Also demonstrate use
DMT competent cells replace actual effects of the demethylase DpnI in rapid molecular clone technology to be gratifying.
In this example, after being circulated at 18, DNA polymerase fidelities guarantee still all right, normal PCR reaction generally requires 30 and follows
Ring, causes the amplification of nonspecific products, it is therefore desirable to the purifying of PCR primer, removes nonspecific products.These are in we
Need not in method.
Claims (9)
1. the rapid molecular cloning process of bioengineered enzyme is independent of, it is characterised in that comprised the following steps：
A, design of primers and synthesis；Two pairs of primers are separately designed and synthesize, first pair is purpose gene forward primer IF and purpose
Gene reverse primer IR, second pair is carrier forward primer VF and carrier reverse primer VR；
In genes of interest forward primer IF and genes of interest reverse primer IR, genes of interest forward primer IF contains Overlap I1
Primer and I2 primer two parts, the sequence of Overlap I1 primer portions are individual with the 12-21 at 5 ' ends of purpose carrier insertion position
The sequence of base is identical, and the sequence of I2 primer portions is identical with the sequence of 10-30, target gene 5 ' end base；IR equally contains
Two parts, respectively Overlap I3 primers and I4 primers, sequence and the carrier insertion position 3 ' of Overlap I3 primers are held
12-21 base sequence reverse complemental of sequence, I4 holds the 10-30 sequence reverse complemental of base with genes of interest 3 '；
Carrier forward primer VF and carrier reverse primer VR are that, as reference, VF is inserted into position with carrier with the 5 ' of carrier to 3 ' directions
Put 3 ' the 24-30 base sequences in end identical, VR is inserted into the 24-30 base sequence reverse complemental in the end of position 5 ' with carrier；B, enter
Performing PCR, obtains linear genes of interest and linear purpose carrier DNA respectively；
C, by twice PCR product mix place, make linear genes of interest and the linear spontaneous connection of purpose carrier DNA circlewise, obtain
To the carrier containing genes of interest.
2. method according to claim 1, it is characterised in that also including step d：With step c products therefrom transform bacterias.
3. method according to claim 2, it is characterised in that also including step e：Select monoclonal bacterium colony and extract plasmid, enter
Performing PCR checking and sequencing identification, obtain the transform bacteria of the carrier containing genes of interest.
4. method according to claim 1, it is characterised in that：The I2 primers of genes of interest forward primer IF described in step a
Partial sequence is identical with the sequence of 20 bases in target gene 5 ' end；Genes of interest reverse primer IR I4 and genes of interest 3 '
The 20 sequence reverse complementals of base in end.
5. method according to claim 2, it is characterised in that the bacterium is Escherichia coli.
6. method according to claim 5, it is characterised in that the Escherichia coli are DMT competence Escherichia coli.
7. method according to claim 1, it is characterised in that the mixing of described step C is placed in and carries out at room temperature,
Time is 30~90 minutes.
8. method according to claim 7, it is characterised in that 30~60 minutes time that described mixing is placed.
9. the method according to any one of claim 1~8, it is characterised in that described purpose carrier is plasmid.
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| CN201410475782.4A CN104212827B (en) | 2014-02-18 | 2014-09-17 | It is independent of the rapid molecular cloning process of bioengineered enzyme |
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| CN201410475782.4A CN104212827B (en) | 2014-02-18 | 2014-09-17 | It is independent of the rapid molecular cloning process of bioengineered enzyme |
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| CN105524919A (en) * | 2015-12-31 | 2016-04-27 | 浙江省农业科学院 | Ligation-independent cloning (LIC) joint sequence and application thereof |
| CN105969784B (en) * | 2016-05-24 | 2019-12-13 | 北京擎科生物科技有限公司 | recombinase-independent DNA seamless cloning method |
| CN108588101B (en) * | 2018-04-27 | 2022-03-04 | 苏丹 | Molecular cloning method for constructing different expression vectors of same gene |
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| US6815185B2 (en) * | 1997-11-17 | 2004-11-09 | Robert D. Klein | Methods of creating constructs useful for introducing sequences into embryonic stem cells |
| 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 |
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