CN108486139A - A kind of site-directed point mutation method and its application based on seamless clone technology - Google Patents
A kind of site-directed point mutation method and its application based on seamless clone technology Download PDFInfo
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Abstract
The present invention relates to a kind of site-directed point mutation method, kit and its applications based on seamless clone technology.Steps are as follows for the method：The forward and reverse mutant primer that 5 ' ends include complementary homology arm sequence is separately designed according to the sequence at mutational site, mutational site can be located at any position in homology arm；The forward and reverse primer that 5 ' ends include another complementary homology arm is separately designed in the non-mutated sites area of plasmid, the segment that two one end that PCR amplification goes out include mutational site need to have the difference in size of 500bp or more；For multisite mutation, the mutant primer of logarithm identical as number of sites is designed according to mutational site number；It is using homologous recombination enzyme that 25 one steps of segment are seamless spliced, build the cyclic plasmid with targeted mutagenesis site.The characteristics of relatively classical Overlap extension PCR method of the method, has fast and convenient, can realize single-point or multiple spot to being less than 40kb plasmids any position, continuously or discontinuously be mutated, and mutation efficiency is up to 100%.
Description
Technical field
The present invention relates to a kind of site-directed point mutation method based on seamless clone technology, the gene prepared based on this method
Site-directed mutagenesis kit and its specific embodiment of application, belong to gene engineering technology field.
Background technology
The site-directed point mutation of based on PCR technology is that gene regulation, albumen are studied in molecular biology and protein engineering
Matter structure and the essential tool of function.Site-directed point mutation mode includes mainly that base is replaced, deletes, is inserted into, multidigit point
Mutation, the random mutation in one or more sites.The site-directed point mutation method of many based on PCR technologies is commercially used,
In the method that most generally uses include following several：
(1) Overlap extension PCR method (overlap extension method).The positive anti-primer used in this method contains
Complementary overlay region.Using overlap extension pcr amplification obtain product be it is a kind of containing target mutation site, have opening,
Loose cyclic plasmid (Sun SH, Huang H, Billy Qi YC, Qiu MS and Dai ZM.Complementary
annealing mediated by exonuclease:a method for seamless cloning and
conditioning site-directed mutagenesis.Biotechnology&Biotechnological
Equipment.2015,29(1):105-110)；
(2) large primer PCR method (megaprimer method).This method generally requires three primers, is drawn first by mutation
Object and corresponding Outside primer carry out first round PCR and big primer are obtained by the reaction, and after purified and another Outside primer carries out second
It takes turns PCR and obtains target fragment (Ke SH and Madison.Rapid and efficient site-directed
mutagenesis by single-tube‘megaprimer’PCR method.Nucleic Acids Research.1997,
25(16):3371-3372)；
(3) quick-replaceable method (Quick Change Method (Agilent Technologies, La Jolla, CA,
USA)).Quick-replaceable method is polymerize in conjunction with PfuTurbo DNA using a pair of primer containing partially or completely complementary overhangs area
Enzymatic amplification cyclic plasmid.The single strand dna that PCR exponential amplifications generate can be generated in annealing by staggered breaks single stranded DNA
Cyclic plasmid (Xia YZ, Chu WQ, Qi QS and Xun LY.New insights into the QuikChange
process guide the use of Phusion DNA polymerase for site-directed
mutagenesis.Nucleic Acids Research.2015,43(2):2-9)。
But there is also following shortcomings for these methods：
(1) when pcr amplification product concentration is low, the structure efficiency of mutant plasmid is very low；
(2) high-fidelity DNA polymerase has faint strand replacement reaction activity, it may cause strand replacement reaction, and
By the product exponential amplification amplificationization；
(3) common tool plasmid is all bigger in Cell Biology Experiment, such as the gene overexpression that tetracycline induces
Carrier pLVX-Tight-Puro sizes are 7791bp, and luciferase assay carrier pmirGLO sizes are 7350bp.It uses
Pair of primers is difficult to amplify the above-mentioned recombinant vector for including target gene sometimes, needs purchasing price costliness, has long segment
The high fidelity enzyme of amplification ability；
(4) due to the use of complementary primer pair, the above method can all be limited by being inserted into or deleting clip size；
(5) large primer PCR carries out rite-directed mutagenesis, and big primer and Outside primer annealing temperature (Tm) must differ notable, and
Mutation efficiency cannot reach 100%.
Most of all, above-mentioned three classes method is primarily adapted for use in unit point gene mutation, and do not have to multisite mutation
Effect.To solve this problem, field waits quietly reporting a kind of method of gene site-directed multi-site mutation, it is to utilize Primers complementary area
To there is the ring plasmid template of opening to complete PCR, to introduce the rite-directed mutagenesis in more than one site.But it should
Method is cumbersome, and a wheel PCR reactions are carried out it is necessary to rejoin a pair of of mutant primer because often introducing a mutational site
(Chinese patent CN101580829B).In addition, a kind of based on PCR of more document reports is limited with Type II s both at home and abroad in recent years
The site-directed point mutation technology of property restriction endonuclease processed can meet multidigit point gene mutation demand.But this method is cumbersome, needs
More steps (Zhang ZQ, Xu K, the Xin Y and Zhang ZY.An efficient such as PCR, digestion, connection are carried out successively
method for multiple site-directed mutagenesis using type IIs restriction
enzymes.Analytical Biochemistry.2015,476:26-28)。
Homologous recombination (Homologous recombination, HR) is the mistake that pair of homologous DNA fragmentation exchanges nucleotide
Journey, it is that DNA plerosis is broken most important mode.Homologous recombination is used for the transformation of inhereditary material in saccharomycete at first.Then, it sends out
Existing one kind phage enzyme can complete homologous recombination reaction independently of bacterium system, and be used for plasmid, bacterial artificial chromosome
With the transformation of bacterial genomes.The seamless clone technology based on external homologous recombination is more and more paid attention in recent years, this method
Do not limited by the restriction enzyme site of carrier and target fragment, be one kind by recombinase, in vitro directly by target fragment and carrier
Seamless spliced gene clone technology.The technology is relatively traditional to carry out gene cloning based on restriction enzyme and ligase
Technology has the characteristics that fast and convenient, one step directed cloning of PCR product, flexible cloning site and efficient.
In conclusion the seamless clone technology of integrated use and overlap extension pcr, establish one kind independent of restricted
Restriction endonuclease and ligase fast and efficiently can realize the technology of one or more site mutations in the arbitrary site of carrier, will
It is a kind of following site-directed point mutation technology most generally used.
Invention content
The purpose of the present invention：For deficiencies of the prior art, it is based on newest seamless clone technology, provides one
Kind of fast and convenient, high efficiency, can realize unit point in carrier any position or multidigit point, the gene that is continuously or discontinuously mutated
Directed mutagenesis method
One of invention content：A kind of site-directed point mutation method based on seamless clone technology
The method of the invention combines high-fidelity by way of introducing mutational site in holding homology arm in primer 5 '
Archaeal dna polymerase expands, and prepares the plasmid fragments for including mutational site.Then, by seamless clone technology, 2-5 both ends are contained
There are the plasmid fragments of homology arm to carry out external homologous recombination reaction, to realize single or multiple sites, continuously or discontinuously position
Point mutation.
1. design of primers
(1) primer includes 5 ' end homology arms and 3 ' end extension areas, and wherein homology arm size is 15-20bp, extension area size
For 0-20bp, mutational site is included in a homology arm；
(2) T of forward and reverse amplimermIt is worth preferably within the scope of 55-65 DEG C, and the two TmDifference had better not
More than 1 DEG C range.
2. design of primers demonstration citing
Mutant forward primer 1 and mutation reverse primer 2 are pair of primers, and mutant forward primer 3 and mutation reverse primer 4 are
Two segments of pair of primers, PCR amplification are spliced in vitro by seamless clone technology, to which structure is prominent comprising single locus
The recombinant plasmid of change.
The two of invention content：A kind of application of the site-directed point mutation method based on seamless clone technology
The present invention using it is above-mentioned based on the site-directed point mutation method of seamless clone technology in people source MEK1 albumen and one kind
In mosaic type disulfurase EH-IscS, 1 and 2 mutational sites are introduced respectively, and sequencing result confirms that mutation efficiency reaches
To 100%, illustrate that the method for the invention is reliable and stable, efficient, a kind of strong genetic analysis is provided for laboratory
With the conventional method of protein function research.
The three of invention content：A kind of site-directed point mutation kit based on seamless clone technology
Patent of the present invention provides a kind of site-directed point mutation kit based on seamless clone technology, except kit package,
Other than liner, Reagent Tube and operation instructions, which includes：(1) high-fidelity DNA polymerase premixed liquid；(2)
Dpn I and 10 × Dpn I buffer；(3) seamless clone enzyme and 5 × seamless clone's buffer solution；(4) DMT competent cells；(5)
The general identification primer of control plasmid and control plasmid；(6) sterile ddH2O。
The wherein described high fidelity enzyme premixed liquid is purchased from following company：Nanjing Vazyme Biotechnology Co., Ltd., 2 ×
Phanta Master Mix, article No. P511-01/02/03,2 × Phanta Max Master Mix, article No. P515-01/02/
03.The Dpn I and 10 × Dpn I buffer can be purchased from following company respectively：Precious bioengineering (Dalian) Co., Ltd,
QuickCutTMDpn I, article No. 1609；Green skies biotechnology research institute, Dpn I, article No. D6257.The DMT competence is thin
The general identification primer of born of the same parents, control plasmid and control plasmid are provided by Wenzhou Medical University's enzyme engineering and medical diagnosis research institute.
The seamless clone enzyme and seamless clone's buffer solution can be purchased from following company respectively：With the limited public affairs of first biotechnology (Shanghai) share
Department, ClonFast is seamless Cloning Kit；Nanjing Vazyme Biotechnology Co., Ltd., homologous recombination one-step cloning kit,
Article No. C112-01/02；Nanjing Genscript Biotechnology Co., Ltd., CloneEZ PCR Cloning Kits, article No. L00339；
Clontech, Seamless Cloning with In-Fusion Cloning Kits, article No. 638911/638918；
Invitrogen,Seamless Cloning and Assembly Enzyme Mix, article No. A14606.It is described
Seamless clone enzyme and seamless clone's buffer solution can also be by reference to document (Zhang Y, Werling U, Edelmann
W.Seamless Ligation Cloning Extract(SLiCE)cloning method.Methods Mol
Biol.2014,1116:235-244) prepare production.The aqua sterilisa is this field conventional reagent.In kit of the present invention
DMT competent cells are placed in -70 DEG C of preservations, other at -20 DEG C of preservations that are placed in.The preparation method of kit of the present invention is such as
It is conventional.
A kind of site-directed point mutation method and its application based on seamless clone technology of the present invention, have following innovation
Point and technical advantage：
1. for the structure of single-site mutant carrier, using two pairs of primer amplification purpose carriers, wherein a forward primer
With base containing mutational site in the complementary homology arm of reverse primer, it is suitable for carrying out rite-directed mutagenesis to 5kb or more carriers, overcomes
Pair of primers expands 5kb or more carriers, and there are success rates and the uncertain defect of fidelity；
2. for the structure of multisite mutation carrier, on the basis of realizing that single-site mutant needs two pairs of primers, often increase
Add a mutational site, is increased by a pair of of mutant primer.The multiple segments amplified, are reacted by external homologous recombination, can be real
The seamless spliced completion multisite mutation of an existing step, without carrying out more wheel PCR amplifications.
Technique effect：
1. the site-directed point mutation method based on seamless clone technology, unit point or more can be realized in carrier any position
Site is continuously or discontinuously mutated；
2.Dpn I digestion external degradation methylates non-mutant plasmid template, in DMT competent cells body degradation methylate
Non-mutant plasmid template, so that it is guaranteed that mutation efficiency reaches 100%.
Description of the drawings
Site-directed point mutation (one or more sites) schematic diagrames of Fig. 1 based on seamless clone technology；
The agarose gel electrophoretogram of Fig. 2 .PCR amplification purpose plasmid MEK1-pCold I products；Quasi- mutational site alkali
Base is located in the homology arm of primer；M:DL 5000Marker；Lane-1:Segment 1 (2898bp)；Lane-2:Segment 2
(2700bp)；
Fig. 3 use general identification primer amplification MEK1 (the Q56P)-pCold I/DH5 α positive colony bacteriums of pCold I plasmids
The agarose gel electrophoretogram of strain PCR product；M:DL 5000Markrer；Lane-1-12:PCR amplification MEK1 (Q56P)-
PCold I/DH5 α positive colony bacterial strain target gene products (1601bp)；
The agarose gel electrophoretogram of Fig. 4 .PCR amplification purpose plasmid EH-IscS-pCold-SUMOa products；Quasi- mutation
Site base is located in the homology arm of primer；M:DL 5000Marker；Lane-1:Build EH-IscS (K206A)-pCold-
The segment 1 (3235bp) of SUMOa plasmids；Lane-2:Build the segment 2 of EH-IscS (K206A)-pCold-SUMOa plasmids
(2416bp)；Lane-3:Build the segment 1 (2791bp) of EH-IscS (C329S)-pCold-SUMOa plasmids；Lane-4:Structure
The segment 2 (2858bp) of EH-IscS (C329S)-pCold-SUMOa plasmids；
Fig. 5 use general identification primer amplification EH-IscS (the K206A)-pCold-SUMOa/DH5 of pCold-SUMOa plasmids
The agarose gel electrophoretogram of α and EH-IscS (C328S)-pCold-SUMOa/DH5 α positive colony bacterial strain PCR products.M:
DL 5000Marker；Lane-1-4:PCR amplification EH-IscS (K206A)-pCold-SUMOa/DH5 α positive colony bacterial strain purposes
Gene outcome (1892bp)；Lane-5-9:PCR amplification EH-IscS (C329S)-pCold-SUMOa/DH5 α positive colony bacterial strains
Target gene product (1892bp).
Fig. 6 .MEK1 (Q56P)-pCold I plasmid rite-directed mutagenesis sequencing results；
Fig. 7 .EH-IscS (K206A)-pCold-SUMOa plasmid rite-directed mutagenesis sequencing results；
Fig. 8 .EH-IscS (C329S)-pCold-SUMOa plasmid rite-directed mutagenesis sequencing results；
Fig. 9 build the principle schematic of site-directed point mutation recombinant plasmid based on seamless clone technology.
Specific implementation mode
Below by embodiment, the invention will be further described.
A kind of application examples one of the site-directed point mutation method based on seamless clone technology of embodiment 1.
1. the 56th glutamine residue of structure MEK1 albumen sports MEK1 (the Q56P)-pCold I weights of proline
Group plasmid
MEK1 is a dual specificity protein kinases in STE7 kinase families.Through Raf, the modification of Mos and Cot tyrosine phosphorylations
There is post activation the MEK1 of kinase activity can make threonine-glutamic acid-junket ammonia in the activation ring of ERK1 and ERK2 protein structure domains
Phosphorylation occurs for the threonine and tyrosine residue of acidic group sequence.MEK1 be in map kinase signal transduction pathway necessary to one at
Point, the signal transduction pathway is related with the bioprocess of various kinds of cell, for example, cell Proliferation, cell differentiation, transcriptional control and hair
It educates.According to the literature, the 56th glutamine residue of MEK1 albumen is sported into proline, constitutes property and activates MEK1 eggs
In vain, so that it is not needed the activation of the phosphorylation modification of upstream kinases just has kinase activity.
MEK1-pCold I plasmids are provided by Wenzhou Medical University's enzyme engineering and medical diagnosis research institute.
1.1 amplification purpose plasmid fragments
1.1.1 PCR reaction systems and condition are as follows：
(1) the F1R1 segments of amplification structure MEK1 (Q56P)-pCold I rite-directed mutagenesis plasmids
Remarks：Primer 1 and a concentration of 10 μM of primer 2.PCR reaction conditions：
(2) the F2R2 segments of amplification structure MEK1 (Q56P)-pCold I rite-directed mutagenesis plasmids
Remarks：4 a concentration of 10 μM of primer 3 and primer.
PCR reaction conditions：
1.2 Dpn I are digested the plasmid template that methylates
Digestion system and condition are as follows：
37 DEG C be incubated 1 hour or longer time.
1.3 glue recycle
PCR product through Dpn I digestions carries out glue recycling with 1% low melting-point agarose gel electrophoresis, and Du- is used in combination
800 nucleic acid/protein analyzer measures recovery product concentration.
1.4 seamless clones build mutant vector
1.4.1 seamless cloning reaction system and condition are as follows：
37 DEG C of incubation 30min, place 5min on ice.
1.4.2 the computational methods of PCR fragment usage amount
When carrying out seamless clone, the mole ratio of long segment and short-movie section is generally 1：2.The calculating of PCR fragment usage amount
Method is as follows：
The bp numbers of usage amount (ng)=number × 660 nmol × PCR fragment of PCR fragment
1.5 conversion
(1) the seamless cloning reaction liquid of 10 μ l is added in 100 μ l DH5 α competent cells, finger flicks mixing, on ice
It is incubated 30min；
(2) 42 DEG C of water-bath heat shocks 90 seconds, place 2-3min on ice；
(3) 900 μ l are added in the LB liquid medium of 37 DEG C of preheatings, 37 DEG C, 200rpm incubations 45min；
(4) 4000rpm centrifuges 5min, discards 900 μ l supernatants, thalline is resuspended with remaining culture medium, and be spread evenly across phase
It answers on the LB tablets of resistance, 37 DEG C of inversion overnight incubations.
The identification of 1.6 positive colonies
A fresh LB tablet containing corresponding antibiotic is taken, 16 small lattice are divided into marking pen.With transfer needle picking
Single bacterium colony distinguishes streak inoculation in each small lattice a little on above-mentioned conversion tablet, inversion culture 10h in 37 DEG C of constant incubators.
Distinguish that the thalline grown in each small lattice on picking tablet is a little with pipette tips, is mixed in the EP pipes containing 50 μ l sterile waters, 100 DEG C are boiled
After 10min, 12 000rpm, centrifugation 5min, takes 9.2 μ l of supernatant to be added in 200 μ l PCR reaction tubes and identify template as PCR.And
Following system is taken to be added in the pipe, mixing.
PCR reaction conditions：
5 μ l PCR products are taken to be identified with 1.0% low melting-point agarose gel electrophoresis.
1.7 sequencing
It selects two bacterium colony PCR and identifies that correct positive colony bacterial strain carries out sequencing identification.It is real
1.8 test result：
As shown in Fig. 2, 1 size of segment of PCR amplification structure MEK1 (Q56P)-pCold I plasmids is 2898bp, segment 2
Size is 2700bp, consistent with expected results；Using general identification primer amplification MEK1 (the Q56P)-pCold of pCold I plasmids
I/DH5 α positive colony bacterial strain PCR product sizes are 1601bp, and wherein MEK1 gene sizes are 1182bp, are drawn using general identification
The PCR product size that object expands pCold I empty carriers is 419bp.From figure 3, it can be seen that bacterium colony PCR identification primer size with it is pre-
Phase result is consistent, tentatively judges the success of MEK1 (Q56P)-pCold I plasmid constructions；Further sequence verification shows the sun selected
Property clone strain in include plasmid be successfully occur site mutation MEK1 (Q56P)-pCold I plasmids, mutational site sequencing
The results are shown in Figure 6.
A kind of application examples two of the site-directed point mutation method based on seamless clone technology of embodiment 2.
EH-IscS is a kind of mosaic type disulfurase, it is by merging Escherichia coli disulfurase
The C-terminal structural domain of the N-terminal structural domain (1-263 amino acids) and people source disulfurase NFS1 albumen of IscS albumen
(316-457 amino acids) are built-up.EH-IscS does not need auxilin and just has with respect to NFS1, protein stability enhancing
There is disulfurase active.
EH-IscS-pCold-SUMOa plasmids are provided by Wenzhou Medical University's enzyme engineering and medical diagnosis research institute.
1. the 206th lysine mutation for building EH-IscS albumen is EH-IscS (K206A)-pCold- of alanine
SUMOa recombinant plasmids
1.1 amplification purpose plasmid fragments
1.1.1 PCR reaction systems and condition are as follows：
(1) the F1R1 segments of amplification structure EH-IscS (K206A)-pCold-SUMOa rite-directed mutagenesis plasmids
Remarks：Primer 1 and a concentration of 10 μM of primer 2.
PCR reaction conditions：
(2) the F2R2 segments of amplification structure EH-IscS (K206A)-pCold-SUMOa rite-directed mutagenesis plasmids
Remarks：4 a concentration of 10 μM of primer 3 and primer.
PCR reaction conditions：
The subsequent experimental method of 1.2 structure EH-IscS (K206A)-pCold-SUMOa plasmids is implemented with step with the present invention
In example 3 described in 1.2 to 1.7 experimental procedures.
2. the 329th cysteine mutation for building EH-IscS albumen is EH-IscS (C329S)-pCold- of serine
SUMOa recombinant plasmids
2.1 amplification purpose plasmid fragments
2.1.1 PCR reaction systems and condition are as follows：
(1) the F1R1 segments of amplification structure EH-IscS (C329S)-pCold-SUMOa rite-directed mutagenesis plasmids
Remarks：Primer 1 and a concentration of 10 μM of primer 2.
PCR reaction conditions：
(2) the F2R2 segments of amplification structure EH-IscS (C329S)-pCold-SUMOa rite-directed mutagenesis plasmids
Remarks：4 a concentration of 10 μM of primer 3 and primer.
PCR reaction conditions：
The subsequent experimental method of 2.2 structure EH-IscS (C329S)-pCold-SUMOa plasmids is implemented with step with the present invention
In example 3 described in 1.2 to 1.7 experimental procedures.
3. experimental result：
As shown in figure 4,1 size of segment of PCR amplification structure EH-IscS (K206A)-pCold-SUMOa plasmids is
3235bp, 2 size of segment are 2416bp.PCR amplification builds 1 size of segment of EH-IscS (C329S)-pCold-SUMOa plasmids
For 2858bp, 2 size of segment is 2791bp, consistent with expected results；Using the general identification primer amplification EH- of pCold I plasmids
IscS (K206A)-pCold-SUMOa/DH5 α and EH-IscS (C329S)-pCold-SUMOa/DH5 α positive colony bacterial strains PCR
Primer size is all 1892bp, and wherein EH-IscS gene sizes are 1197bp, using general identification primer amplification pCold-
The PCR product size of SUMOa empty carriers is 695bp.From fig. 5, it can be seen that bacterium colony PCR identification primer sizes and expected results one
Cause, tentatively judge EH-IscS (K206A)-pCold-SUMOa and EH-IscS (C329S)-pCold-SUMOa plasmid constructions at
Work(；Further sequence verification shows that the plasmid for including in the positive colony bacterial strain selected is the EH- that site mutation successfully occurs
IscS (K206A)-pCold-SUMOa and EH-IscS (C329S)-pCold-SUMOa plasmids, mutational site sequencing result difference
As shown in Figure 7 and Figure 8.
A kind of site-directed point mutation kit based on seamless clone technology of embodiment 3.
1. a kind of site-directed point mutation kit specification based on seamless clone technology
1.1 kit brief introductions
This site-directed point mutation kit uses two pairs of primer amplification target gene, two of which primer alkali containing mutational site
Base is suitable for carrying out rite-directed mutagenesis to 5kb or more plasmids.Overcome using common high fidelity enzyme when, pair of primers expand 5kb with
There are success rates and the uncertain defect of fidelity for upper plasmid；Amplified production is using the cyclic annular position containing mutation of seamless clone technology structure
Point plasmid, positive clone rate are more than 90%；Dpn I digestion, external degradation methylate non-mutant plasmid template, DMT competence
Degradation methylates non-mutant plasmid template in cell body, so that it is guaranteed that mutant plasmids positive clone rate is up to 100%.
1.2 application range
(original plasmid template need to use the host strain of no methylase deficient to expand to DNA rite-directed mutagenesis, such as DH5 α, TOP10
With XL10 bacterial strains)
1.3 rite-directed mutagenesis principles
As shown in Figure 9.
1.4 Kit components
* control plasmid is used to build rite-directed mutagenesis plasmid, mutant plasmids positive clone rate ＞ 90%
1.5 are illustrated using step
1.5.1 expanding purpose plasmid fragments
1.5.1.1 recommend PCR reaction systems and condition as follows：
1.5.1.2 PCR reaction conditions：
1.5.2 electrophoresis detection
Take 2 μ l PCR products, 1% low melting-point agarose detected through gel electrophoresis.
1.5.3 Dpn I are digested the plasmid template that methylates
Digestion system and condition are as follows：
37 DEG C be incubated 1 hour or longer time.
1.5.4 glue recycles
PCR product through Dpn I digestions carries out glue recycling with 1% low melting-point agarose gel electrophoresis, and Du- is used in combination
800 nucleic acid/protein analyzer measures recovery product concentration.
1.5.5 seamless clone builds mutant vector
1.5.5.1 seamless cloning reaction system and condition are as follows：
37 DEG C of incubation 30min, place 5min on ice.
Points for attention：Seamless cloning reaction liquid can be placed in -20 DEG C of long-term preservations if not being immediately available for converting；If you need to simultaneously
Multiple carriers are built, eight connecting legs or 96 orifice plates can be used to carry out high-throughput operation.
1.5.5.2 the computational methods of PCR fragment usage amount
When carrying out seamless clone, the mole ratio of long segment and short-movie section is generally 1：2.The calculating of PCR fragment usage amount
Method is as follows：
The bp numbers of usage amount (ng)=number × 660 nmol × PCR fragment of PCR fragment
1.5.6 conversion
(1) the seamless cloning reaction liquid of 10 μ l is added in 100 μ l DMT competent cells, finger flicks mixing, on ice
It is incubated 30min；
(2) 42 DEG C of water-bath heat shocks 90 seconds, place 2-3min on ice；
(3) 900 μ l are added in the LB liquid medium of 37 DEG C of preheatings, 37 DEG C, 200rpm incubations 45min；
(4) 4000rpm centrifuges 5min, discards 900 μ l supernatants, thalline is resuspended with remaining culture medium, and be spread evenly across phase
It answers on the LB tablets of resistance, 37 DEG C of inversion overnight incubations.
1.5.7 the identification of positive colony
A fresh LB tablet containing corresponding antibiotic is taken, 16 small lattice are divided into marking pen.With transfer needle picking
Single bacterium colony distinguishes streak inoculation in each small lattice a little on above-mentioned conversion tablet, inversion culture 10h in 37 DEG C of constant incubators.
Distinguish that the thalline grown in each small lattice on picking tablet is a little with pipette tips, is mixed in the EP pipes containing 50 μ l sterile waters, 100 DEG C are boiled
After 10min, 12 000rpm, centrifugation 5min, takes 9.2 μ l of supernatant to be added in 200 μ l PCR reaction tubes and identify template as PCR.And
Following system is taken to be added in the pipe, mixing.
1.5.7.1 PCR reaction conditions：
5 μ l PCR products are taken to be identified with 1.0% low melting-point agarose gel electrophoresis.
1.5.8 sequencing
It selects two bacterium colony PCR and identifies that correct positive colony bacterial strain carries out sequencing identification.
1.5.9 kit preservation condition
DMT competent cells are placed in -70 DEG C of preservation half a year, other to be preserved 1 year at being placed in -20 DEG C.
To those skilled in the art, the present invention can be improved or converted according to the embodiment above, and institute
There are these to improve or convert the protection domain that should all belong to the claims in the present invention.
Claims (6)
1. a kind of site-directed point mutation method based on seamless clone technology, it is characterised in that：By designing two pairs of primer amplifications
Build single-site mutant plasmid, one pair of which primer be according to mutational site at sequence separately design 5 ' ends comprising complementary homologous
The forward mutation assay primer and inverse transition primer of arm sequence, mutational site can be located at any position in homology arm；Another pair primer
It is to separately design the forward primer and reverse primer that 5 ' ends include another complementary homology arm, primer in the non-mutated sites area of plasmid
Middle homology arm size is 15-20bp；Then it includes mutation to use seamless clone technology, two one end that high fidelity enzyme is amplified
The segment in site carries out seamless spliced, cyclic plasmid of the structure with targeted mutagenesis site.
2. the site-directed point mutation method according to claim 1 based on seamless clone technology, it is characterised in that：PCR expands
Increase the segment that two one end include mutational site, there are when the difference in size of 500bp or more, it can be ensured that the height of seamless clone
Effect property.
3. the site-directed point mutation method according to claim 1 based on seamless clone technology, it is characterised in that：For more
Site mutation, the 5 ' ends that logarithm identical as number of sites is designed according to mutational site number include the mutation of complementary homology arm sequence
Primer, can be seamless spliced by 2-5 one step of segment using homologous recombination enzyme, builds the cyclic plasmid with multiple mutational sites.
4. the site-directed point mutation method according to claim 1 based on seamless clone technology, it is characterised in that：PCR expands
After increasing plasmid template, the non-mutant plasmid template that is methylated using Dpn I enzyme external degradations；Seamless clone technology structure will be utilized
The cyclic plasmid in the site containing targeted mutagenesis built is transformed into DMT competent cells, and degrading in DMT competent cells body methylates
Non-mutant plasmid template, to Effective selection mutant clon.
5. a kind of site-directed point mutation kit based on seamless clone technology, it is characterised in that：The kit includes packaging
Box, liner, Reagent Tube and operation instructions, constituent include：One, high-fidelity DNA polymerase premixed liquid；Two, Dpn I and
10×Dpn I buffer；(3) seamless clone enzyme and 5 × seamless clone's buffer solution；(4) DMT competent cells；(5) matter is compareed
The general identification primer of grain and control plasmid；(6) sterile ddH2O。
6. a kind of site-directed point mutation method based on seamless clone technology is with kit in molecular biology, science of heredity and albumen
Application in the research fields such as matter engineering.
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| CN108424907A (en) * | 2018-05-09 | 2018-08-21 | 北京大学 | A kind of high throughput DNA multidigit point exact base mutation methods |
| CN110305861A (en) * | 2019-07-18 | 2019-10-08 | 深圳市菲鹏生物治疗股份有限公司 | The method for constructing multipoint mutation nucleic acid sequence |
| CN111996188A (en) * | 2020-08-25 | 2020-11-27 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Method for performing gene site-directed mutagenesis by using In-Fusion PCR technology |
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| CN115927546A (en) * | 2022-12-29 | 2023-04-07 | 北京海创科业生物科技有限责任公司 | Novel plasmid site-directed mutagenesis method based on seamless cloning technology |
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| CN108424907A (en) * | 2018-05-09 | 2018-08-21 | 北京大学 | A kind of high throughput DNA multidigit point exact base mutation methods |
| CN108424907B (en) * | 2018-05-09 | 2021-10-15 | 北京大学 | High-throughput DNA multi-site accurate base mutation method |
| CN110305861A (en) * | 2019-07-18 | 2019-10-08 | 深圳市菲鹏生物治疗股份有限公司 | The method for constructing multipoint mutation nucleic acid sequence |
| CN111996188A (en) * | 2020-08-25 | 2020-11-27 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Method for performing gene site-directed mutagenesis by using In-Fusion PCR technology |
| CN113005141A (en) * | 2021-01-05 | 2021-06-22 | 温州医科大学 | Gene editing tool composed of high-activity mutant, preparation method and method for repairing congenital retinoschisis disease pathogenic gene |
| CN114703212A (en) * | 2022-03-01 | 2022-07-05 | 东华大学 | Method for modifying laccase by using random mutation method of specific section and laccase strain LAC123 |
| CN114703212B (en) * | 2022-03-01 | 2024-03-29 | 东华大学 | Method for modifying laccase by using specific segment random mutation method and laccase strain LAC123 |
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