CN109266644A - A method of gene site-directed saturation mutation is carried out using dI primer - Google Patents
A method of gene site-directed saturation mutation is carried out using dI primer Download PDFInfo
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- CN109266644A CN109266644A CN201811225909.1A CN201811225909A CN109266644A CN 109266644 A CN109266644 A CN 109266644A CN 201811225909 A CN201811225909 A CN 201811225909A CN 109266644 A CN109266644 A CN 109266644A
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- primer
- target gene
- plasmid vector
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
Abstract
The invention discloses a kind of methods for carrying out gene site-directed saturation mutation using dI primer to obtain amplified production with plasmid vector of the primer amplification containing target gene that mutational site base is dI base；Amplified production restriction endonuclease Dpn I is digested, then cut off the DNA chain at thio-modification by tincture of iodine thermal cracking, obtain pyrolysis product；Pyrolysis product is stood, the single stranded DNA of the 3 ' jags at pyrolysis product both ends is made to hybridize pairing each other, obtains the band notch ring-type recombinant plasmid vector of the target gene of the point mutation containing saturation；After the band notch ring-type recombinant plasmid vector conversion Escherichia coli of target gene containing saturation mutation, the notch of the recombinant plasmid vector containing target gene is repaired, and obtains the complete recombinant plasmid vector containing target gene.Gene mutation method of the invention substantially increases the quantity that single introduces gene mutation, and simultaneous mutation site controllable precise can be used for extensive site-directed point mutation.
Description
Technical field
The present invention relates to genetic engineering fields, carry out gene site-directed saturation mutation using dI primer more particularly to a kind of
Method.
Background technique
Genetic engineering and genetic modification are most important for the protein and functional gene that obtain particular characteristic.Traditional base
Because mutating technology has random mutation, rite-directed mutagenesis etc..These technologies in the saturation that specific site carries out 4 kinds of bases it is difficult to ensure that dash forward
Become.The mutation type and site randomness of random mutation are too big, substantially completely uncontrollable, and the later period needs a large amount of screenings to obtain mesh
Mutant.Although rite-directed mutagenesis can introduce particular bases mutation in specific site, a kind of mutation alkali can only be once introduced
Base causes workload and cost to greatly increase if carrying out saturation mutation needs to synthesize a plurality of mutant primer.
Summary of the invention
The invention mainly solves the technical problem of providing a kind of sides that gene site-directed saturation mutation is carried out using dI primer
Method, substantially increases the quantity that single introduces gene mutation, and simultaneous mutation site controllable precise can be used for extensive gene site-directed
Mutation.
In order to solve the above technical problems, one technical scheme adopted by the invention is that: provide it is a kind of utilize dI primer carry out
The method of gene site-directed saturation mutation, including step are as follows: (1) with the primer amplification base containing target that mutational site base is dI base
The plasmid vector of cause, obtains amplified production；(2) disappear to the amplified production restriction endonuclease Dpn I that step (1) obtains
Change, then carry out thermal cracking, cuts off the DNA chain at thio-modification, obtain pyrolysis product；(3) pyrolysis product for obtaining step (2)
It stands, so that the single stranded DNA of the 3 ' jags at the pyrolysis product both ends is hybridized pairing each other, obtain the target base containing saturation mutation
The band notch ring-type recombinant plasmid vector of cause；(4) the band notch ring-type weight for the target gene containing saturation mutation that step (3) obtains
After group plasmid vector conversion Escherichia coli, the notch of the recombinant plasmid vector containing target gene is repaired, and is contained
The complete recombinant plasmid vector of target gene.
In a preferred embodiment of the present invention, mutational site base described in step (1) is the sequence of the primer of dI base
Column feature are as follows: (a) expands the forward primer of target gene and the 5 ' of the reverse primer of amplification target gene and hold preceding 10-20 base
Pairing complimentary to one another；(b) the mutational site base of the primer is deoxyinosine dI base；(c) forward direction of target gene is expanded
Contain the existing thio-modification of dispersion in 5 ' end complementary pairing areas of the reverse primer of primer and amplification target gene.
In a preferred embodiment of the present invention, using polymerase chain reaction in amplification described in step (1)
PCR includes the forward primer for expanding target gene, the reverse primer for expanding target gene, target in amplified reaction buffer
Nucleic acid template molecules, archaeal dna polymerase, 4 kinds of deoxynucleoside triphosphates.
In a preferred embodiment of the present invention, thermal cracking described in step (2) is that thermal cracking is carried out using the tincture of iodine, described
Thermal cracking is carried out thermal cracking 1-30 minutes under 60-80 degree.
In a preferred embodiment of the present invention, standing described in step (3) is that the pyrolysis product is first made to be cooled to room
Temperature, then place 1~15 minute.
In a preferred embodiment of the present invention, the method for carrying out gene site-directed saturation mutation using dI primer is also wrapped
The identification to the complete recombinant plasmid vector containing target gene is included, the identification is picking single colonie, and extracting contains target after culture
The complete recombinant plasmid vector of gene, sequencing identification mutation type.
The beneficial effects of the present invention are:
(1) method for carrying out gene site-directed saturation mutation using dI primer does not need to synthesize a plurality of primer, utilizes dI and 4 kinds
Normal basepairing rule a, as long as primer can be realized as the saturation mutation of gene specific site；
(2) method for carrying out gene site-directed saturation mutation using dI primer can do the saturation mutation in continuous multiple sites,
Reduce primer quantity and mutation operation number, accelerate mutation process, time cost is greatly saved；
(3) method for carrying out gene site-directed saturation mutation using dI primer can be in specific site selectivity DNA breakage chain, shape
At the controllable single stranded tails of length, saturation mutation and carrier are coupled together from ring, the clone behaviour after eliminating gene mutation
Make, greatly improves mutation efficiency；
(4) method for carrying out gene site-directed saturation mutation using dI primer can overcome random mutation in the prior art and determine
The defect of point gene mutation, in terms of can be applied to gene mutation body building, albumen Evolution in vitro.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing, in which:
Fig. 1 is the flow chart of method one preferred embodiment of the invention that gene site-directed saturation mutation is carried out using dI primer.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1: the random saturated mutant of S69 and the D74 residue of bacillus subtilis single-stranded DNA binding protein SSB
Building
Referring to Fig. 1, providing a kind of method for carrying out gene site-directed saturation mutation using dI primer, including step are as follows:
(1) design synthesizes the primer for expanding target gene and plasmid vector, the sequence signature of the primer are as follows: (a) amplification
5 ' end preceding 12 bases pairings complimentary to one another of the reverse primer of the forward primer and amplification target gene of target gene；(b) institute
The mutational site base for stating primer is 3 continuous deoxyinosine dI bases；(c) forward primer of target gene and reversed is expanded
6,12nd phosphodiester bond at 5 ' ends of primer is thio-modification.
The primer sequence that S69 and the D74 residue of amplification SSB is saturated point mutation at random is as follows:
D74-F:5 ' CTTGCA-GGCGTA-dIdIdIGGCCGTTTACAAACAAGAAA
S69-R: 5’TACGCC-TGCAAG-dIdIdITCCTTTTTTCAAGAAGTTTG
Wherein capitalization is DNA base, and dI is deoxyinosine base ,-it is thio-modification.
(2) the linearization plasmid DNA fragmentation containing target gene is expanded using polymerase chain reaction PCR.Target will be expanded
The forward primer of gene, reverse primer, target nucleic acid template molecule (the wild type mesh of SSB containing hay bacillus for expanding target gene
Mark the plasmid vector DNA of gene), high fidelity high temperature-resisting DNA polymerase, 4 kinds of deoxynucleoside triphosphates (dNTPs) PCR are added
Reaction buffer is reacted.(50 microlitres) compositions of PCR reaction system are as follows: forward primer (0.2 μM), reverse primer (0.2 μ
M), wild plasmid template molecule (5 nanogram), archaeal dna polymerase, 4 kinds of deoxynucleoside triphosphates (0.2mM), the archaeal dna polymerase
For KOD archaeal dna polymerase (1 unit).PCR condition be 98 degree 3 minutes；(95 degree 0.5 minute, 52 degree 0.5 minute, 72 degree 3 points
Clock) × 30 circulations；72 degree × 6 minutes；16 degree 3 minutes.Wherein the high fidelity high temperature-resisting DNA polymerase can be KOD
Archaeal dna polymerase.
(3) the restriction enzyme DpnI digestion of linearized plasmid vector DNA fragmentation.To the linearized plasmid vector of amplification
PCR product digests wild type ring plasmid template with restriction endonuclease Dpn I.Add directly in 20 microlitres of PCR products
Entering DpnI is 10 units, and 37 degree of reaction time are 5 minutes to 2 hours.The restriction endonuclease Dpn I can specificity
Identify the G of A methylation in cutting board plasmid double-stranded DNAmATC sequence, the processing of Dpn I is so that plasmid template becomes many sections
Short DNA double chain, these short DNA double chain conversion Escherichia coli can not generate clone, to eliminate wild plasmid mould
The false positive mutant that plate generates.
(4) cyclisation of PCR fragment.2 microlitres of tincture of iodine are added in 20 microlitres of PCR products, divide in 70 degree of progress thermal crackings 10
Clock, cuts off DNA chain at thio-modification, the short segment DNA chain of fracture and complementary chain separation, thus the two of double chain DNA fragment
End generates the 3 ' jags that length is 12 nucleotide.
(5) hybridize pairing between target gene and linearized plasmid vector DNA segment.Tincture of iodine pyrolysis product slow cooling is extremely
Room temperature is placed 5 minutes, and the single stranded DNA of the 3 ' jags at both ends is made to hybridize pairing each other, forms the target base containing saturation mutation
The band notch ring-type recombinant plasmid vector of cause.
(6) the band notch ring-type recombinant plasmid vector of the target gene containing saturation mutation converts Escherichia coli.It is prominent containing saturation
The band notch ring-type recombinant plasmid vector of the target gene of change converts competent escherichia coli cell, 37 degree on solid medium
Overnight incubation.The notch of recombinant plasmid vector containing target gene can be repaired by Escherichia coli, be formed containing the complete of target gene
Whole recombinant plasmid vector.
(7) identification of the sequencing of saturation mutation plasmid vector.Picking single colonie, extracting is containing the complete of target gene after culture
Recombinant plasmid vector, sequencing identification mutation type.
The present invention is the primer pair of dI base using mutational site base, to carry the cyclic plasmid or gene of target gene
DNA is as template for group, and PCR amplification target gene, PCR product is cracked through the tincture of iodine, and generating 3 ' single stranded DNA tails, (length can be with
It is determined in design of primers, i.e., the location of thio-modification farthest apart from 5 ' end of primer distance, general long 10-15 core
Thuja acid).Due to the base sequence complementary pairing of 3 ' single stranded DNA tails of generation, the two forms stable complete pairing double-strand,
It is cyclized into notched cyclic plasmid, the specific site of the target gene on plasmid contains the saturation mutation of 4 kinds of bases, and conversion is big
After enterobacteria, the DNA notch on plasmid vector is repaired by e. coli dna repair system, is formed and is completely contained saturation mutation
Target gene recombinant DNA molecules.It can be containing at one or the dI nucleotide of the random saturation mutation in a few places on primer sequence.
The present invention expands target gene using deoxyinosine dI Mdification primer, will based on dI base and four kinds of normal bases
The base all standing of mutated site is at 4 kinds of normal bases, to achieve the purpose that realize fixed point saturation mutation using single primer.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (6)
1. a kind of method for carrying out gene site-directed saturation mutation using dI primer, which is characterized in that including step are as follows: (1) with prominent
Displacement point base is plasmid vector of the primer amplification containing target gene of dI base, obtains amplified production；(2) step (1) is obtained
The amplified production arrived is digested with restriction endonuclease Dpn I, then carries out thermal cracking, is cut off the DNA chain at thio-modification, is obtained
To pyrolysis product；(3) pyrolysis product for obtaining step (2) is stood, and makes the single-stranded of the 3 ' jags at the pyrolysis product both ends
DNA hybridizes pairing each other, obtains the band notch ring-type recombinant plasmid vector of the target gene containing saturation mutation；(4) step (3) obtains
It is described to contain target base after the band notch ring-type recombinant plasmid vector conversion Escherichia coli of the target gene containing saturation mutation arrived
The notch of the recombinant plasmid vector of cause is repaired, and obtains the complete recombinant plasmid vector containing target gene.
2. the method according to claim 1 for carrying out gene site-directed saturation mutation using dI primer, which is characterized in that step
(1) mutational site base described in is the sequence signature of the primer of dI base are as follows: (a) expands forward primer and the expansion of target gene
Increase the 5 ' of the reverse primer of target gene and holds the pairing complimentary to one another of preceding 10-20 base；(b) the mutational site base of the primer
For deoxyinosine dI base；(c) 5 ' ends for expanding the forward primer of target gene and the reverse primer of amplification target gene are complementary
Contain the existing thio-modification of dispersion in collochore.
3. the method according to claim 1 for carrying out gene site-directed saturation mutation using dI primer, which is characterized in that step
It (1) include amplification target gene using polymerase chain reaction PCR in amplification described in, in amplified reaction buffer
Forward primer, reverse primer, target nucleic acid template molecule, archaeal dna polymerase, the 4 kinds of deoxynucleoside triphosphates for expanding target gene.
4. the method according to claim 1 for carrying out gene site-directed saturation mutation using dI primer, which is characterized in that step
(2) thermal cracking described in is that thermal cracking is carried out using the tincture of iodine, and the thermal cracking is that thermal cracking 1-30 points are carried out under 60-80 degree
Clock.
5. the method according to claim 1 for carrying out gene site-directed saturation mutation using dI primer, which is characterized in that step
(3) standing is so that the pyrolysis product is cooled to room temperature, then place 1~15 minute described in.
6. the method according to claim 1 for carrying out gene site-directed saturation mutation using dI primer, which is characterized in that described
Utilize the mirror that the method that dI primer carries out gene site-directed saturation mutation further includes to the complete recombinant plasmid vector containing target gene
Fixed, the identification is picking single colonie, and the complete recombinant plasmid vector containing target gene, sequencing identification mutant variety are extracted after culture
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| CN201811225909.1A CN109266644A (en) | 2018-10-21 | 2018-10-21 | A method of gene site-directed saturation mutation is carried out using dI primer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951762A (en) * | 2019-12-25 | 2020-04-03 | 苏州博睐恒生物科技有限公司 | Method for carrying out gene random mutation by using dITP |
Citations (2)
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| CN105420225A (en) * | 2014-09-17 | 2016-03-23 | 苏州旷世骏弛生物科技有限公司 | Iodine dissociation type gene cloning method based on sulfo-modifiers |
| CN106754875A (en) * | 2016-12-12 | 2017-05-31 | 中国科学院天津工业生物技术研究所 | Gene saturation mutation storehouse and its construction method, application |
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2018
- 2018-10-21 CN CN201811225909.1A patent/CN109266644A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420225A (en) * | 2014-09-17 | 2016-03-23 | 苏州旷世骏弛生物科技有限公司 | Iodine dissociation type gene cloning method based on sulfo-modifiers |
| CN106754875A (en) * | 2016-12-12 | 2017-05-31 | 中国科学院天津工业生物技术研究所 | Gene saturation mutation storehouse and its construction method, application |
Non-Patent Citations (3)
| Title |
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| TOBIAS BAUMANN ET AL: "Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V", 《BMC BIOTECHNOLOGY》 * |
| 付畅等: "蛋白质体外定向进化策略研究进展", 《生物技术通报》 * |
| 陶苏丹等: "点饱和突变技术及其在蛋白质工程中的应用", 《中国生物工程杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951762A (en) * | 2019-12-25 | 2020-04-03 | 苏州博睐恒生物科技有限公司 | Method for carrying out gene random mutation by using dITP |
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