CN109439682A - Utilize the method for the gene cloning of dU and archaeal archaeal dna polymerase - Google Patents
Utilize the method for the gene cloning of dU and archaeal archaeal dna polymerase Download PDFInfo
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- CN109439682A CN109439682A CN201811255456.7A CN201811255456A CN109439682A CN 109439682 A CN109439682 A CN 109439682A CN 201811255456 A CN201811255456 A CN 201811255456A CN 109439682 A CN109439682 A CN 109439682A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
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- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Abstract
The invention discloses a kind of methods of gene cloning using dU and archaeal archaeal dna polymerase, utilize dU Mdification primer and archaeal archaeal dna polymerase amplification target gene and cloning vector, generate the PCR fragment that 5 ' ends are single stranded DNA tail, target gene and the single stranded tails of carrier PCR fragment are paired with each other, any enzyme is not needed to handle PCR product, it simply mixes, it will be able to form recombinant plasmid.Therefore this method greatly simplifies operating procedure; it eliminates the low caused seamless gene cloning of PCR product treatment effeciency and convenient restriction inscribe enzyme clone is easy the technological deficiency of failure; the recombination efficiency between target gene and plasmid is significantly improved, is highly convenient for carrying out extensive gene cloning.Meanwhile this method can be applied to point mutation etc., it can also be used to the DNA library building in the fields such as accurate medical treatment.
Description
Technical field
The present invention relates to genetic engineering fields, more particularly to a kind of gene cloning using dU and archaeal archaeal dna polymerase
With point mutation process.
Background technique
The appearance of gene clone technology is greatly promoted the development of modern genetic engineering, protein engineering.Traditional gene
Clone technology digests target gene DNA fragmentation and plasmid vector first with restriction endonuclease, and then DNA ligase connects
Cyclisation target gene and plasmid are connect, complete recombinant plasmid is formed.Since the digestion of restriction endonuclease digestion reaction is endless
Entirely, the positive recombinant plasmid percentage containing target gene is often resulted in lower than 10%, or even clone's failure.In order to overcome routine
The defect of gene clone method develops the cloning process independent of ligase.These methods all rely on target gene with
Recombining reaction between plasmid vector DNA forms cyclic annular (band DNA notch) plasmid.However recombining reaction efficiency is difficult to control, and has
When will cause the failure of recombining reaction, eventually lead to gene cloning failure.
Summary of the invention
The invention mainly solves the technical problem of providing a kind of gene clonings and point using dU and archaeal archaeal dna polymerase
Mutation method, simplifies operating procedure, can significantly improve target gene and matter the problem of can solve existing recombinant clone technology
Recombination efficiency between grain carrier.
In order to solve the above technical problems, one technical scheme adopted by the invention is that: a kind of utilization dU and archaeal DNA is provided
The method of the gene cloning of polymerase, including step are as follows: (1) carried using the primer amplification target gene containing dU base and plasmid
Body；(2) digestion process is carried out to the ring plasmid template in the plasmid vector after amplification with restriction endonuclease Dpn I；
(3) by target gene and the step (2) after amplification that step (1) obtains, with Dpn I, treated that plasmid vector mixes and each other
Hybridization pairing forms the band notch ring-type recombinant plasmid vector containing target gene；(4) step (3) obtain it is notched containing
After the recombinant plasmid vector conversion Escherichia coli of target gene, the notch of the recombinant plasmid vector containing target gene is repaired
It answers, obtains the complete recombinant plasmid vector containing target gene.
In a preferred embodiment of the present invention, the sequence of the primer containing dU base described in step (1) are as follows: (a) expands
The 1-30 bit base sequence that the forward primer 5 ' of the forward primer and amplification plasmid vector that increase target gene is held is complimentary to one another to match
It is right；(b) expand target gene reverse primer and amplification plasmid vector reverse primer 5 ' hold 1-30 bit base sequence that
This complementary pairing；(c) primer length is 40-50 base, 3 ' end base pair downstream sequences and the target gene and plasmid vector
DNA fragmentation both ends to be amplified base sequence pairing；(d) dU base distance 5 ' holds 10-25 base, and distance 3 ' holds 25-35
Base.
In a preferred embodiment of the present invention, it is expanded described in step (1) using polymerase chain reaction PCR,
Include in amplified reaction buffer for expanding the forward primer of target gene, the forward primer for expanding plasmid vector,
It is poly- for expanding the reverse primer of target gene, the reverse primer for expanding plasmid vector, target nucleic acid template molecule, DNA
Synthase, 4 kinds of deoxynucleoside triphosphates.
In a preferred embodiment of the present invention, hybridizing pairing process described in step (3) each other is transferred in 4-50 degree
It sets 1-15 minutes.
In a preferred embodiment of the present invention, the method for the gene cloning using dU and archaeal archaeal dna polymerase is also
Including the identification to the complete recombinant vector containing target gene: picking single colonie utilizes the primer and Taq of amplification target gene
Archaeal dna polymerase carries out bacterium colony PCR, identifies the positive colony of the complete recombinant plasmid vector containing target gene, cultivates positive colony,
And extract the complete recombinant plasmid vector containing target gene.
In a preferred embodiment of the present invention, the method for the gene cloning using dU and archaeal archaeal dna polymerase is used
In point mutation, including step are as follows: (1) using the plasmid vector of the primer PCR amplification wild type gene containing dU base, obtain
To the plasmid vector of coding mutation type gene；(2) plasmid of the DpnI coding mutation type gene obtained to step (1) is utilized
The plasmid vector of wild type gene in carrier carries out digestion process, obtains the plasmid with 5 ' complementary single stranded DNA tails and carries
Body；(3) plasmid vector with complementary single strand tail for obtaining step (2) carries out self hybridization pairing, is formed notched
The recombinant plasmid vector of the target gene containing saltant type；(4) recombination for the notched target gene containing saltant type that step (3) obtains
After plasmid vector converts Escherichia coli, the notch of the recombinant plasmid vector of the target gene containing saltant type is repaired, extracting
The complete recombinant plasmid vector of the target gene containing saltant type is sequenced, and is verified point mutation, is obtained the target gene containing saltant type
Complete recombinant plasmid vector.
The beneficial effects of the present invention are:
One, the characteristics of present invention can be stagnated using archaeal archaeal dna polymerase at dU base, PCR amplification, which generates, has 5 '
The linearization plasmid and target gene segment of single stranded tails, single stranded tails between the two are paired with each other, form cyclic annular heavy constituent
Son does not need to carry out recombining reaction between target gene and the PCR fragment of linearization plasmid, so that operating procedure is simplified,
Shorten the operating time；
Two, the method operation flux is big, is suitable for multiple target genes while cloning.Due to only being needed in entire cloning procedure
PCR amplification is wanted, therefore can simply expand clone's flux, carries out extensive gene cloning；
Three, it since the formation of recombinant plasmid only needs to mix target gene and linearization plasmid, greatly improves
The formation efficiency of recombinant plasmid, greatly improves recombination efficiency；
Four, the positive clone rate containing target gene is high.Due to using PCR amplification plasmid vector, eliminate in restriction nuclease
Enzyme cutting digested plasmid is not thorough caused false positive clones, to greatly improve positive colony ratio.
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 one preferred embodiment of method of the gene cloning of the invention using dU and archaeal archaeal dna polymerase；
Fig. 2 is the flow chart of one preferred embodiment of method of the point mutation of the invention using dU and archaeal archaeal dna polymerase.
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 gene cloning of bacillus subtilis single-stranded DNA binding protein SSB
Referring to Fig. 1, providing a kind of method of gene cloning using dU and archaeal archaeal dna polymerase, including step are as follows:
(1) primer of the design synthesis containing dU base, wherein the sequence feature to be met of the primer containing dU base are as follows:
(a) it is dU base that the forward primer 5 ' for expanding bacillus subtilis SSB gene, which holds the 15th, and it is dU that reverse primer 5 ', which holds the 15th,
Base；It is dU base that the forward primer 5 ' for expanding pUC18 plasmid, which holds the 15th, and it is dU base that reverse primer 5 ', which holds the 15th,；(b)
The 1-14 bit base sequence for expanding the end of forward primer 5 ' of target gene and plasmid vector is complementary pairing, expands target base
The 1-14 bit base sequence at the reverse primer 5 ' of cause and plasmid vector end is complementary pairing.The target gene expanded is withered
Careless bacillus SSB gene, the carrier of insertion are pUC18 carrier.
Expand the primer sequence of bacillus subtilis SSB gene are as follows:
Gene-F:5 ' CAAAATAGCAGGCTXTCCCATATGCTTAACCGAGTTGTATT
Gene-R:5 ' CAAGAAAGCXGGGTXGGATCCTTAGAATGGA AGATCATCAT
Expand the primer sequence of pUC18 are as follows:
PUC18-F:5 ' AGCCTGCTATTTTGXGATATGGTCGACCTGCAGGCATGCAA
PUC18-R:5 ' ACCCAGCXTTCTTGXGTAATGTCTAGAGGATCCCCGGGTAC
Wherein X is dU base.
(2) target gene bacillus subtilis is expanded using polymerase chain reaction PCR using the primer containing dU base
SSB gene and pUC18 plasmid vector, wherein including 0.2 μM in 50 microlitres of pcr amplification reaction buffers for expanding target base
The forward primer of cause and plasmid vector, 0.2 μM for expanding reverse primer, the 0.2 μM of target core of target gene and plasmid vector
4 kinds of template polynucleotide molecule, archaeal dna polymerase, 0.2mM deoxynucleoside triphosphates, the target nucleic acid template molecule are target gene mould
Plate is 50 nanogram Bacillus subtilis genes group DNA, and plasmid vector template molecule is 5 nanogram pUC18 cyclic plasmids, the DNA
Polymerase is 2.5 unit high fidelity Pfu archaeal dna polymerases, PCR reaction condition in the present invention are as follows: pUC18 is carried in the present invention
Body PCR reaction condition are as follows: 98 degree 3 minutes；(95 degree 0.5 minute, 64 degree 0.5 minute, 72 degree 5 minutes) × 30 circulations；72 degree × 5
Minute；16 degree 3 minutes.The PCR reaction condition of target gene bacillus subtilis SSB gene in the present invention are as follows: 98 degree 3 minutes；
(95 degree 0.5 minute, 52 degree 0.5 minute, 72 degree 1 minute) × 30 circulations；72 degree × 2 minutes；16 degree 3 minutes.
(3) it is deposited in the PCR product of the linearized plasmid vector pUC18 of restriction endonuclease Dpn I digest amplification
Ring plasmid template.It is 10 units that DpnI is added directly in 20 microlitres of PCR products, and 37 degree of reaction time are 1 hour.
The G of A methylation in the restriction endonuclease Dpn I energy specific recognition cutting board plasmid double-stranded DNAmATC sequence,
So that plasmid template becomes the short DNA double chain of many sections, these short DNA double chain conversion Escherichia coli cannot for the processing of Dpn I
It is enough to generate clone, to eliminate the false positive clones of wild plasmid template generation.
(4) with 14 length of nucleotides 5 ' jags target gene and linearized plasmid vector DNA segment between into
Hybridization of going is matched, and the hybridization pairing is after mixing two kinds of DNA segments, to be placed at room temperature for 0.5~15 minute, keeps the 5 ' of the two prominent
Outlet hybridization pairing forms band notch recombinant plasmid of the stable complete pairing double-strand containing target gene, does not need to carry out DNA company
It is reversed to answer.
It (5) include the notched recombinant plasmid vector containing target gene in the hybridization mixture that step (4) obtains,
The notched recombinant plasmid vector containing target gene converts competent escherichia coli cell, 37 on solid medium
Spend overnight incubation.In the reproductive process of Escherichia coli, in notch recombinant plasmid vector between target gene and plasmid vector
Notch can be repaired, and form the complete recombinant plasmid vector containing target gene.
(6) identification of the complete recombinant plasmid vector containing target gene: picking single colonie utilizes drawing for amplification target gene
Object and Taq archaeal dna polymerase carry out bacterium colony PCR, identify the positive colony of the complete recombinant plasmid containing target gene.Culture is positive
Clone, and extract the complete recombinant plasmid dna containing target gene.
Embodiment 2: the building of the site-directed point mutation body S69A of bacillus subtilis single-stranded DNA binding protein SSB
Referring to Fig. 2, providing a kind of site-directed point mutation method using dU specific nucleic acid enzyme endoQ, including step are as follows:
(1) primer of the design synthesis containing dU base, wherein the sequence feature to be met of the primer containing dU base are as follows:
(a) base at 5 ' the 13rd, the ends of forward primer S69A-F is dU base, the base at 5 ' the 13rd, the ends of reverse primer S69A-R
For dU base；(b) the 1-12 bit base sequence at 5 ' ends of forward primer and reverse primer is complementary pairing；(c) forward direction is drawn
The downstream object dU 3 ' holds 26 base sequences and the wild type SSB target gene to match, and 26 alkali are held in the downstream reverse primer dU 3 '
Basic sequence and the wild type SSB target gene match.
The primer sequence for expanding the S69A point mutation of SSB is as follows:
S69A-F:5 ' aaaaggaGCcctdUgcaggcgtagatggccgtttacaaac
S69A-R: 5’aggGCtccttttdUtcaagaagtttgcaacgttttcggct
Wherein capitalization is base after mutation, and lowercase is wild-type base, and dU is Brdurd base.
(2) it is carried using the primer containing dU base using the plasmid of polymerase chain reaction PCR amplification wild type SSB gene
Body.Wherein in 50 microlitres of pcr amplification reaction buffers include 0.2 μM and is used for the forward primer of amplification, 0.2 μM of reverse primer, 5
4 kinds of nanogram wild plasmid template molecule, Pfu archaeal dna polymerase, 0.2mM deoxynucleoside triphosphates, the wild plasmid mould
Plate molecule is the cyclic plasmid molecule pUC18 of the SSB of target gene containing wild type, and the archaeal dna polymerase is 2.5 unit Pfu
Archaeal dna polymerase, the present invention in PCR reaction condition are as follows: 98 degree 3 minutes；(95 degree 0.5 minute, 55 degree 0.5 minute, 72 degree 6 minutes)
× 30 circulations；72 degree × 6 minutes；16 degree 3 minutes.
(3) ring-type present in the linearized plasmid vector PCR product of restriction endonuclease Dpn I digest amplification
Plasmid template.It is 10 units that DpnI is added directly in 20 microlitres of PCR products, and 37 degree of reaction time are 5 minutes to 2 hours.
The G of A methylation in the restriction endonuclease Dpn I energy specific recognition cutting board plasmid double-stranded DNAmATC sequence,
So that plasmid template becomes the short DNA double chain of many sections, these short DNA double chain conversion Escherichia coli cannot for the processing of Dpn I
It is enough to generate clone, to eliminate the false positive mutant of wild plasmid template generation.
(4) self single-stranded hybridization of 5 ' jags of 12 length of nucleotides of the linearization plasmid of saltant type SSB gene is matched
It is right, 5 minutes are placed at room temperature for, makes 5 ' the jags hybridization pairing of the two, forms stable complete pairing double-strand target containing saltant type
The band notch recombinant plasmid of gene.
(5) the notched recombinant plasmid vector containing saltant type target gene that step (4) obtains converts Escherichia coli
Competent cell, 37 degree of overnight incubations on solid medium.In the reproductive process of Escherichia coli, band notch recombinant plasmid is carried
The notch of body can be repaired, and form the complete recombinant plasmid vector of the target gene of SSB containing saltant type.
(6) identification of the complete recombinant plasmid vector of the SSB of target gene containing saltant type: picking single colonie, culture clone, and
Extracting plasmid is sequenced, and the S69A point mutation of saltant type SSB is verified.
The present invention generates 5 ends using dU Mdification primer and archaeal archaeal dna polymerase amplification target gene and cloning vector ' be
The single stranded tails of the PCR fragment of single stranded DNA tail, target gene and carrier PCR fragment are paired with each other, do not need any enzyme pair
PCR product is handled, and is simply mixed, it will be able to form recombinant plasmid.Therefore this method greatly simplifies behaviour
Make step, eliminates the technological deficiency of the low caused gene cloning failure of PCR treatment effeciency, significantly improve target gene and matter
The recombination efficiency of intergranular is highly convenient for carrying out extensive DNA clone.
The present invention carries out gene cloning and point mutation using dU modified nucleoside acid and archaeal archaeal dna polymerase, first with specific
Primer amplification carrier and target fragment, make the both ends of carrier and genetic fragment generate the single-stranded homology region 20-40bp, then
Linear carrier and target fragment are mixed, makes the two both ends single stranded zone that pairing occur and connect, forms notched recombination
Then DNA converts Escherichia coli, the reparation of DNA notch is completed in Escherichia coli body, forms complete recombinant plasmid.Pass through
Target gene can be inserted into any site of carrier by this method, easy to operate, be a kind of novel not need any DNA modification
Enzyme carries out the universal clone technology of DNA recombining reaction to target gene and cloning vector.
The present invention can be used for gene cloning, the fields such as genetic fragment splicing, target gene rite-directed mutagenesis.Target gene and matter
Grain carrier is digested without restriction endonuclease, therefore can substantially reduce false positive clones (empty plasmid) percentage, is improved
Target gene clones success rate.
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 of the gene cloning using dU and archaeal archaeal dna polymerase, which is characterized in that including step are as follows: (1) utilize
Primer amplification target gene and plasmid vector containing dU base；(2) with restriction endonuclease Dpn I to the matter after amplification
Ring plasmid template in grain carrier carries out digestion process；(3) target gene and step after the amplification for obtaining step (1)
(2) it is mixed with Dpn I treated plasmid vector and hybridization pairing each other, forms the band notch ring-type recombination matter containing target gene
Grain carrier；(4) described after the notched recombinant plasmid vector conversion Escherichia coli containing target gene that step (3) obtains
The notch of recombinant plasmid vector containing target gene is repaired, and obtains the complete recombinant plasmid vector containing target gene.
2. the method for the gene cloning according to claim 1 using dU and archaeal archaeal dna polymerase, which is characterized in that step
Suddenly the sequence of the primer containing dU base described in (1) are as follows: (a) expands the forward primer of target gene and expand plasmid vector
The 1-30 bit base sequence pairing complimentary to one another that forward primer 5 ' is held；(b) reverse primer and amplification matter of target gene are expanded
The 1-30 bit base sequence pairing complimentary to one another that the reverse primer 5 ' of grain carrier is held；(c) primer length is 40-50 base,
The DNA fragmentation both ends to be amplified base sequence of 3 ' end base pair downstream sequences and the target gene and plasmid vector matches；(d) dU
Base distance 5 ' holds 10-25 base, and distance 3 ' holds 25-35 base.
3. the method for the gene cloning according to claim 1 using dU and archaeal archaeal dna polymerase, which is characterized in that step
Suddenly amplification described in (1) includes for expanding target base in amplified reaction buffer using polymerase chain reaction PCR
The forward primer of cause, the forward primer for expanding plasmid vector, the reverse primer for expanding target gene, for expanding matter
Reverse primer, target nucleic acid template molecule, archaeal dna polymerase, the 4 kinds of deoxynucleoside triphosphates of grain carrier.
4. the method for the gene cloning according to claim 1 using dU and archaeal archaeal dna polymerase, which is characterized in that step
Suddenly hybridizing pairing process described in (3) each other is placed 1-15 minutes under 4-50 degree.
5. the method for the gene cloning according to claim 1 using dU and archaeal archaeal dna polymerase, which is characterized in that institute
Stating using the method for the gene cloning of dU and archaeal archaeal dna polymerase further includes mirror to the complete recombinant vector containing target gene
Fixed: picking single colonie carries out bacterium colony PCR using the primer and Taq archaeal dna polymerase of amplification target gene, and identification contains target gene
Complete recombinant plasmid vector positive colony, cultivate positive colony, and extract the complete recombinant plasmid vector containing target gene.
6. gene clone method according to claim 1, which is characterized in that described using dU and archaeal archaeal dna polymerase
The method of gene cloning is used for point mutation, including step are as follows: (1) expands wild type using the primer PCR containing dU base
The plasmid vector of gene obtains the plasmid vector of coding mutation type gene；(2) nucleosides that step (1) is obtained using DpnI
The plasmid vector of wild type gene in the plasmid vector of acid mutation type gene carries out digestion process, obtains single with complementary 5 '
The plasmid vector of chain DNA tail；(3) plasmid vector with complementary single strand tail for obtaining step (2) carries out self hybridization
Pairing forms the recombinant plasmid vector of the notched target gene containing saltant type；(4) what step (3) obtained is notched containing prominent
After the recombinant plasmid vector conversion Escherichia coli of modification target gene, the recombinant plasmid vector of the target gene containing saltant type
Notch is repaired, and the complete recombinant plasmid vector of extracting target gene containing saltant type is sequenced, and is verified point mutation, is contained
The complete recombinant plasmid vector of saltant type target gene.
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