CN102321660A - Method for cloning deoxyribonucleic acid (DNA) - Google Patents
Method for cloning deoxyribonucleic acid (DNA) Download PDFInfo
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Abstract
The invention relates to a method for cloning deoxyribonucleic acid (DNA). The method comprises the following steps of: digesting a carrier by using a restriction enzyme at an insertion site of the carrier so as to obtain DNA of a linear carrier; synthesizing a polymerase chain reaction (PCR) amplification primer according to the sequence design of a target DNA fragment and the insertion site of the carrier, and performing PCR amplification to obtain the target DNA fragment; purifying the DNA of the linear carrier and the target DNA fragment, mixing, adding exonuclease III and an exonuclease III reaction buffer solution to form a reaction system, continuing to react, adding ethylene diamine tetraacetic acid to terminate the reaction, reacting, and annealing; taking competent cells of transformed E. coli from the reaction system; coating a transformed bacterial liquid on a plate of an Luria-Bertani (LB) culture medium containing corresponding antibiotics and culturing; and selecting a monoclonal bacterial colony from the culture plate, and extracting a plasmid and identifying to confirm the successful cloning of the target DNA fragment. Independent of restriction enzyme cutting sites and ligase, the quick and efficient cloning of DNA fragments can be realized.
Description
Technical field
The present invention relates to a kind of dna clone method, especially relate to a kind of target DNA fragment high-efficient cloning method that need not ligation by exonuclease III mediation.
Background technology
The molecular cloning method that adopts in the genetically engineered at present is the method that depends on restriction enzyme and ligase enzyme, is promptly obtained with complementary terminal viscosity or flat terminal target DNA fragment and carrier DNA fragment by restriction enzyme earlier; Then, the ligation through ligase enzyme connects into a cyclic DNA again with carrier and target DNA fragment, at last with this cyclic plasmid DNA transformed into escherichia coli to obtain to contain the clone of target DNA fragment.The molecular cloning method of this routine relies on restriction endonuclease recognition sequence, because behind the being limited property endonuclease digestion, carrier DNA and target DNA fragment must produce ability complementary sticky end or flat terminal, could accomplish follow-up ligation.Yet; Because the restriction enzyme site that carrier DNA and target DNA fragment can utilize jointly is often less; This just makes the clone of some dna fragmentation need could obtain required recombinant plasmid through a few step subclone processes owing to can not find proper restriction site, and whole process takes time and effort.If target DNA fragment is a gene order; Often also need painstakingly to add some bases for the goal gene reading frame that inserts carrier is not changed so, more this makes expressed albumen n end often add several amino acid and proteic characteristic is changed.Moreover, because conventional molecular cloning method connection carrier relies on the catalytic ligation of ligase enzyme with target DNA fragment, thus increased carrier connect probability certainly, strengthened the screening operation amount.In addition; Traditional cloning process is difficult to the lengthy motion picture segment DNA is inserted in the macromolecule carrier; This mainly is generally only to contain four, five bases via the terminal viscosity that restriction enzyme handle to obtain, and these several bases are difficult to keep the meta open-circle DNA complex body that forms between big fragment and larger vector and to cause large fragment DNA to be connected difficult; Adopt conventional molecular cloning method to be difficult to once accomplish how segmental ligation usually; This makes how segmental connection often need experience a few step subclone processes; Even experience a few step subclone processes, goal gene also often inserts carrier and causes false positive with opposite direction.Therefore,, save clone's required time in order to improve target DNA fragment clone's success ratio, necessary set up a kind of fast, efficient, stable, can improve long segment gene clone rate simultaneously and realize the gene clone method of the disposable seamless connection of multi-disc segment DNA.
Exonuclease III (is Exonuclease III; Hereinafter to be referred as ExoIII or excision enzyme III) be a kind of nonspecific excision enzyme; It has from double-stranded DNA 5 ' protruding terminus or the flat terminal 5 prime excision enzyme activity that progressively cuts mononucleotide along 3 ' → 5 ' direction; But do not have 5 prime excision enzyme activity from 3 ' protruding terminus digestion double-stranded DNA [1, Wu R, Ruben G Siegel B, et al.Synchronous digestion of SV40 DNA by exonuclease III.Biochemistry; 1976,15:734-740; 2, Thomas K R, Olivera B M.Processivity of DNA exonucleases.J Biol Chem, 1978,253:424-429; 3, Li C, Tucker P W.Exoquence DNA sequencing.Nucleic Acids Res, 1993,21:1239-1244.].Utilize this 5 prime excision enzyme activity of this enzyme that the linear carrier and the target DNA fragment that contain homologous sequence are handled; Can on carrier DNA and target DNA fragment, produce the long terminal viscosity of ability complementary respectively; The carrier and the target DNA fragment of the complementary long terminal viscosity of this band; After annealing, can form stable open-circle DNA complex body, this dna complex often be with breach or outstanding sequence [4, Aslanidis C, de Jong P J.Ligation-independent cloning of PCR products (LIC-PCR) .Nucleic Acid Res; 1990,18:6069-6074; 5, Yang Y S, Watson W J, Tucker P W et al.Construction of recombinant DNA by exonuclease recession.Nucleic Acids Res.1993,21:1889-1893; 6, Li C and Ronald M.Evans.Ligation independent cloning irrespective of restriction site compatibility.Nucleic Acids Research, 1997,25 (20): 4165-4166].But behind the dna complex transformed into escherichia coli of this band breach and outstanding sequence; Can be by the repair system reparation of Bacillus coli cells; Form complete cyclic plasmid DNA; Thereby accomplish target DNA fragment the clone (6, Li C and Ronald M.Evans.Ligation independent cloning irrespective of restriction site compatibility.Nucleic Acids Research, 1997,25 (20): 4165-4166; 7, Baogong Zhu; Guifang Cai, Emily O.Hall, et al.In-Fusion assembly:seamless engineering of multidomain fusion proteins; Modular vectors; And mutations.BioTechniques, 2007,43 (3): 354-359).
Summary of the invention
The present invention aims to provide a kind of dna clone method, and this method does not rely on restriction enzyme site and ligase enzyme, can realize that dna fragmentation rapidly and efficiently clones.
The present invention includes following steps:
1) inserts site at carrier and use the digestion with restriction enzyme carrier, obtain linear carrier DNA;
2) insert the synthetic pcr amplification primer of site sequences Design according to target DNA fragment and carrier;
3) carry out pcr amplification with institute's synthetic pcr amplification primer by ordinary method, obtain target DNA fragment;
4) the method difference purifying that passes through the recovery of electrophoresis and glue is by the linear carrier DNA of step 1) acquisition and the target DNA fragment of step 3) acquisition;
5) target DNA fragment and the mixing of linear carrier DNA that the step 4) purifying obtains be will pass through, and exonuclease III and exonuclease III reaction buffer, anabolic reaction system added;
6) reaction system with step 5) continues reaction;
7) in the reaction system of step 6), add EDTA Disodium (EDTA) with termination reaction;
8) reaction system with step 7) places 60 ℃ of reaction 5～10min;
9) reaction system of step 8) is annealed;
10) from the reaction system of step 9), get 5～10 μ L transformed into escherichia coli E.coli competent cells;
11) the transformed bacteria liquid coating with step 10) contains corresponding antibiotic LB culture medium flat plate, cultivates 10～15h in 37 ℃;
12) picking mono-clonal bacterium colony from the culture plate of step 11) extracts plasmid by ordinary method and identifies, to confirm the successful clone of target DNA fragment.
In step 1), saidly insert site at carrier and use the digestion with restriction enzyme carrier, be meant that carrier produces 5 ' protruding terminus down or puts down end in corresponding restriction enzyme effect; Said carrier can be selected from any carrier that dna clone uses in pUC series, pBOB series, the genetically engineereds such as pcDNA is serial, pBKS is serial, pCMV is serial, pLV series.
In step 2) in; The synthetic pcr amplification primer of said design is made up of carrier homologous sequence and target DNA fragment homologous sequence two portions: 1. primer 5 ' end contains the homologous sequence that the plan of having an appointment about 8～20bp is inserted carrier, and this sequence can produce the long sticky end that inserts site sequence complementary pairing with carrier under the effect of exonuclease III; 2. primer 3 ' end then is the primer of 15～40bp target DNA fragment to be amplified, is used for and the target DNA fragment complementary pairing amplification target DNA fragment.
In step 5), said target DNA fragment can be 10～100ng, and the content of said linear carrier DNA can be 10～100ng, and wherein the optimum content of linear carrier DNA is 25ng; Molecule ratio between said target DNA fragment and the linear carrier DNA can be (1～4): 1, and optimum proportion is 2: 1; The enzyme of said exonuclease III concentration alive is 10～100units, and best enzyme concentration alive is 20units.
In step 6), the condition of said reaction can be: temperature is ice bath or 4 ℃, and the time is 10～240min; The condition of optimum response is 4 ℃ of reaction 1h.
In step 7), the add-on of said EDTA can be 1 μ L, 0.5mol/L, pH8.0.
In step 9), said annealed temperature can be ice bath or 4 ℃, and the annealed time can be 5min.
In step 10), said transformed into escherichia coli competent cell can be selected from E.coli DH5 α competent cell, E.coli M15 competent cell, E.coli Top10F ' competent cell, E.coli XL-blue competent cell, E.coli JM109 competent cell, E.coli HB101 competent cell or the used E.coli competent cell of other genetically engineered operation.
The present invention has following outstanding advantage:
1) designed primer is made up of the homologous sequence of 8～20bp carrier and the homologous sequence of 15～40bp target DNA fragment in step 3); So both guaranteed target DNA fragment effectively, specifically the amplification; Guaranteed that also target DNA fragment and the seamless of carrier are connected, and are particularly advantageous in the connection of long segment.
2) be reflected at ice bath or 4 ℃ of exonuclease III are in order to reduce the activity of enzyme in step 6); Be convenient to controlling reaction time and make sticky end that carrier and target DNA fragment produce via exonuclease I II in colibacillary repair coverage, can be formed the closed loop plasmid by the maintenance of intestinal bacteria maintenance system with breach and the outstanding sequence that guarantees to produce behind the complementary pairing.
3) in step 9), taking 4 ℃ of annealing rather than 30～40 ℃ of conventional annealing, is because low temperature helps annealed particularity and accuracy, helps to improve clone's accuracy and efficient.
4) the present invention does not need ligase enzyme to carry out ligation when carrying out the dna fragmentation clone, thereby does not exist carrier from the problem that connects yet.
5) the present invention need not consider restriction enzyme site when the PCR of purpose of design dna fragmentation primer, and the restriction of unrestricted restriction endonuclease need not added the protection base, does not worry reading frame and changes, and can realize seamless connection the between goal gene and carrier.
6) owing to exonuclease III under condition of the present invention can produce the complementary sticky end greater than 8bp respectively at carrier DNA and target DNA fragment two ends; The complementary sticky end of this length helps forming stable open loop complex body between lengthy motion picture segment DNA and macromolecular carrier, thereby can improve clone's success ratio of lengthy motion picture segment DNA greatly.
7) the inventive method sequence-dependent complementary pairing when the clone, each fragment all only contains specific sequence, can only match with specific fragment complementation, thereby can realize the seamless connection of how segmental orientation.
This shows, carry out the target DNA fragment clone with the present invention and not only have the advantage that the reaction times is short, the positive colony rate is high, and the clone of long segment and multi-disc segment DNA is had clear superiority.
Description of drawings
Fig. 1 cuts synoptic diagram for carrier pBOB enzyme in the embodiment of the invention 1.
Fig. 2 is goal gene hPRAK primer and a pcr amplification synoptic diagram in the embodiment of the invention 1.
Fig. 3 be in the embodiment of the invention 1 behind goal gene and the linear carrier DNA purifying agarose gel electrophoresis detect figure.
Fig. 4 under the effect of exonuclease III, produces sticky end for goal gene hPRAK in the embodiment of the invention 1 and linear carrier DNA and the complementation of annealing after the synoptic diagram in intestinal bacteria, repaired.
Fig. 5 is the optimization figure of dna content among the inventive method embodiment 1.In Fig. 5, X-coordinate is a linear carrier DNA concentration (ng), and ordinate zou is clone's number (individual).
Fig. 6 is the optimization figure of goal gene hPRAK and vector dna molecule ratio in the embodiment of the invention 1.In Fig. 6, X-coordinate is the molecular ratio of goal gene/carrier DNA, and ordinate zou is clone's number (individual).
Fig. 7 cuts the optimization figure of time in the embodiment of the invention 1 to exonuclease III enzyme.In Fig. 7, X-coordinate be the excision enzyme III treatment time (minute), ordinate zou for clone number (individual).
Fig. 8 is the optimization figure of the embodiment of the invention 1 amplifying nucleic acid excision enzyme III enzyme concentration alive.In Fig. 8, X-coordinate is an excision enzyme III activity unit (U), and ordinate zou is clone's number (individual).
Fig. 9 carries out the positive colony bacterium colony that gene clone is produced for the embodiment of the invention 1 on resistant panel.
Figure 10 carries out the figure as a result that plasmid enzyme restriction is identified for the positive colony bacterium colony that the embodiment of the invention 1 is produced.
Figure 11 cuts synoptic diagram for carrier enzyme in the embodiment of the invention 8.
Figure 12 is the design of primers and the pcr amplification synoptic diagram of target DNA fragment in the embodiment of the invention 9.
Figure 13 under the effect of exonuclease III, produces sticky end for target DNA fragment in the embodiment of the invention 9 with linear carrier DNA and the complementation of annealing after the synoptic diagram in intestinal bacteria, repaired.
Embodiment
Embodiment 1
Present embodiment is inserted into BamHI and the XhoI site of virus vector pBOB with human source gene PRAK, and concrete operations are following:
1) with BamHI and XhoI digestion circular vectors pBOB, obtains the linear carrier (Fig. 1) that two ends all contain 5 ' protruding terminus;
2) the BamHI upper reaches 5 ' end and the XhoI downstream 3 ' terminal sequence according to goal gene hPRAK and pBOB designs two primers with amplifying target genes.Article two, the long respectively 35bp of primer, 5 ' end contains the homologous sequence (representing with capitalization) of 15bp carrier pBOB, and 3 ' end contains 20bp goal gene hPRAK homologous sequence (indicating with lowercase).
HPRAK forward direction primer sequence: 5 ' AGA GAA TTC GGA TCC atgtcggaggagagcgacat-3 ';
(capitalization is the pBOB carrier B amHI upper reaches 5 ' end homologous sequences, and lowercase is a target gene 5 ' end forward direction amplimer)
HPRAK reverse primer sequence: 5 ' CTT CCA TGG CTC GAG ttattgggattcgtgggacg-3 '.
(capitalization is pBOB carrier XhoI downstream 3 ' end homologous sequences, and lowercase is goal gene a 3 ' end reverse primer)
3) with step 2) designed primer, carry out pcr amplification by ordinary method and obtain hPRAK gene fragment (Fig. 2);
4) the linear carrier DNA fragment of step 1) acquisition and the goal gene hPRAK of step 3 acquisition are carried out agarose gel electrophoresis; Reclaim linear carrier DNA fragment of purifying and goal gene hPRAK fragment through digging glue, and linear carrier DNA fragment and goal gene hPRAK concentration (Fig. 3) behind the mensuration purifying;
5) reaction system of foundation and optimization exonuclease III.The reaction system TV is 10uL, wherein contains exonuclease III reaction buffer, exonuclease III, goal gene hPRAK and linear carrier pBOB.In order to obtain the peak optimization reaction condition, we are provided with a series of exonuclease III reaction systems, respectively the blending ratio of DNA concentration, goal gene and linear carrier in the reaction system, enzyme concentration alive and the reaction times of exonuclease III are optimized.
6) each reaction system with the step 5) setting places 4 ℃ of reaction different time down, so that exonuclease III produces the 5 ' sticky end (Fig. 4) of appropriate length respectively at linear carrier and goal gene two ends;
7) (0.5mol/L is pH8.0) to stop endonuclease reaction in each reaction system of step 6), to add the EDTA of 1 μ L;
8) after the termination reaction, each reaction system of step 7) is placed 60 ℃ of water-bath 5min;
9) change each reaction system of step 8) over to 4 ℃ of annealing 5min again;
10) from each reaction system of step 9), get 5 μ L and transform 50～100 μ L intestinal bacteria E.coli DH5a competent cells respectively;
11) the Escherichia coli bacteria liquid separate application that transforms is contained corresponding antibiotic LB culture plate, in 37 ℃ of cultivation 15h, so that (Fig. 4) repaired and duplicated to intestinal bacteria divided ring plasmid;
12) add up the positive colony number that grows in the flat board of each experimental group incubated overnight.Experimental result shows; DNA concentration is at 10～100ng in the 10uL reaction system, and the ratio of goal gene and carrier DNA is in (1～4): between 1, the reaction times of exonuclease III can both produce the positive colony number greater than 100 on resistant panel when 10～240min; Wherein the optimum concn of carrier DNA is 25ng (Fig. 5); The The Best Mixed ratio of goal gene and carrier DNA is 2: 1 (Fig. 6), and the optimum reacting time of exonuclease III is 60min (Fig. 7), and this mainly is owing to the reaction times is too short; The sticky end that exonuclease III produces is shorter, is not enough to form stable DNA open loop complex body between goal gene and linear carrier; And the reaction times is long; The sticky end that produces is oversize; Though can form stable DNA open loop complex body between goal gene and linear carrier, breach that produces between the two or suspension sequence have surpassed the repair coverage of intestinal bacteria repair systems, and cause the positive colony number to reduce.Reaction system is at 4 ℃; Reaction 60min can both produce on the resistance culture plate greater than 300 positive colony numbers when exonuclease III was 10～100units, when wherein excision enzyme III is 20units, can on flat board, produce above 2000 positive colony bacterium colonies (Fig. 8 and 9); Therefore; Optimum reaction condition is in the inventive method: in the 10uL reaction system, contain the 10ng goal gene, the linear carrier of 25ng, 20units exonuclease III; 1 μ L10X exonuclease III reaction buffer, this reaction system transformed into escherichia coli behind 4 ℃ of reaction 60min can grow on the resistance culture plate and surpass 2000 positive colony bacterium colony.
13) picking part mono-clonal bacterium colony at random from each flat board that step 12) obtains; Extract plasmid on a small quantity by ordinary method the successful cloning efficiency of goal gene hPRAK is identified, the result shows that it is 100% (Figure 10) that goal gene hPRAK is cloned into the success ratio of carrier pBOB.
Embodiment 2
Basic step is identical with embodiment 1, and difference is:
1) the insertion site of goal gene in carrier pBOB is SmaI, and the linear carrier two ends that produced by Restriction enzyme Sma I in the step 1 are flat terminal;
2) by the long respectively 35bp of two primers of the pcr amplification hPRAK of step 3 design, 5 ' end contains the homologous sequence (representing with capitalization) of 15bp carrier pBOB, and 3 ' holds and contain 20bp goal gene hPRAK homologous sequence (indicating with lowercase).
HPRAK forward direction primer sequence: 5 ' TCC AAT ATT CCC GGG atgtcggaggagagcgacat-3 ';
(capitalization is the pBOB carrier S maI upper reaches 5 ' end homologous sequences, and lowercase is a target gene 5 ' end forward direction amplimer)
HPRAK reverse primer sequence: 5 ' TGG CTC GAG CCC GGG ttattgggattcgtgggacg-3 '.
(capitalization is pBOB carrier S maI downstream 3 ' end homologous sequences, and lowercase is goal gene downstream reverse primers)
Basic step is identical with embodiment 1, and difference is: step 3 designed primer length can change, and the length of primer 5 ' end carrier homologous sequence can be between 8～20bp.
Embodiment 4
Basic step is identical with embodiment 1, and difference is: can be changed by step 3 designed primer length, primer 3 ' end goal gene homologous sequence length is between 15～40bp.
Basic step is identical with embodiment 1, and difference is: the temperature of reaction in the step 7,9,12 all can use ice bath to replace for 4 ℃.
Embodiment 6
Basic step is identical with embodiment 1, and difference is: also can from reaction system, get 6～10 μ L reaction solutions in the step 13 and transform.
Embodiment 7
Owing to all have the DNA repair system in all Bacillus coli cells; The general character of this repair system is can repair the breach in the double-stranded DNA or excise the outstanding sequence in the double-stranded DNA; Therefore all Bacillus coli cells all can be used as the host cell of open-circle DNA complex body, and the divided ring dna complex is repaired and formed the closed loop plasmid.Therefore basic step is identical with embodiment 1 in the present embodiment, and difference is: the competent escherichia coli cell that transforms in the step 13 can be the used any E.coli competent cells of genetically engineered operation such as E.coli M15 competent cell, E.coli Top10F ' competent cell, E.coli XL-blue competent cell, E.coli JM109 competent cell, E.coliHB101 competent cell, E.coli BL21.
Embodiment 8
Basic step is identical with embodiment 1; Difference is: step 1 used carrier can be any carrier that uses in the genetically engineered dna clone, like: pUC series, pGEX is serial, pcDNA is serial, pBKS is serial, pCMV is serial, pLV is serial, pET is serial or the like.Owing to having from double-stranded DNA 5 ' protruding terminus or flat terminal edge 3 ' → 5 ' direction, exonuclease III progressively cuts the 5 prime excision enzyme activity that mononucleotide produces long sticky end; And each carrier that is used for dna clone in the genetically engineered all contain can the effect of being limited property restriction endonuclease and produce 5 ' protruding terminus or flat terminal site; The linear dna vector that these sites produce after by suitable digestion with restriction enzyme can be as the effect substrate of the inventive method amplifying nucleic acid excision enzyme III, so used carrier can be any carrier (Figure 11) of dna clone in the genetically engineered in the inventive method step 1.
Embodiment 9
Basic step is identical with embodiment 1, and difference is: goal gene can be any one section dna fragmentation that is produced by pcr amplification in the step 3,4,5.Because the pcr amplification primer can add the non-specific dna sequence dna of certain-length and not influence the specificity that target DNA increases at 5 ' end; Insert site upstream and downstream homologous sequence so when the amplimer of design and synthetic target DNA fragment, can add carrier at primer 5 ' end easily; The homologous sequence that a bit of carrier inserts site is just respectively contained at the target DNA fragment two ends of therefore being come out by this primer amplification; Simultaneously the dna fragmentation that produces of pcr amplification is generally the double-stranded DNA that contains flat terminal or 5 ' protruding terminus, and the double-stranded DNA that contains these two kinds of ends just in time all is the effect substrate of exonuclease III.Therefore any can all can carrying out design of primers, synthesize and pcr amplification (Figure 12) by the step 3,4,5 of the inventive method by the dna fragmentation that pcr amplification obtains.The purpose D N A fragment two ends that amplification obtains are under the effect of exonuclease III; Generation can be inserted 5 ' long sticky end of site sequence complementary pairing with carrier; This sticky end with form the open-circle DNA complex body that contains breach or suspension sequence through the linear carrier complementary pairing of suitable digestion with restriction enzyme, can in Bacillus coli cells, repair behind this open loop complex body transformed into escherichia coli and be the closed loop plasmid and duplicate (Figure 13).
Claims (10)
1. dna clone method is characterized in that may further comprise the steps:
1) inserts site at carrier and use the digestion with restriction enzyme carrier, obtain linear carrier DNA;
2) insert the synthetic pcr amplification primer of site sequences Design according to target DNA fragment and carrier;
3) carry out pcr amplification with institute's synthetic pcr amplification primer by ordinary method, obtain target DNA fragment;
4) the method difference purifying that passes through the recovery of electrophoresis and glue is by the linear carrier DNA of step 1) acquisition and the target DNA fragment of step 3) acquisition;
5) target DNA fragment and the mixing of linear carrier DNA that the step 4) purifying obtains be will pass through, and exonuclease III and exonuclease III reaction buffer, anabolic reaction system added;
6) reaction system with step 5) continues reaction;
7) in the reaction system of step 6), add EDTA Disodium with termination reaction;
8) reaction system with step 7) places 60 ℃ of reaction 5～10min;
9) reaction system of step 8) is annealed;
10) from the reaction system of step 9), get 5～10 μ L transformed into escherichia coli E.coli competent cells;
11) the transformed bacteria liquid coating with step 10) contains corresponding antibiotic LB culture medium flat plate, cultivates 10～15h in 37 ℃;
12) picking mono-clonal bacterium colony from the culture plate of step 11) extracts plasmid by ordinary method and identifies, to confirm the successful clone of target DNA fragment.
2. a kind of dna clone method as claimed in claim 1 is characterized in that in step 1), saidly inserts site at carrier and use the digestion with restriction enzyme carrier, is meant that carrier produces 5 ' protruding terminus down or puts down end in corresponding restriction enzyme effect.
3. a kind of dna clone method as claimed in claim 1; It is characterized in that in step 1) any carrier that said carrier is selected from pUC series, pBOB series, pcDNA is serial, pBKS is serial, pCMV is serial, dna clone uses in the pLV serial genes engineering.
4. a kind of dna clone method as claimed in claim 1; It is characterized in that in step 2) in; The synthetic pcr amplification primer of said design is made up of carrier homologous sequence and target DNA fragment homologous sequence two portions: 1. primer 5 ' end contains the homologous sequence that the plan of having an appointment about 8～20bp is inserted carrier, and this sequence can produce the long sticky end that inserts site sequence complementary pairing with carrier under the effect of exonuclease III; 2. primer 3 ' end then is the primer of 15～40bp target DNA fragment to be amplified, is used for and the target DNA fragment complementary pairing amplification target DNA fragment.
5. a kind of dna clone method as claimed in claim 1 is characterized in that in step 5) said target DNA fragment is 10～100ng, and the content of said linear carrier DNA is 10～100ng; Molecule ratio between said target DNA fragment and the linear carrier DNA is (1～4): 1; The enzyme of said exonuclease III concentration alive is 10～100units.
6. a kind of dna clone method as claimed in claim 5, the content that it is characterized in that said linear carrier DNA is 25ng; Molecule ratio between said target DNA fragment and the linear carrier DNA is 2: 1; The enzyme of said exonuclease III concentration alive is 20units.
7. a kind of dna clone method as claimed in claim 1 is characterized in that in step 6) the condition of said reaction is: temperature is ice bath or 4 ℃, and the time is 10～240min; The condition of optimum response is 4 ℃ of reaction 1h.
8. a kind of dna clone method as claimed in claim 1 is characterized in that in step 7), and the add-on of said EDTA is 1 μ L, 0.5mol/L, pH8.0.
9. a kind of dna clone method as claimed in claim 1 is characterized in that in step 9), and said annealed temperature is ice bath or 4 ℃, and the annealed time is 5min.
10. a kind of dna clone method as claimed in claim 1; It is characterized in that in step 10) said transformed into escherichia coli competent cell is selected from E.coli DH5 α competent cell, E.coli M15 competent cell, E.coli Top10F ' competent cell, E.coli XL-blue competent cell, E.coli JM109 competent cell, E.coli HB 101 competent cells or the used E.coli competent cell of other genetically engineered operation.
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| CN111349638A (en) * | 2020-03-17 | 2020-06-30 | 深圳市泽龙生物技术有限公司 | Method for constructing vector containing large-fragment reverse complementary sequence |
| CN112877324A (en) * | 2021-01-28 | 2021-06-01 | 杭州师范大学 | DNA cloning method |
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| CN104774861A (en) * | 2015-03-10 | 2015-07-15 | 江苏康为世纪生物科技有限公司 | DNA recombination method using blunt end ligation for efficient screening of positive transformant |
| CN107760706A (en) * | 2017-10-11 | 2018-03-06 | 吴江近岸蛋白质科技有限公司 | The application of DNA excision enzymes and the method for seamless clone |
| CN111349638A (en) * | 2020-03-17 | 2020-06-30 | 深圳市泽龙生物技术有限公司 | Method for constructing vector containing large-fragment reverse complementary sequence |
| CN112877324A (en) * | 2021-01-28 | 2021-06-01 | 杭州师范大学 | DNA cloning method |
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