CN101016551B - Method of introducing a plurality of DNA fragments simultaneously into DNA vector - Google Patents
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Abstract
The invention discloses a method to introducing multiple DNA fragment in DNA carrier, which comprises the following steps: augmenting two or more goal DNA fragment with oligonucleotide isogeneic limbthrough polyose chain-reaction; transforming to permissive state cell with recombinant enzyme vector and armed reforming carrier; getting the reforming carrier with one step through consangnuinity reconstruction of oligonucleotide; making the first fragment 5' end of goal DNA fragment possesses the same sequence with armed replacing position left side of target carrier; setting the last 3' end possesses the same sequence with armed replacing position left side of target carrier; making each fragment 5' end possesses the reversing complementary sequence with the last fragment 3' end from the second fragment. The method of this invention is simple, which can be a general method to reform DNA carrier.
Description
Technical field
The present invention relates to the genetically engineered field, specifically relate to a kind of method of in dna vector, introducing a plurality of dna fragmentations simultaneously.
Background technology:
Existing dna vector is transformed, as the sequence that changes multiple clone site, introduce one or more exogenous dna fragments, be one of the most basic operation of molecular biology with external fragment displacement original vector part (as changing the formation of resistant gene) etc.
The basic step of classical dna vector remodeling method is: (1) with original vector and wait to introduce fragment with produce identical cohesive end or generation can complementary cohesive end or the restriction enzyme that produces blunt ends simultaneously handle; (2) two or more purpose fragments are carried out agarose gel electrophoresis, after the purpose fragment is cut down, DNA is reclaimed; (3) two or more purpose fragments connect under the effect of T4 ligase enzyme; (4) connect the electricity conversion of product transformed into escherichia coli or the competent cell of chemical conversion, transform solution coat and containing accordingly on the antibiotic flat board, incubation is cultivated; (5) the some bacterium colonies that grow of picking are to containing in the antibiotic liquid nutrient medium shaking culture; (6) centrifugal collection thalline, plasmid extract, and carry out enzyme with restriction enzyme and cut, and agarose gel electrophoresis judges whether to obtain the whether success of transformation that recombinant clone is a dna vector according to electrophoresis result.
In this classical experimentation, seek and on carrier, to carry out suitable cutting can not destroy the segmental restriction enzyme of external source simultaneously again be an individual critical step, yet this is usually complicated, consuming time even be difficult to carry out.The sudden change that the enzyme of dna fragmentation restriction enzyme is cut in the experiment, glue recovery etc. all might be introduced base.Especially when needs are introduced an above fragment, be multiplied all corresponding of the time that is spent with the base mutation that may introduce.Therefore, above-mentioned classical way is loaded down with trivial details, consuming time, efficient is low, limiting factor is many.
For seek a kind of easy, fast, dna vector remodeling method efficiently, especially simultaneously two purpose fragments are introduced simultaneously the method for original vector.The present invention has utilized the recombinant technology that adopts between a kind of oligonucleotide in intestinal bacteria to realize this purpose.Its action principle is based on two genes: the red α of lambda particles phage and red β gene.Red α is 5 ' of DNA chain-＞a 3 ' exonuclease, and it acts on double-stranded DNA and produces the dna molecular of 3 ' strand overhang; Red β is a single strand binding protein, is combined in 3 outstanding ' end of DNA and the right DNA digestion of prevention single stranded DNA nuclease, and red β shows recombinase active simultaneously, promptly impels the annealing between the strand homologous dna.Recombination system also comprises gam gene and recA gene, and the former suppresses the exonuclease activity of the RecBCD of host bacterium, stops its degraded to external source linear dsdna molecule, and the latter then can improve the activity of recombinase.
Summary of the invention
The purpose of this invention is to provide two above fragments of a kind of introducing and replaced the method for directly dna vector being transformed of original vector top sequence simultaneously.
The said method of in dna vector, introducing a plurality of dna fragmentations simultaneously, the steps include: target DNA fragment by two or more band oligonucleotide homology arms of polymerase chain reaction (PCR) amplification, be converted into the competent cell that contains recombinase carrier and carrier to be transformed simultaneously, the direct improved carrier of acquisition of one step by the homologous recombination of oligonucleotide; The target DNA fragment of said two or more band oligonucleotide homology arms, wherein first segmental 5 ' end has with targeting vector and treats the identical sequence in alternative site left side, last 3 ' end has with targeting vector and treats the sequence that the alternative site right side is identical, from each segmental 5 ' end of second fragment sequence with a last fragment 3 ' end reverse complemental is arranged.
When in dna vector, introducing two dna fragmentations simultaneously, the steps include: target DNA fragment by two bands of polymerase chain reaction (PCR) amplification oligonucleotide homology arm, be converted into the competent cell that contains recombinase carrier and carrier to be transformed simultaneously, the direct improved carrier of acquisition of one step by the homologous recombination of oligonucleotide; The target DNA fragment of said two band oligonucleotide homology arms, wherein first segmental 5 ' end has with targeting vector and treats the identical sequence in alternative site left side, second segmental 5 ' end has the sequence with first fragment 3 ' end reverse complemental, 3 ' hold to have and treats the sequence that the alternative site right side is identical with targeting vector.
The target DNA fragments of said two band oligonucleotide homology arms in the aforesaid method, wherein the homology arm with the exchange of carrier homology is 50 bases, the homology arm between two fragments is 30 bases.
Obtain the target DNA fragment of said two band oligonucleotide homology arms among the present invention, can design primer according to following principle:
Primer 1: about 70 bases, preceding 50 is to treat alternative site left side identical sequence with targeting vector, back about 20 for amplification first waits to introduce segmental 5 ' terminal sequence.Like this, primer 1 promptly contains and identical 50 bases " homology arm " in carrier left side.
Primer 2: 30 bases, first waits to introduce segmental 3 ' terminal sequence in order to increase;
By primer 1 and primer 2 first fragment to be introduced that increases.
Primer 3: about 50 bases, preceding 30 is the reverse complementary sequence of primer 2, back about 20 is that second of amplification waits to introduce segmental 5 ' terminal sequence.Like this, primer 3 promptly has " homology arm " of 30 bases with primer 2.
Primer 4: about 70 bases, preceding 50 is the reverse complementary sequence that targeting vector is treated the alternative site right direction, back about 20 is that second of amplification waits to introduce segmental 3 ' terminal sequence.Like this, primer 4 promptly contains 50 bases " homology arm " identical with the carrier right side.
By primer 3 and second fragment to be introduced of primer 4 amplifications.
When the fragment that will introduce is two when above, those skilled in the art can design primer with reference to aforesaid method equally.
Said in the present invention targeting vector treats that the base number of alternative site can be zero, when the base number is zero, is actually and does not remove any fragment of targeting vector, and directly insert the purpose fragment.
Known recombinase carrier may be used to invent described method, preferred recombinase plasmid pSC101-gba-tet in the concrete scheme of the present invention.
Above-mentioned said carrier to be transformed includes but not limited to pBluescript KS (-), and other dna vector can be transformed with method of the present invention equally.
An example of the present invention is: with oriT-Am box gene and rpsL gene clone to pBluescript KS (-), the ampicillin resistance gene of removing pBluescript KS (-) simultaneously partly is concrete content.PBluescript KS (-) is one of the most frequently used gene clone carrier.In order to be structured in the gene disruption carrier that uses in the actinomycetes, need oriT-Am box gene and rpsL gene clone to pBluescript KS (-), the oriT fragment is responsible for conjugal transfer function between the DNA of bacteria, the Am gene is the A Pu mycin resistant gene, be selected marker commonly used in the actinomycetes genetic manipulation, the rpsL gene is the ribosomal S12 albumen of 30S (gene in streptomyces coelicolor Streptomyces coelicolor A3 (2) genome is numbered SCO4659), is streptomycin resistance gene.Use method of the present invention can one the step be cloned into pBluescriptKS (-) with these two sections, time from about ten days required time of two segmental substeps clones (suppose experimental design talk about smoothly) foreshortens at three days and finishes with interior.Need seven days if the designed Oligonucleolide primers of the present invention is synthetic in the calculating, and conventional short primer needs three days, the time of the present invention has reduced four days than classical way.
Therefore the present invention has avoided the restriction of restriction enzyme, and gel recovery etc. may be introduced the step of base mutation.Easy to be quick, recombination efficiency is high.
Description of drawings
Fig. 1 is a homologous recombination synoptic diagram of the present invention.
Fig. 2 is the plasmid map of pLS205, wherein:
21-327: strand forms district (fl (-) ori);
460-816::LacZ (beta galactosidase enzyme α fragment);
1851-2658: conjugal transfer fragment (oriT);
2862-3638: the open reading frame of A Pu mycin (Am) resistant gene;
3879-4250: the open reading frame of Streptomycin sulphate (rpsL) resistant gene;
1158-1825: the duplicate field of carrier (pUCori);
SacI (652)-KpnI (754): multiple clone site (MCS);
SacI, NotI, XbaI, SpeI, BamHI, EcoRI, EcoRV, HindIII, ClaI, XhoI, KpnI: be restriction endonuclease sites.
Fig. 3 is the agarose gel electrophoresis result that the pLS205 enzyme is cut
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
Used plasmid among the embodiment is openly carrier:
pBluescript?KS(-)(Alting-Mees，M.A.and?Short，J.M.(1989)pBluescript?II：gene?mappingvectors。Nucleic Acids Res.17 (22), 9494.) purchase company in Novagen.
POJ446 (Bierman, M., R.Logan, et al. (1992). " Plasmid cloning vectors for the conjugaltransfer of DNA from Escherichia coli to Streptomyces spp. " Gene 116 (1): 43-9.), used pOJ446 is from Britain John Innes Institute in the present embodiment, and David Hopwood teaches.
PSC101-gba-tet (Zhang, Y., F.Buchholz, et al. (1998). " A new logic for DNA engineeringusing recombination in Escherichia coli. " Nat Genet 20 (2): 123-8.); Used pSC101-gba-tet is from German Dresden University, Youming doctor Zhan in the present embodiment.
The rpsL gene is from streptomyces coelicolor Streptomyces coelicolor A3 (2), be type strain, gene order-checking document Bentley, S.D., K.F.Chater, et al. (2002). " Complete genome sequence ofthe modelactinomycete Streptomyces coelicolor A3 (2). " Nature 417 (6885): 141-7.Used Streptomyces coelicolor A3 (2) is from Britain John Innes Institute in the present embodiment, and David Hopwood teaches.
Embodiment 1: oriT-Am box gene and rpsL gene clone to pBluescript KS (-), are removed the ampicillin resistance gene part of pBluescript KS (-) simultaneously
The oriT-Am box gene of amplified band homology arm
Primer CT1:5 '-
AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA
CTGCAGGTCCCCGGGGATCGGTC-3′
50 bases in front are 1801 to 1850 parts of pBluescript KS (-), and about 23 bases in back are 5 ' terminal sequence of 2517-2540 amplification oriT-Am box gene among the plasmid pOJ446;
Primer CT2:5 '-TCAGCCAATCGACTGGCGAGCGGCATCGCA-3 '
3 ' sequence of the 4255-4284 amplification oriT-Am box gene that 30 bases are pOJ446;
With pOJ446 is template, and a pair of primer of CT1 and CT2 amplifies the oriT-Am box gene of the band homology arm of 2.0kb by PCR (polymerase chain reaction).
The rpsL gene of amplified band homology arm
Primer CT3:
5′-TGCGATGCCGCTCGCCAGTCGATTGGCTGACGGTGTCGCCCATTTGTTTTG-3′
The reverse complementary sequence that 30 bases in front are CT2,21 bases in back are amplification rpsL gene 5 ' terminal sequence;
Primer CT4:
5′-TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCT
GCCCTTACGAGGCATTCTTAC-3′
The reverse complementary sequence that 50 bases in front are 2911-2960 of pBluescript KS (-), about 21 bases in back are amplification rpsL gene 3 ' terminal sequence.
Genomic dna with streptomyces coelicolor Streptomyces coelicolor A3 (2) is a masterplate, and a pair of primer of CT3 and CT4 amplifies the rpsL fragment of the band homology arm of 0.6kb by PCR (polymerase chain reaction).
More than 50 μ l PCR reaction systems:
33μl?dH
2O
5 μ l 10xPCR reaction buffers
1.25μl?10mM?dNTP
1.5 μ l upstream primer (final concentration 0.75 μ M)
1.5 μ l downstream primer (final concentration 0.75 μ M)
2 μ l templates (plasmid～100ng, genomic dna～200ng)
0.5 μ l pfu polysaccharase (5U/ μ l)
The PCR reaction conditions: 95 ℃ of sex change 5 minutes, (95 ℃ 45 seconds, 60 ℃ 1 minute, 72 ℃ 3 minutes), totally 30 circulations, 72 ℃ were extended 10 minutes.Use the PCR instrument of instrument as the PTC-200 model of MJ researcher company.
After reaction finishes, detect with 0.7% agarose gel electrophoresis of ethidium bromide (EB) dyestuff that adds 20mg/ml.After identifying correctly, the dehydrated alcohol of 4 ℃ of precoolings of the sodium acetate soln of the pH5.2 of 1/10 times of volume of adding and 2.5 times of volumes in solution, the mixing precipitation, the centrifugal supernatant that goes, after the drying at room temperature, be dissolved in the Tris.Cl (Tutofusin tris is regulated pH to 8.0 with concentrated hydrochloric acid) of 10mM pH8.0, OD260 measures DNA concentration, and regulating concentration is 300ng/ μ l.
Contain the preparation of electric transformed competence colibacillus of the intestinal bacteria DH10B of recombinase plasmid pSC101-gba-tet:
To line on the LB solid plate that contains 10 μ g/ml tsiklomitsins at-70 ℃ of frozen DH10B/pSC101-gba-tet, cultivate more than 20 hours for 30 ℃.Picking list bacterium colony is to the LB liquid nutrient medium of 10ml 10 μ g/ml tsiklomitsins, cultivate in 30 ℃ of overnight shakings, 12,000rpm, 4 ℃ centrifugal 3 minutes, abandon supernatant, the glycerine washing precipitation twice with 10% is suspended in 10% the glycerine of 100 μ l at last.
The conversion of pBluescript KS (-):
Will about 100ng pBluescript KS (-) add in the electric transformed competence colibacillus cell of the DH10B/pSC101-gba-tet that 50 μ l melt on ice, flick mixing.The 1mm electricity that mixed solution is transferred to precooling on ice changes in the pond, and electric shock transforms.Electricity conversion condition: 1mm electricity changes pond, 200 Ω, 1300V, the Gene Pulser II of Bio-Rad company
RThe electricity conversion instrument.Add 1ml LB liquid nutrient medium to electricity and change the pond, the piping and druming mixing, solution is transferred in the aseptic 1.5ml eppendorf pipe, 37 ℃ of shaking culture are after 60 minutes, conversion fluid is coated with and contains on the LB resistant panel of 100 μ g/ml penbritins and 10 μ g/ml tsiklomitsins, cultivates more than 20 hours for 30 ℃.The bacterial strain that obtains is DH10B/pSC101-gba-tet+pBluescript KS (-).
The preparation of the electric transformed competence colibacillus cell of DH10B/pSC101-gba-tet+pBluescript KS (-):
To line the LB resistant panel that contains 100 μ g/ml penbritins and 10 μ g/ml tsiklomitsins at-70 ℃ of frozen bacterial strains, cultivate more than 20 hours for 30 ℃.Choose single bacterium colony and contain the LB liquid nutrient medium of 100 μ g/ml penbritins and 10 μ ug/ml tsiklomitsins to 2ml, cultivate in 30 ℃ of shaken overnight, the volume by 1/50 is forwarded to the same substratum of 20ml, 30 ℃ of shaking culture, to OD260 be 0.2 o'clock, add final concentration and be 0.15% L-arabinose, 37 ℃ of shaking culture to OD260 be 0.4 o'clock, 12,000rpm, 4 ℃ centrifugal 3 minutes, abandon supernatant, glycerine washing precipitation twice with 10% finally is suspended in the glycerine of 200 μ l 10%.
Two segmental conversions:
The oriT-Am box gene dna fragmentation of 0.3 μ g 2.0kb and 0.3 μ g 0.6kb rpsL dna fragmentation are added in the electric transformed competence colibacillus cell that 50 μ lDH10B/pSC101-gba-tet+pBluescript KS (-) melt on ice, flick mixing.The 1mm electricity that mixed solution is transferred to precooling on ice changes in the pond, and electric shock transforms.Electricity conversion condition: 1mm electricity changes pond, 200 Ω, 1300V, the Gene Pulser II of Bio-Rad company
RThe electricity conversion instrument.Adding 1ml LB liquid nutrient medium to electricity changes the pond, and the piping and druming mixing is transferred to solution in the aseptic 1.5ml eppendorf pipe, and 37 ℃ of shaking culture were coated in the LB substratum that contains 50 μ g/ml A Pu mycins 37 ℃ of incubated overnight after 70 minutes.Its homology exchange synoptic diagram as shown in Figure 1.
On flat board, grow 100 bacterium colonies approximately, provoking 12 at random is seeded in the liquid LB substratum that contains 50 μ g/ml A Pu mycins, 37 ℃ of shaking culture are spent the night, by the alkaline lysis method of extracting plasmid, restriction enzyme digestion is identified, proves that 11 is correct clone, and clone's accuracy is 92%, with correct clone's called after pLS205, its structure such as Fig. 2 show.The agarose gel electrophoresis that enzyme is cut the results are shown in Figure 3 (runic be single endonuclease digestion site).
Claims (4)
1. method of in dna vector, introducing a plurality of dna fragmentations simultaneously, the steps include: target DNA fragment by two or more band oligonucleotide homology arms of polymerase chain reaction (PCR) amplification, be converted into the competent cell of recombinase carrier pSC101-gba-tet and carrier to be transformed simultaneously, the direct improved carrier of acquisition of one step by the homologous recombination of oligonucleotide; The target DNA fragment of said two or more band oligonucleotide homology arms, wherein first segmental 5 ' end has the homology arm for the treatment of the homology exchange of alternative site left side with targeting vector, last 3 ' end has the homology arm for the treatment of homology exchange in alternative site right side with targeting vector, from each segmental 5 ' end of second fragment sequence with a last fragment 3 ' end reverse complemental is arranged; Wherein the homology arm with carrier homology exchange is 50 bases, and the homology arm between two fragments is 30 bases.
2. as the said method of claim 1, it is characterized in that: said target DNA fragment is two, and wherein first fragment is obtained by primer 1 and primer 2 amplification, and its 5 ' end has the homology arm for the treatment of the homology exchange of alternative site left side with targeting vector; Second fragment obtained by primer 3 and primer 4 amplifications, and its 5 ' end has the sequence with first fragment 3 ' end reverse complemental, and 3 ' end has the homology arm for the treatment of the homology exchange of alternative site right side with targeting vector; Wherein
A primer 1:70 base, preceding 50 is to treat alternative site left side identical sequence with targeting vector, back about 20 for amplification first waits to introduce segmental 5 ' terminal sequence;
Primer 2: 30 bases, first waits to introduce segmental 3 ' terminal sequence in order to increase;
A primer 3:50 base, preceding 30 is the reverse complementary sequence of primer 2, back about 20 is that second of amplification waits to introduce segmental 5 ' terminal sequence;
A primer 4:70 base, preceding 50 is the reverse complementary sequence that targeting vector is treated the alternative site right direction, back about 20 is that second of amplification waits to introduce segmental 3 ' terminal sequence.
3. as the said method of claim 2, it is characterized in that, said target DNA is oriT-Am box gene and rpsL gene, and said targeting vector is pBluescript KS (-), and the said alternative site for the treatment of is the ampicillin resistance gene part of pBluescript KS (-).
4. as the said method of claim 3, it is characterized in that the primer of amplification oriT-Am box gene:
Primer CT1:5 '-AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAACTGCAG GTCCCCGGGGATCGGTC-3 ',
Primer CT2:5 ' TCAGCCAATCGACTGGCGAGCGGCATCGCA-3 ';
The primer of amplification rpsL gene:
Primer CT3:5 '-TGCGATGCCGCTCGCCAGTCGATTGGCTGACGGTGTCGCCCATTTGTTTTG-3 ',
Primer CT4:5 '-TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTGCCCTT ACGAGGCATTCTTAC-3 '.
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| CN101921800B (en) * | 2010-06-08 | 2012-07-04 | 南京师范大学 | Escherichia coli protein expression vector using trigger factor as fusion tag and construction method and application thereof |
| CN102719481B (en) * | 2012-06-14 | 2014-11-05 | 中国科学院微生物研究所 | Method for simultaneously transferring multiple genes into microbial genome |
| CN103484492A (en) * | 2013-09-30 | 2014-01-01 | 湖南农业大学 | Method for simultaneously ligating multiple DNA (deoxyribonucleic acid) fragments to same carrier |
| CN105483188B (en) * | 2015-12-21 | 2019-04-23 | 生工生物工程（上海）股份有限公司 | A kind of joining method of DNA fragmentation |
| CN106636213A (en) * | 2016-12-23 | 2017-05-10 | 西安建筑科技大学 | Method for improving acquisition efficiency of electroporated positive recombinants of escherichia coli |
| CN114908111B (en) * | 2021-02-08 | 2024-02-09 | 中国科学院分子植物科学卓越创新中心 | Method and system for continuous cloning of long DNA fragments |
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