CN103160495A - Disconnected dependent rapid cloning method - Google Patents
Disconnected dependent rapid cloning method Download PDFInfo
- Publication number
- CN103160495A CN103160495A CN2011104184509A CN201110418450A CN103160495A CN 103160495 A CN103160495 A CN 103160495A CN 2011104184509 A CN2011104184509 A CN 2011104184509A CN 201110418450 A CN201110418450 A CN 201110418450A CN 103160495 A CN103160495 A CN 103160495A
- Authority
- CN
- China
- Prior art keywords
- dna
- donor
- receptor
- donor dna
- cloning
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000010367 cloning Methods 0.000 title claims abstract description 37
- 230000001419 dependent effect Effects 0.000 title description 5
- 239000012634 fragment Substances 0.000 claims abstract description 71
- 102000005962 receptors Human genes 0.000 claims abstract description 49
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 claims abstract description 16
- 230000008569 process Effects 0.000 claims abstract description 14
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 6
- 108060002716 Exonuclease Proteins 0.000 claims abstract 4
- 102000013165 exonuclease Human genes 0.000 claims abstract 4
- 108020004414 DNA Proteins 0.000 claims description 144
- 102000004190 Enzymes Human genes 0.000 claims description 41
- 108090000790 Enzymes Proteins 0.000 claims description 40
- 238000006243 chemical reaction Methods 0.000 claims description 37
- 241000894006 Bacteria Species 0.000 claims description 35
- 230000000968 intestinal effect Effects 0.000 claims description 30
- 108091008146 restriction endonucleases Proteins 0.000 claims description 25
- 238000012408 PCR amplification Methods 0.000 claims description 15
- 238000002744 homologous recombination Methods 0.000 claims description 11
- 230000006801 homologous recombination Effects 0.000 claims description 11
- 230000006798 recombination Effects 0.000 claims description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 108010055016 Rec A Recombinases Proteins 0.000 claims description 10
- 102000001218 Rec A Recombinases Human genes 0.000 claims description 10
- 238000005215 recombination Methods 0.000 claims description 10
- 239000000543 intermediate Substances 0.000 claims description 6
- 108091009282 enzyme binding proteins Proteins 0.000 claims description 5
- 241000206602 Eukaryota Species 0.000 claims description 4
- 108010052305 exodeoxyribonuclease III Proteins 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 3
- 230000000692 anti-sense effect Effects 0.000 claims description 3
- 101000669895 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) Replication factor A Proteins 0.000 claims description 2
- 101710150114 Protein rep Proteins 0.000 claims description 2
- 101710152114 Replication protein Proteins 0.000 claims description 2
- 101150104425 T4 gene Proteins 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 108010076804 DNA Restriction Enzymes Proteins 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 5
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 5
- 238000001976 enzyme digestion Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract 1
- 238000002741 site-directed mutagenesis Methods 0.000 abstract 1
- 230000001131 transforming effect Effects 0.000 abstract 1
- 108020003175 receptors Proteins 0.000 description 34
- 239000013612 plasmid Substances 0.000 description 12
- 238000013461 design Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 7
- 239000003292 glue Substances 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000005457 ice water Substances 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 238000005267 amalgamation Methods 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 230000001351 cycling effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000594182 Sarcophaga sigma Species 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000012868 site-directed mutagenesis technique Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a rapid cloning method for donor DNA fragments. The method includes: first introducing additional sequences to the 5' ends of amplification primers so as to endow both terminals of donor DNA and both terminals of receptor DNA respectively with at least 12bp homologous sequences; then linearizing the receptor DNA, finally making the donor DNA and the linearized receptor DNA form a recombinant intermediate efficiently in vitro under catalysis of exonuclease and single-strand binding protein, and transforming the intermediate directly to a competent cell, thus finishing the cloning process from the donor DNA to the receptor DNA. With no need for an enzyme digestion treatment on the donor DNA, the method provided in the invention can realize rapid directed cloning of a single donor DNA fragment, also can realize one-step splicing cloning of a plurality of donor DNA fragments, and can be used for DNA site-directed mutagenesis. The method can provide a rapid and efficient operation platform for research in genetics, molecular biology, biochemistry and the like as well as high-throughput cloning.
Description
Technical field
The present invention relates to the genetically engineered field, specifically relate to a kind of method of quick clone.
Background technology
The foundation of the dependent DNA directed cloning of ligase enzyme technology is landmark achievement in the field of biology in last century.The foundation of this technology and application make people can revise within the specific limits the genetic recombination matter DNA, thereby provide possibility for exploring gene concrete function in vivo.Along with the development of biological development, particularly round pcr and the foundation of DNA site-directed mutagenesis technique, the DNA directed cloning has now become the basis of whole biology field.The DNA directed cloning refers to the donor dna fragment is cloned into specific position on receptor dna, such as multiple clone site.Traditional DNA directed cloning comprises following steps usually:
1) the primer amplification donor dna fragment of using 5 ' end to add the specific limited restriction enzyme site;
2) be connected in the T carrier after donor dna fragment amplification product purification;
3) use pre-designed restriction enzyme site with target fragment from the T carrier enzyme cutting from out and carry out purifying;
4) use with the same restriction enzyme site of step 3) the receptor dna linearizing, and carry out purifying;
5) use step 3) and 4) middle the purifying donor dna and the receptor dna that obtain carry out ligation by a certain percentage;
6) with step 5) in the connection product be converted in competent cell;
7) recombinant DNA of exact connect ion is identified, extracted to mono-clonal.
This shows, traditional DNA directed cloning is a job that takes time and effort very much, and cloning efficiency is poor, and success ratio is low.In addition, owing to usually comprising multiple restriction enzyme site in the donor dna fragment, therefore can choose the biggest obstacle that the suitable restriction enzyme that is used for the clone becomes this technical development of restriction.In today of biology develop rapidly, be accompanied by the very big development of DNA sequencing technology and synthetic biology, inefficient traditional cloning process can't satisfy and becomed more diverse and complicated clone's demand, especially can't satisfy the high-flux clone demand.
The novel cloning process based on the restructuring principle can effectively be eliminated restriction enzyme site to the restriction of Strategies For The Cloning, has many potential advantages such as suitability is wide, flux is high.At present existing several recombinant clone systems that can satisfy the high-flux clone demand come out one after another: universal support plasmid emerging system UPS, GateWay entry clones system and MAGIC cloning system.Wherein, universal support plasmid emerging system UPS, GateWay entry clones system is based on the exploitation of external locus specificity restructuring principle, and the MAGIC cloning system is based on homologous recombination and bacterium Hybridization principle in body and develop.Above three cloning systems can be completed seamless shuttle back and forth of any DNA fragment under different background, and operating process has versatility, can satisfy to a certain extent variation, high-throughout clone's demand.Yet regrettably, these disconnected dependent cloning systems all exist some self unavoidable defective.At first, compare with traditional cloning process, the other two kinds of methods except the Gateway cloning system all do not have clear superiority to initial gene clone.And Gateway clone need to complete initial gene clone with extremely expensive enzyme, and need to add the sequence that comprises recombination site of specific a section very long during design of primers.Secondly, these cloning systems require initial gene clone to carry out on fixing carrier, specify in carrier if goal gene need to be cloned into, and these systems are out of use.The 3rd, these cloning systems can only be allowed donor dna fragment of a time cloning, are not suitable for the disposable amalgamation clone of complicated many donors fragment.
Due to above-mentioned many disadvantages, these three kinds of locus specificity recombinant clone systems can't be widely used.Homologous recombination is the another kind of DNA recombinant forms that occurs in vivo.The locus specificity of comparing, the dependent restructuring of homology has great advantage.At first, the DNA restructuring that homology relies on does not need specific DNA sequence dna, only need donor dna and receptor dna to have the homologous sequence that can mate fully of certain-length to get final product at recombination site, and the locus specificity restructuring need specific DNA sequence dna to realize.Therefore, if replace the locus specificity restructuring to carry out community's DNA clone with homologous recombination, can eliminate its initial gene clone and can only carry out this restriction to specifying on carrier, make the donor dna clone have higher handiness and selectivity.Secondly, based on the Strategies For The Cloning of homologous recombination, allow the amalgamation clone who a plurality of donor fragments is carried out a step, namely complete simultaneously the connection amalgamation of a plurality of donor fragments and be cloned into receptor dna.This advantage provides a kind of solution of single step for the gene of complexity or carrier transformation.
This shows with the locus specificity restructuring and compare, the homologous recombination cloning system has unrivaled advantage.The In-FusionTM PCR clone test kit of Clontech just is based on the exploitation of homologous recombination principle and forms.Use this test kit, but donor dna PCR product Direct Cloning need not the loaded down with trivial details steps such as enzyme is cut, connection, end-filling to any receptor dna.When using this system, need to introduce at least 15 base homologous sequences at 5 ' end of PCR primer.These 15 bases must be respectively can be mated fully with 5 ' and 3 ' end of linearizing destination carrier.After the PCR reaction finished, the donor dna PCR product that purifying is crossed and linearizing receptor dna reacted 30 minutes under the catalysis of In-FusionTM recombinase, and the homologous sequence of end can merge.Reaction product directly is converted into the intestinal bacteria competence can complete clone's process.The single step clone that In-FusionTM PCR cloning system has really been realized the donor dna fragment has simplified clone's process greatly, and has significantly improved to a certain extent clone's success ratio.Yet, due to this recombinase is difficult to obtain, the cost costliness and character unstable, this system also fails to be promoted widely at present
Technology contents
For the problems referred to above, the invention provides a kind of method that realizes the directed restructuring of external DNA by DNA excision enzyme and single strand binding protein.At first design donor dna fragments specific amplimer, add respectively 12bp at least and sequence receptor dna end homology at its forward and reverse primer 5 ' end, can also add for the linearizing restriction enzyme site of donor dna if need.Use this primer pair amplification donor dna fragment, amplified production is reclaimed test kit by glue or the ethanol precipitation is carried out the purifying recovery.Secondly, receptor dna is used suitable restriction enzyme carry out linearizing and purifying recovery.At last, donor dna and linearizing receptor dna fragment can be at external efficient formation recombination intermediates under the co-catalysis of DNA excision enzyme and single strand binding protein.The direct transformed competence colibacillus cell of this recombination intermediate can be completed clone's process (seeing accompanying drawing 1).This inventive method can be used for realizing the directed cloning of single donor dna fragment, also can realize the one-step cloning of a plurality of target dna fragments, completes the seamless spliced of DNA, can also be used as the DNA rite-directed mutagenesis.The pcr amplification primer principle of design that the present invention preferably is used for single donor dna directed cloning is: forward primer: 5 '-15bp receptor dna, 5 ' end homologous sequence+restriction enzyme site+donor dna specificity forward amplimer-3 '; Reverse primer: 5 '-15bp receptor dna, 3 ' end homologous sequence+restriction enzyme site+reverse amplimer-3 ' of donor dna specificity.Described 15bp receptor dna 5 ' end homologous sequence refers to receptor dna 5 ' the last 15bp DNA sequence dna of end antisense strand end, and direction is 5 ' to 3 '; Described 15bp receptor dna 3 ' end homologous sequence refers to the proper last 15bp DNA sequence dna of adopted chain end of acceptor 3 ', and direction is 5 ' to 3 '.The length of described homologous sequence is 12bp at least, and its length is 15bp in a preference of the present invention.Described restriction enzyme site is total length or the part of receptor dna linearizing restriction enzyme digestion sites used, and direction is 5 ' to 3 '.Described donor dna specificity just (instead) refers to donor dna PCR amplimer to amplimer, and direction is 5 ' to 3 ' (seeing accompanying drawing 2).
The pcr amplification primer principle of design that the present invention preferably is used for the directed splicing clone of many donor dnas is (take two donor fragment assembly clones as example, set the splicing order and be respectively by 5 ' to 3 ' direction: donor Segment A, donor fragment B):
Donor A forward primer: 5 '-15bp receptor dna, 5 ' end homologous sequence+restriction enzyme site+donor A fragments specific forward amplimer-3 '; Donor A reverse primer: the reverse amplimer-3 ' of 5 '-donor A fragments specific; Donor B forward primer: 5 '-15bp donor A fragment, 3 ' end homologous sequence+donor B fragments specific forward amplimer-3 '; Donor B reverse primer: 5 '-15bp receptor dna, 3 ' end homologous sequence+restriction enzyme site+reverse amplimer-3 ' of donor dna specificity.Described 15bp receptor dna 5 ' end homologous sequence refers to receptor dna 5 ' the last 15bp DNA sequence dna of end antisense strand end, and direction is 5 ' to 3 '; Described 15bp donor A fragment 3 ' end homologous sequence confession under directions body A fragment 3 ' the last 15bp DNA sequence dna of end positive-sense strand, direction 5 ' to 3 '; Described 15bp receptor dna 3 ' end homologous sequence refers to the proper last 15bp DNA sequence dna of adopted chain end of acceptor 3 ', and direction is 5 ' to 3 '.The length of described homologous sequence is 12bp at least, and its length is 15bp in a preference of the present invention.Described restriction enzyme site is total length or the part of receptor dna linearizing restriction enzyme digestion sites used, and direction is 5 ' to 3 '.Described donor A fragment (B fragment) specificity just (instead) refers to donor dna PCR amplimer to amplimer, and direction is 5 ' to 3 '.
The present invention preferably is used for DNA rite-directed mutagenesis pcr amplification primer principle of design for (introducing single base mutation as example take plasmid DNA, setting take the sudden change position on M and the plasmid vector 3 ' direction with its apart the another location more than 500bp as N, be divided into two sections by 5 ' to 3 ' direction M and N two plasmid vector of naming a person for a particular job, donor MN fragment and acceptor N fragment M section): donor MN fragment forward primer and the reverse complete complementary of acceptor NM fragment reverse primer or part are complementary, and between two primers, the overlapping region is 15bp; Donor MN fragment reverse primer and acceptor NM fragment forward primer complete complementary or part are complementary, and between two primers, the overlapping region is 15bp.The single base mutation of introducing in described donor MN fragment forward primer and acceptor NM fragment reverse primer complementary region.Between described primer, the overlapping region is at least 12bp, and its length is 15bp in a preference of the present invention.
The archaeal dna polymerase that in the present invention, the amplification of donor dna fragment PCR is used can be any type of archaeal dna polymerase.In view of the requirement to the donor dna fidelity of reproduction, select high-fidelity DNA polymerase in a preference of the present invention.Described receptor dna refers to any DNA double chain link that can be used for DNA clone, includes but are not limited to following several: plasmid, BAC (bacterial artificial chromosome), YAC (artificial yeast's karyomit(e)) and Phagemid (phagemid) etc.Described receptor dna linearizing can use restriction enzyme to carry out, and also can directly use with the pcr amplification product of receptor dna as template.Donor dna fragment and linearizing receptor dna fragment need be carried out purifying and be reclaimed, the purifying mode comprise but be not limited only to following several, pillar DNA purification system, ethanol precipitation etc.In view of the DNA purity requirement, in a preference of the present invention, use the pillar DNA purification system to carry out the purifying recovery to donor dna and receptor dna.
In the present invention, the recombining reaction of donor dna fragment and receptor dna fragment is completed external under DNA excision enzyme and DNA single chain binding protein co-catalysis.Described DNA excision enzyme refers to the general name of class of enzymes that can the cutting DNA end, can be the combination of any one 5 ' → 3 ' DNA excision enzyme or several this kind excision enzymes, includes but not limited to following several: intestinal bacteria excision enzyme VII, T7 excision enzyme gene 6 etc.; Also can be the combination of any one 3 ' → 5 ' DNA excision enzyme or several this kind excision enzymes, include but not limited to following several: intestinal bacteria excision enzyme I, intestinal bacteria exonucleaseⅢ, T4 polysaccharase etc.; Also one or several combination of two class excision enzymes.Described DNA excision enzyme can derive from prokaryotic organism, for example intestinal bacteria; Also can derive from eukaryote, for example yeast, nematode, mouse, rat or people.Select the DNA excision enzyme in intestinal bacteria source in preference of the present invention.Described single strand binding protein can be any albumen that can be combined with single stranded DNA, includes but not limited to following several: intestinal bacteria SSB, eukaryote replication protein RPA, RecA, T4 gene 32 albumen etc.Described single strand binding protein can derive from prokaryotic organism, for example intestinal bacteria; Also can derive from eukaryote, for example yeast, nematode, mouse, rat or people.Select the single strand binding protein in intestinal bacteria source in preference of the present invention.
the enzyme liquid that can be used for the recombining reaction of catalysis donor dna fragment and receptor dna fragment in the present invention can be the combination of any or several excision enzyme and any or several DNA single chain binding proteins: include but not limited to following several: intestinal bacteria excision enzyme I and intestinal bacteria SSB, intestinal bacteria exonucleaseⅢ and intestinal bacteria SSB, intestinal bacteria excision enzyme VII and intestinal bacteria SSB, T7 excision enzyme gene 6 and intestinal bacteria SSB, T4 polysaccharase and intestinal bacteria SSB, intestinal bacteria excision enzyme I and RecA, intestinal bacteria exonucleaseⅢ and RecA, intestinal bacteria excision enzyme VII and RecA, T7 excision enzyme gene 6 and RecA, T4 polysaccharase and RecA etc.Select intestinal bacteria T4 polysaccharase and intestinal bacteria RecA in preference of the present invention, this combination can efficiently mediate the donor dna fragment and the receptor dna fragment is reacted in external generation homologous recombination.
In the present invention, after donor dna fragment and the purified recovery of receptor dna fragment, and after mixing, DNA excision enzyme and single strand binding protein can carry out recombining reaction.Need comprise damping fluid, salt ion and ATP in reaction system, PH can be 5.0-9.0.In preference of the present invention, the recombining reaction system comprises following component: 500mM Tris-HCL, 200mM Mg (AC) 2,10mM DTT, 1M NaAc, 1mM ATP, 10mM BSA, reaction solution PH is 8.0.Above component can replace with other similar reagent of physicochemical property, such as NaAc can replace with KAc.After donor dna and receptor dna can use respectively the DNA excision enzyme to process, remix also added single strand binding protein to carry out recombining reaction; Add DNA excision enzyme and single strand binding protein to carry out recombining reaction after also can mixing.Reaction times can be 10-60 minute, but the reaction times is too short or oversizely all will cause the recombining reaction decrease in efficiency.Temperature of reaction can be 18 ℃-50 ℃, along with the rising reaction times of temperature of reaction is answered corresponding shortening, otherwise also will cause the recombining reaction decrease in efficiency.Temperature of reaction is 30 ℃ in preference of the present invention, and the reaction times is 30 minutes.
Donor dna fragment and receptor dna fragment form recombination intermediate after recombining reaction, because of the repair mechanism that cell self has, recombination intermediate directly transformed competence colibacillus cell can be completed the homologous recombination of donor dna fragment and receptor dna fragment.One section 500bp λ DNA sequence dna of clone to the result of PUC19 plasmid can draw the following conclusions from embodiment 1: DNA excision enzyme (T4 polysaccharase) and single strand binding protein (RecA) can be in external efficient mediated dna fragment homologous recombination, thereby complete the DNA directed cloning, and clone's positive rate surpasses 90%.
The invention has the advantages that:
1. cloning efficiency is high, and clone's number is many and positive rate is high;
2. step is simple, need not to the donor dna fragment shuttle back and forth clone or enzyme cut processing;
3. the donor dna fragment does not need restriction enzyme to process, and has therefore thoroughly eliminated the restriction that the inherent restriction enzyme site of donor dna is selected for Strategies For The Cloning;
4. the inventive method not only can be used as the quick clone of single donor dna fragment, and can be used for realizing the seamless spliced and single step clone of a plurality of donor dna fragments.
Above-mentioned explanation is not limited only to the present invention and described embodiment, and those skilled in the art can implement and use technical solutions according to the invention by modifying, adjust, changing, and these technical schemes all fall within the scope of protection of the present invention.
Description of drawings
Fig. 1: present patent application methods experiment schematic flow sheet.
Fig. 2: present patent application method design of primers schematic diagram.
Fig. 3: embodiment 1 mono-clonal PCR qualification result electrophorogram.
The M:DL2000 molecular weight standard, 2.0kbp, 1.0kbp, 0.75kbp, 0.5kbp, 0.25kbp, 0.1kbp; 1-14:14 different mono-clonal bacterium colony PCR products, positive colony amplified production size should be 700bp.
Fig. 4: embodiment 1 mono-clonal EcoRI and BamHI double digestion product electrophorogram.
The M:DL2000 molecular weight standard, 2.0kbp, 1.0kbp, 0.75kbp, 0.5kbp, 0.25kbp, 0.1kbp; 1-8:8 different mono-clonal enzymes are cut product, and positive colony should produce 2.6kbp and two bands of a spectrum of 500bp.
Embodiment
The term that uses in the present invention unless other explanation is arranged, generally has the implication that those of ordinary skills understand usually.
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 and the method do not described in detail are ordinary methods as known in the art.The source of all reagent, trade(brand)name and be necessary to list its moiety person are all indicated when occurring first, thereafter identical reagent used if no special instructions, all identical with the content of indicating first.
Experiment reagent used and plasmid in embodiment, its source is as follows:
1.λDNA。Available from U.S. Promega company;
2.PrimeSTAR HS DNA Polymerase。Available from Japanese Takara company;
3. restriction enzyme EcoRI, BamHI.Available from Japanese Takara company;
4.T4 polysaccharase, intestinal bacteria RecA.Available from NEB company;
5. glue reclaims test kit.Available from sky, Beijing root company limited;
6. the inorganic salt that use in the experiment are all available from U.S. Sigma company.
1. design primer
At first design cloned sequence pcr amplification primer
Forward primer: 5 '-ATCAGTCATTTCTCGCACATTG-3 ', (SEQ ID NO.1)
Reverse primer: 5 '-TTACGGGGTTGGAGGTCA-3 ', (SEQ ID NO.2)
15bp carrier homologous sequence and restriction enzyme site that forward primer 5 ' end needs to add are: 5 '-AAAACGACGGCCAGT
GAATTC(EcoRI)-3 ', (SEQ ID NO.3)
15bp carrier homologous sequence and restriction enzyme site that reverse primer 5 ' end needs to add are: 5 '-GACGATATCTCTAGA
GGATCC(BamHI)-3 ', (SEQ ID NO.4)
Therefore the forward primer ultimate sequence is: 5 '-AAAACGACGGCCAGT
GAATTCATCAGTCATTTCTCGCACATTG-3 ', (SEQ ID NO.5)
The reverse primer ultimate sequence is: 5 '-GACGATATCTCTAGA
GGATCCTTACGGGGTTGGAGGTCA-3 ', (SEQ ID NO.6)
2.PCR amplification
The pcr amplification system:
| PrimeSTAR | 0.5 | μl |
| 5X PrimeSTAR Buffer | 10 | μl |
| λDNA(1ng/ul) | 1 | μl |
| Forward primer (10um) | 1 | μl |
| Reverse primer (10um) | 1 | μl |
| Sterile purified water | 36.5 | μl |
| Amount to | 50 | μl |
The PCR reaction conditions is: at first 94 ℃ of sex change are 5 minutes, and cycling condition is: 98 ℃ 10 seconds, 68 degree 30 seconds, totally 35 circulations.Thoroughly extended 10 minutes at 72 ℃ at last.Use instrument to be 2720 model PCR instrument of AB company.Reaction product is carried out glue and is reclaimed after 1% agarose electrophoresis.
3.PUC19 carrier linearizing
1 μ g PUC19 plasmid uses 1 μ l EcoRI and 1 μ l BamHI, cuts 2 hours at 37 ℃ of lower enzymes.Product carries out glue and reclaims after 1% agarose electrophoresis.
4. vitro recombination reaction
The following reaction system of configuration in ice-water bath:
The 10X reaction buffer comprises following component: 5M Tris-HCL, 2M Mg (AC)
2, 100mM DTT, 10M NaAc, 100mM BSA, PH is 8.0.Reaction mixture reacts under 30 ℃ and was placed on the ice-water bath stopped reaction in 30 minutes.
5. transform the intestinal bacteria competence
The recombining reaction system is added (CaCL in 200ul bacillus coli DH 5 alpha competent cell
2Method is made, and efficient approximately has 10
6～10
7Cfu/ug), be coated on the dull and stereotyped upper 37 ℃ of incubated overnight of LB of ammonia benzyl resistance after conversion.
6. clone identification
Grow on the LB flat board of incubated overnight～1000 mono-clonals.Identify through bacterium colony PCR, positive rate is 100% (14/14) (seeing accompanying drawing 2).After getting wherein 8 mono-clonals and extracting plasmid, enzyme is cut evaluations (EcoRI+BamHI), is all correctly to clone (8/8) (seeing accompanying drawing 3).Get wherein that 4 positive colonies send order-checking, sequence is all consistent with expection.
The sequence (Segment A) of one section 500bp of embodiment 2. clone λ DNA and the sequence of one section 700bp (fragment B), two DNA fragmentations are sequentially seamless spliced, and directed cloning is to the carrier PUC19 between EcoRI and BamHI.
1. design primer
Segment A forward amplimer is: 5 '-ATCAGTCATTTCTCGCACATTG-3 ', (SEQ ID NO.7)
The reverse amplimer of Segment A is: 5 '-TTACGGGGTTGGAGGTCA-3 ', (SEQ ID NO.8)
15bp carrier homologous sequence and restriction enzyme site that Segment A forward primer 5 ' end needs to add are:
5’-AAAACGACGGCCAGT
GAATTC(EcoRI)-3’，(SEQ ID NO.9)
Segment A reverse primer 5 ' end needs the 15bp and the fragment B 5 ' that add to hold the sequence of homology to be:
5’-GAAGCGGCGCAATAT-3’，(SEQ ID NO.10)
Therefore Segment A forward primer ultimate sequence is: 5 '-AAAACGACGGCCAGT
GAATTCATCAGTCATTTCTCGCACATTG-3 ', (SEQ ID NO.11)
The reverse primer ultimate sequence is: 5 '-GAAGCGGCGCAATATTTACGGGGTTGGAGGTCA-3 ', (SEQ ID NO.12)
Fragment B forward amplimer is: 5 '-ATATTGCGCCGCTTCAGGATGC-3 ', (SEQ ID NO.13)
The reverse amplimer of fragment B is: 5 '-TCACATTCAAACATTAATTT-3 ', (SEQ ID NO.14)
The 15bp homologous sequence that needs because of Segment A and fragment B restructuring has been included in the Segment A reverse primer, and fragment B forward amplimer 5 ' end need not to introduce additional sequences.
15bp carrier homologous sequence and restriction enzyme site that fragment B reverse primer 5 ' end needs to add are:
5’-GACGATATCTCTAGA
GGATCC(BamHI)-3’，(SEQ ID NO.15)
Therefore fragment B reverse primer ultimate sequence is: 5 '-GACGATATCTCTAGA
GGATCCTCACATTCAAACATTAATTT-3 ', (SEQ ID NO.16)
2.PCR amplification
The pcr amplification system:
| PrimeSTAR | 0.5 | μl |
| 5X PrimeSTAR Buffer | 10 | μl |
| λDNA(1ng/ul) | 1 | μl |
| Forward primer (10um) | 1 | μl |
| Reverse primer (10um) | 1 | μl |
| Sterile purified water | 36.5 | μl |
| Amount to | 50 | μl |
The PCR reaction conditions is: at first 94 ℃ of sex change are 5 minutes, and cycling condition is: 98 ℃ 10 seconds, 68 degree 30 seconds, totally 35 circulations.Thoroughly extended 10 minutes at 72 ℃ at last.Use instrument to be 2720 model PCR instrument of AB company.Reaction product is carried out respectively glue and is reclaimed after 1% agarose electrophoresis.
3.PUC19 carrier linearizing
1 μ g PUC19 plasmid uses 1 μ l EcoRI and 1 μ l BamHI, cuts 2 hours at 37 ℃ of lower enzymes.Product carries out glue and reclaims after 1% agarose electrophoresis.
4. vitro recombination reaction
The following reaction system of configuration in ice-water bath:
The 10X reaction buffer comprises following component: 5M Tris-HCL, 2M Mg (AC)
2, 100mM DTT, 10M NaAc, 100mM BSA, PH is 8.0.Reaction mixture reacts under 30 ℃ and was placed on the ice-water bath stopped reaction in 30 minutes.
5. transform the intestinal bacteria competence
The recombining reaction system is added (CaCL in 200ul bacillus coli DH 5 alpha competent cell
2Method is made, and efficient approximately has 10
6～10
7Cfu/ug), be coated on the dull and stereotyped upper 37 ℃ of incubated overnight of LB of ammonia benzyl resistance after conversion.
6. clone identification
Grow on the LB flat board of incubated overnight～400 mono-clonals.Identify through bacterium colony PCR, positive rate is 89% (16/18).After getting wherein 6 mono-clonals and extracting plasmid, enzyme is cut evaluations (EcoRI+BamHI), is all correctly to clone (6/6).Get wherein that 4 positive colonies send order-checking, sequence is all consistent with expection.
Experimental strategy is: plasmid is divided into two sections of 2362-967 (Segment A) and 968-2361 (fragment B) increases with PCR respectively, required point mutation is introduced when design of primers.The reverse amplimer of Segment A and the complete reverse complemental of fragment B forward amplimer, and all introduce 967 T to the point mutation of G; The complete reverse complemental of reverse amplimer of the forward amplimer of Segment A and fragment B, and all introduce the G of 2631 to the point mutation of A.
1. design primer
Segment A forward amplimer: 5 '-CTTTTACTTTCACCA
ACGTTTCTGGGTGAG-3 ', (SEQ ID NO.17)
The reverse amplimer of Segment A: 5 '-GCTTCCAGGGGGAA
CCGCCTGGTATCTTTA-3 ', (SEQ ID NO.18)
Fragment B forward amplimer: 5 '-TAAAGATACCAGGCG
GTTCCCCCTGGAAGC-3 ', (SEQ ID NO.19)
The reverse amplimer of fragment B: 5 '-CTCACCCAGAAACG
TTGGTGAAAGTAAAAG-3 ', (SEQ ID NO.20)
The base of underscore mark is the point mutation of introducing in primer.
2.PCR amplification
The pcr amplification system:
| PrimeSTAR | 0.5 | μl |
| 5X PrimeSTAR Buffer | 10 | μl |
| λDNA(1ng/ul) | 1 | μl |
| Forward primer (10um) | 1 | μl |
| Reverse primer (10um) | 1 | μl |
| Sterile purified water | 36.5 | μl |
| Amount to | 50 | μl |
The PCR reaction conditions is: at first 94 ℃ of sex change are 5 minutes, and cycling condition is: 98 ℃ 10 seconds, 68 degree 2 minutes, totally 35 circulations.Thoroughly extended 10 minutes at 72 ℃ at last.Use instrument to be 2720 model PCR instrument of AB company.Reaction product is carried out respectively glue and is reclaimed after 1% agarose electrophoresis.
3. vitro recombination reaction
The following reaction system of configuration in ice-water bath:
The 10X reaction buffer comprises following component: 5M Tris-HCL, 2M Mg (AC)
2, 100mM DTT, 10M NaAc, 100mM BSA, PH is 8.0.Reaction mixture reacts under 30 ℃ and was placed on the ice-water bath stopped reaction in 30 minutes.
4. transform the intestinal bacteria competence
The recombining reaction system is added (CaCL in 200ul bacillus coli DH 5 alpha competent cell
2Method is made, and efficient approximately has 10
6～10
7Cfu/ug), be coated on the dull and stereotyped upper 37 ℃ of incubated overnight of LB of ammonia benzyl resistance after conversion.
5. clone identification
Grow on the LB flat board of incubated overnight～800 mono-clonals.Identify through bacterium colony PCR, positive rate is 100% (8/8).Get wherein 2 positive colony complete sequences surveys logical, sequence is all consistent with expection.
Claims (10)
1. one kind is utilized the external homologous recombination reaction of exonuclease and single strand binding protein mediation the donor dna fragment to be carried out the method for quick clone, it is characterized in that: first make donor dna two ends and receptor dna two ends that the homologous sequence of 12bp at least be arranged respectively by introducing appended sequence at amplimer 5 ' end, again with the receptor dna linearizing, donor dna and linearizing receptor dna can be at external efficient formation recombination intermediates under the catalysis of exonuclease and single strand binding protein, the direct transformed competence colibacillus cell of this intermediate just can be completed donor dna to clone's process of receptor dna.
2. the method for claim 1 it is characterized in that a kind of method of quick clone, wherein donor dna need not restriction endonuclease just process can Direct Cloning to receptor dna.
3. method as claimed in claim 2, is characterized in that, described donor dna needs to introduce appended sequence at pcr amplification primer 5 ' end.
4. method as claimed in claim 3, is characterized in that, the pcr amplification forward primer 5 ' end of donor dna need add 12bp receptor dna 5 ' end homologous sequence at least, the i.e. last 12bp DNA sequence dna of its 5 ' end antisense strand end; Donor dna pcr amplification reverse primer 5 ' end need add 12bp receptor dna 3 ' end homologous sequence at least, i.e. its 3 ' proper last 12bp DNA sequence dna of adopted chain end.
5. method as claimed in claim 2, is characterized in that, described receptor dna need to carry out linearizing.
6. method as claimed in claim 5, is characterized in that, described linearizing is to use the DNA restriction enzyme to carry out, or directly uses receptor dna total length pcr amplification product.
7. the method for claim 1, it is characterized in that, described DNA excision enzyme is 5 ' DNA excision enzyme, or 3 ' DNA excision enzyme, include but are not limited to following several: intestinal bacteria excision enzyme VII, T7 excision enzyme gene 6, intestinal bacteria excision enzyme I, intestinal bacteria exonucleaseⅢ, T4 polysaccharase.
8. the method for claim 1, it is characterized in that, described single strand binding protein refer to can with the albumen of single stranded DNA (ssDNA) combination, include but are not limited to following several: intestinal bacteria SSB, eukaryote replication protein RPA, RecA, T4 gene 32 albumen.
9. the method for claim 1, is characterized in that, comprises at least a DNA excision enzyme and at least a single strand binding protein in the enzyme liquid of the outer homologous recombination reaction of catalytic body.
10. the method for claim 1, is characterized in that, after donor dna and receptor dna used respectively exonuclease to process, remix also added single strand binding protein to carry out recombining reaction; Or with donor dna with add simultaneously DNA excision enzyme and single strand binding protein to carry out recombining reaction after receptor dna mixes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104184509A CN103160495A (en) | 2011-12-15 | 2011-12-15 | Disconnected dependent rapid cloning method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104184509A CN103160495A (en) | 2011-12-15 | 2011-12-15 | Disconnected dependent rapid cloning method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103160495A true CN103160495A (en) | 2013-06-19 |
Family
ID=48584030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011104184509A Pending CN103160495A (en) | 2011-12-15 | 2011-12-15 | Disconnected dependent rapid cloning method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103160495A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805600A (en) * | 2014-03-06 | 2014-05-21 | 福州大学 | Molecular cloning method for deoxyribonucleic acid (DNA) |
| CN104178477A (en) * | 2013-05-28 | 2014-12-03 | 中国人民解放军军事医学科学院生物工程研究所 | Gene synthesis method |
| CN104195161A (en) * | 2014-09-12 | 2014-12-10 | 刘喜朋 | Preparing method for DNA recombinase based on controllable DNA polymerase and exonuclease activity and application thereof |
| CN104498451A (en) * | 2015-01-06 | 2015-04-08 | 苏州泓迅生物科技有限公司 | Recombinase with nucleic acid exterior contact and single-chain DNA exchange activity and application of recombinase |
| CN104630178A (en) * | 2015-01-28 | 2015-05-20 | 苏州神洲基因有限公司 | Mixed protease system, kit containing mixed protease system and use method of kit |
| CN106119222A (en) * | 2016-07-04 | 2016-11-16 | 翌圣生物科技（上海）有限公司 | A kind of protease composition for external homologous recombination, test kit and method |
| 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 |
| CN109439684A (en) * | 2018-12-27 | 2019-03-08 | 尚科生物医药（上海）有限公司 | A method of improving foreign gene efficiency of evolution in eukaryocyte |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331748A (en) * | 1998-10-30 | 2002-01-16 | 默多克研究所 | Recombinant method and reagents therefor |
| EP1034260B1 (en) * | 1997-12-05 | 2003-06-04 | Europäisches Laboratorium Für Molekularbiologie (Embl) | Novel dna cloning method relying on the e. coli rece/rect recombination system |
| WO2007124065A1 (en) * | 2006-04-21 | 2007-11-01 | The Brigham And Women's Hospital, Inc. | Generation of recombinant dna by sequence-and ligation-independent cloning |
| WO2010030776A1 (en) * | 2008-09-10 | 2010-03-18 | Genscript Corporation | Homologous recombination-based dna cloning methods and compositions |
-
2011
- 2011-12-15 CN CN2011104184509A patent/CN103160495A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1034260B1 (en) * | 1997-12-05 | 2003-06-04 | Europäisches Laboratorium Für Molekularbiologie (Embl) | Novel dna cloning method relying on the e. coli rece/rect recombination system |
| CN1331748A (en) * | 1998-10-30 | 2002-01-16 | 默多克研究所 | Recombinant method and reagents therefor |
| WO2007124065A1 (en) * | 2006-04-21 | 2007-11-01 | The Brigham And Women's Hospital, Inc. | Generation of recombinant dna by sequence-and ligation-independent cloning |
| WO2010030776A1 (en) * | 2008-09-10 | 2010-03-18 | Genscript Corporation | Homologous recombination-based dna cloning methods and compositions |
| CN102124112A (en) * | 2008-09-10 | 2011-07-13 | 金斯瑞公司 | Homologous recombination-based DNA cloning methods and compositions |
Non-Patent Citations (3)
| Title |
|---|
| CHARALAMPOS ASLANIDIS ET AL.: "Ligation-independent cloning of PCR products (LIC-PCR)", 《NUCLEIC ACIDS RESEARCH》 * |
| MAMIE Z LI ET AL.: "Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC", 《NATURE METHODS》 * |
| YOUMING ZHANG ET AL.: "DNA cloning by homologous recombination in Escherichia coli", 《NATURE BIOTECHNOLOGY》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178477A (en) * | 2013-05-28 | 2014-12-03 | 中国人民解放军军事医学科学院生物工程研究所 | Gene synthesis method |
| CN103805600A (en) * | 2014-03-06 | 2014-05-21 | 福州大学 | Molecular cloning method for deoxyribonucleic acid (DNA) |
| CN104195161A (en) * | 2014-09-12 | 2014-12-10 | 刘喜朋 | Preparing method for DNA recombinase based on controllable DNA polymerase and exonuclease activity and application thereof |
| CN104195161B (en) * | 2014-09-12 | 2020-06-09 | 刘喜朋 | Preparation method and application of DNA recombinase based on controllable DNA polymerase and exonuclease activity |
| CN104498451A (en) * | 2015-01-06 | 2015-04-08 | 苏州泓迅生物科技有限公司 | Recombinase with nucleic acid exterior contact and single-chain DNA exchange activity and application of recombinase |
| CN104630178A (en) * | 2015-01-28 | 2015-05-20 | 苏州神洲基因有限公司 | Mixed protease system, kit containing mixed protease system and use method of kit |
| CN106119222A (en) * | 2016-07-04 | 2016-11-16 | 翌圣生物科技（上海）有限公司 | A kind of protease composition for external homologous recombination, test kit and method |
| 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 |
| CN109439684A (en) * | 2018-12-27 | 2019-03-08 | 尚科生物医药（上海）有限公司 | A method of improving foreign gene efficiency of evolution in eukaryocyte |
| CN109439684B (en) * | 2018-12-27 | 2023-09-01 | 尚科生物医药（上海）有限公司 | Method for improving evolution efficiency of exogenous gene in eukaryotic cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103160495A (en) | Disconnected dependent rapid cloning method | |
| CN102124112B (en) | Homologous recombination-based DNA cloning methods and compositions | |
| Tseng et al. | A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method | |
| CN107488656B (en) | Nucleic acid isothermal self-amplification method | |
| US6372429B1 (en) | Method for assembly of multiple DNA fragments | |
| US20130046083A1 (en) | Oligonucleotide ligation | |
| WO1998038299A1 (en) | Single step assembly of multiple dna fragments | |
| CN1427079A (en) | Construction method of multigene carrier and its application | |
| CN101368188A (en) | A fast and efficient method for constructing plant artificial microRNA expression vector | |
| CN113717960A (en) | Novel Cas9 protein, CRISPR-Cas9 genome directed editing vector and genome editing method | |
| CN117778377A (en) | Efficient synthesis and assembly of large DNA fragments based on the novel programmable nuclease Argonaute | |
| CN105969784B (en) | A recombinase-independent DNA seamless cloning method | |
| JP2024509047A (en) | CRISPR-related transposon system and its usage | |
| CN113817731A (en) | Targeted reduction drug resistance gene blaTEMgRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof | |
| Zumkeller et al. | Modular assembly of synthetic secondary chromosomes | |
| CN109957029B (en) | Recombinant protein gp32-UvsX, and preparation method and application thereof | |
| Dong et al. | A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method | |
| CN116640765A (en) | Engineering-modified sgRNA design and preparation method | |
| Walker et al. | A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs | |
| EP1362101B1 (en) | Orientation-directed construction of plasmids | |
| CN116200366A (en) | Bst DNA polymerase mutant strain with XNA synthesis and reverse transcription activity | |
| Balbás et al. | Molecular cloning by plasmid vectors | |
| CN104357438B (en) | DNA assembling and cloning method | |
| CN116135982A (en) | Application method and kit for gene fragment ligation | |
| JP2024509048A (en) | CRISPR-related transposon system and its usage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Zhang Lijun Document name: the First Notification of an Office Action |
|
| DD01 | Delivery of document by public notice | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130619 |
|
| WD01 | Invention patent application deemed withdrawn after publication |