WO2017066943A1 - 一种dna片段体外组装方法和试剂盒 - Google Patents

一种dna片段体外组装方法和试剂盒 Download PDF

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WO2017066943A1
WO2017066943A1 PCT/CN2015/092438 CN2015092438W WO2017066943A1 WO 2017066943 A1 WO2017066943 A1 WO 2017066943A1 CN 2015092438 W CN2015092438 W CN 2015092438W WO 2017066943 A1 WO2017066943 A1 WO 2017066943A1
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dna
fragment
assembly
fragments
vector
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French (fr)
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陈泰
赵宏翠
范楚珧
陈世宏
王云
沈玥
徐讯
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深圳华大基因研究院
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Priority to CN201580083695.7A priority Critical patent/CN108138189B/zh
Priority to PCT/CN2015/092438 priority patent/WO2017066943A1/zh
Publication of WO2017066943A1 publication Critical patent/WO2017066943A1/zh
Priority to HK18113473.9A priority patent/HK1254396A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the invention relates to the technical field of DNA assembly, and in particular to a method and a kit for assembling a DNA fragment in vitro.
  • DNA chemical synthesis methods can synthesize a certain length of DNA sequences from scratch, DNA synthesis of larger fragments must be completed by DNA assembly, so DNA assembly technology has become the most important basic experiment in the fields of synthetic biology and metabolic engineering.
  • DNA assembly technology has become the most important basic experiment in the fields of synthetic biology and metabolic engineering.
  • Another branch of the digestion and ligation method can assemble and link one or more linear or circular double-stranded DNA fragments to the assembled vector by enzymatic cleavage, ligation and simultaneous reaction. It is also a common assembly.
  • One of the methods there are still some insurmountable defects in the methods of digestion and ligation, such as restriction of restriction sites, number of fragments to be joined, and limitation of length.
  • the Gibson assembly method is especially convenient and has a wide range of applications.
  • a double-stranded linear DNA fragment carrying a homologous region can be assembled into a double-stranded linear DNA fragment by a one-step assembly reaction.
  • Gibson assembly also enables assembly and cloning of multiple single-stranded DNA fragments.
  • the Gibson assembly one-step splicing method has high synthesis cost and low splicing efficiency.
  • the present invention provides a method for in vitro assembly of DNA fragments which depends on overlapping sequences, and can assemble and clone a plurality of DNA fragments. Accordingly, the present invention also provides kits for in vitro assembly of DNA fragments.
  • the present invention provides a method for in vitro assembly of a DNA fragment, comprising the steps of assembling a plurality of DNA fragments and an assembly vector; wherein the plurality of DNA fragments are in a set fragment sequence, the first fragment The upstream and the last segment respectively have the same overlapping sequence as the above-mentioned assembled carrier, the other segments have the same overlapping sequence upstream of the previous segment, and the other segments have the same overlapping sequence upstream of the next segment;
  • the assembled vector comprises two overlapping sequences, optionally with an enzymatic cleavage site on its inner side, optionally with a screening marker between the cleavage sites; the above method allows the plurality of DNA fragments and the above-described assembled vector to be recognized
  • the endonuclease, exonuclease, DNA polymerase and DNA ligase of the above-mentioned cleavage site are brought into contact with each other, and the plurality of DNA fragments are assembled to the above-de
  • a kit for in vitro assembly of a DNA fragment comprising an assembly buffer and an assembly carrier; the kit for assembling a plurality of DNA fragments and an assembly carrier, wherein the above The DNA fragments are arranged in the same sequence of fragments, and the upstream of the first fragment and the downstream of the last fragment respectively have the same overlapping sequence as the above-mentioned assembled vector, and the other fragments have the same overlapping sequence upstream of the previous fragment, and other fragments.
  • the above-described assembled vector comprises two overlapping sequences, optionally with an enzyme cleavage site on its inner side, optionally with a screening marker between the cleavage sites;
  • the kit further comprises at least one of a positive control test fragment, an endonuclease, an exonuclease, a DNA polymerase, and a DNA ligase.
  • FIG. 1 is a schematic diagram showing the principle of assembly of a double-stranded circular DNA fragment according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the principle of assembling a mixed type DNA fragment according to an embodiment of the present invention
  • Figure 3 is a diagram showing the assembly test of a double-stranded circular DNA fragment according to an embodiment of the present invention
  • Figure 4 is a diagram showing the assembly of a double-stranded circular DNA fragment according to an embodiment of the present invention.
  • Figure 5 is a flat view of a mixed type DNA fragment assembly test according to an embodiment of the present invention.
  • Fig. 6 is a diagram showing the assembly of a mixed type DNA fragment and an enzyme digestion electrophoresis according to an embodiment of the present invention.
  • Embodiments of the present invention describe a method of in vitro assembly of DNA fragments that rely on overlapping sequences, which can be carried out in the same reaction system to complete assembly and cloning of multiple DNA fragments.
  • an embodiment of the present invention for a plurality of double-stranded DNA fragments cloned on a vector (i.e., fragment 1, fragment 2, and fragment 3, in other embodiments, the number of fragments may not be limited to three)
  • the set segment order ie, the order of segment 1, segment 2, and segment 3 in turn
  • the first segment (segment 1) upstream (01) and the last segment (segment 3) downstream (02) are respectively assembled and assembled
  • the same overlapping sequences (01 and 02) of the vector, the other fragments (segment 2) upstream (03) carry the same overlapping sequence as the downstream (03) of the previous fragment (segment 1), and the other fragments (fragment 2) are downstream (04) ) with the same overlapping sequence as the upstream (04) of the next segment (segment 3), with the overlapping sequences (01, 03, 04, 02) of each segment (segment 1, segment 2, and segment 3)
  • the cleavage site (E); the assembled vector has two
  • fragment and vector shown in Figure 1 are both intact double-stranded circular DNA structures and thus carry an enzyme cleavage site. If the fragment and vector are linear DNA fragments, there may be no restriction sites, and thus the restriction sites are "optionally", i.e., with or without restriction sites, as appropriate. Similarly, there may or may not be a screening marker on the vector, preferably a screening marker to increase screening efficiency.
  • the assembled carrier has two overlapping sequences (01 and 02), it should be understood that the method of the present invention does not limit that the assembled carrier can only include two overlapping sequences, but at least Two (such as three, four or more) overlapping sequences may be present, and other overlapping sequences may be located, for example, adjacent to the screening marker.
  • the above plurality of DNA fragments and the assembled vector are added to the reaction system, and a DNA endonuclease, an exonuclease, a DNA polymerase, and a DNA ligase which recognize the cleavage site (E) are simultaneously added.
  • the DNA endonuclease acts, and the DNA fragment and the assembled vector backbone to be assembled are excised from the plasmid (Fig. 1B), and then the DNA exonuclease acts to cleave the terminal overlapping region of the fragment and the assembled vector to reveal complementarity.
  • the cohesive ends, while the cohesive ends are complementary paired, the fragments to be assembled and the assembled vector are sequentially ligated into a loop (Fig.
  • the reaction product can be directly used for bacterial transformation such as Escherichia coli, and a monoclonal antibody is grown on the assembled vector resistant medium, the empty vector is removed by screening the label on the vector, the positive clone is picked, and the plasmid is obtained to obtain a large number of successfully assembled DNA. Fragment.
  • the method of the present invention is preferably in the assembly buffer of the same reaction system, the DNA fragment and the assembly vector are simultaneously combined with the endonuclease, exonuclease, and DNA polymerase which recognize the cleavage site.
  • the reaction with DNA ligase is carried out, that is, a one-step reaction is preferred; however, based on the spirit of the present invention, it can also be carried out stepwise, that is, the above-mentioned DNA endonuclease, exonuclease, DNA polymerase and DNA ligase are sequentially added to the reaction.
  • the reaction was carried out separately in the system.
  • the method of the present invention can be used not only for (a) assembly of double-stranded DNA fragments (i.e., double-stranded circular DNA fragments) which are both cloned on a vector, in which case, double-stranded DNA fragments are
  • double-stranded DNA fragments are The outer side of the overlapping sequence has a restriction site that can be recognized by the above DNA endonuclease; and can also be used for (b) assembly of double-stranded linear DNA fragments, and (c) assembly of single-stranded linear DNA fragments.
  • the number of single-stranded linear DNA fragments is even, and (d) any two or three types of DNA in a double-stranded DNA fragment, a double-stranded linear DNA fragment, and a single-stranded linear DNA fragment cloned on a vector a mixed assembly of fragments, in the presence of a double-stranded DNA fragment cloned on a vector, the outer side of the overlapping sequence on both sides of the double-stranded DNA fragment carries an enzyme cleavage site which is recognized by the above-mentioned DNA endonuclease;
  • the number of single-stranded linear DNA fragments is an even number. It is only necessary to have overlapping sequences as described above and as shown in FIG. The assembly principle is similar to the assembly of double-stranded circular DNA fragments.
  • efficient assembly can be achieved with overlapping sequences greater than 15 bp in length, preferably 20-40 bp.
  • the selection marker in the assembled vector can be any marker useful in bacteria, such as an antibiotic resistance gene, a fluorescent protein gene, a lethal gene, a lacZ gene, etc., or any combination thereof, A combination of a red fluorescent protein gene or a lacZ gene and a red fluorescent protein group gene is preferred.
  • the restriction site may be any restriction endonuclease recognition site or polyclonal restriction site (MCS), preferably a homeoendase (such as I-SceI) or a high density.
  • MCS polyclonal restriction site
  • Cloning sites eg, GGCCGCGGCGGCGC, GTCGACCGGACCGCGGGTC, GGGCCCGCCGGCGCGCC, CGCCGGCGCGCCGCCGCCGC, GACGACGTCGACCGCGGGTC, ACCGGACCGCGGGTCACC, GGCCGCCGGCGCC, etc.).
  • the cleavage site of the assembled fragment and the assembled vector requires that the cleavage site is not present on all of the assembled fragments and the assembled vector, and the cleavage sites may be the same or different. Since the reaction is carried out in the same system, and it is required that these cleavage sites must be present on all the assembled fragments and the assembled vector, otherwise the fragments or vectors are cleaved, so the same restriction sites are preferred. In order to reduce the risk of the assembled fragments and the assembled carrier being cut, and also simple and easy to operate, the requirements for the reaction system are also the simplest.
  • restriction enzymes can be used as the endonuclease of the present invention, and rare restriction enzymes with low probability of occurrence of cleavage sites, such as I-SceI, FseI, AscI, NotI, SfiI.
  • all double-stranded DNA exonucleases can be used in the present invention, preferably T5 exonuclease; all DNA polymerases can be used in the present invention, preferably DNA polymerases having 3'-exo-activity, such as phusion DNA polymerase, etc. All DNA ligases can be used in the present invention, preferably Taq DNA ligase.
  • the assembly vector only needs to have an overlapping sequence and an enzyme cleavage site, preferably the assembled vector described in the above embodiment of the invention.
  • the assembled vector is a linear DNA fragment, it may not have an enzyme cleavage site.
  • the invention can be used for the assembly of DNA fragments of any number and length.
  • double-stranded circular DNA fragments preferably used to assemble 1-20 DNA fragments larger than 200 bp
  • the total length of the fragments is less than 20 kbp
  • double-stranded linear DNA fragments it is preferably used to assemble 1-10 DNA fragments larger than 200 bp
  • the total length of the fragments is less than 20 kbp
  • single-stranded linear DNA fragments it is preferably used to assemble 2-20 (even) DNA fragments larger than 40 nt.
  • the total length is less than 1.5 kbp.
  • the preferred reaction conditions are 50 ° C reaction for 1 h. If some restriction enzymes have low enzymatic activity at 50 ° C, they can be reacted at their optimum temperature (such as 37 ° C) for 0.5-1 h, and then reacted at 50 ° C for 1 h.
  • the assembly buffer in one embodiment of the present invention contains Tris-acetic acid, magnesium ion, potassium ion, dNTPs, BSA (bovine serum albumin) and PEG 8000; preferably, the above assembly buffer further contains DTT (dithiothreitol), NAD + (nicotinamide adenine dinucleotide, coenzyme I); Change within a certain range.
  • the assembly buffer contains Tris-acetic acid 100 mmol/L, magnesium acetate 10 mmol/L, potassium acetate 20 mmol/L, dNTPs 0.2 mmol/L, DTT 10 mmol/L, NAD + 1 mmol/ L, BSA 100 ⁇ g/mL, PEG 8000 5% (mass/volume), pH 7.5.
  • the inventors have confirmed that the content of each of the above components varies within the range of 10% above and below the above value, and the pH is in the range of 7.2 to 7.8, and can be used as the assembly buffer of the present invention.
  • each component described above is an equal-magnification relationship (for example, 2, 3, 5, 6, or 8 times) in the upper and lower 10% range of the above numerical value
  • the pH may be in the range of 7.2 to 7.8, and may be configured as a mother liquid.
  • the assembly buffer is diluted in the corresponding multiples at the time of use to obtain an assembly buffer as a working solution.
  • a kit for in vitro assembly of DNA fragments comprising an assembly buffer and an assembly vector, optionally further comprising a positive control test fragment, an endonuclease, an exonuclease, a DNA polymerase, and At least one of DNA ligase; the kit is used to assemble a plurality of DNA fragments and an assembly vector, wherein the plurality of DNA fragments are respectively arranged in a sequence of fragments, upstream of the first fragment and downstream of the last fragment.
  • the other fragments are upstream with the same overlapping sequence as the downstream of the previous fragment, and the other fragments are downstream with the same overlapping sequence as the upstream of the next fragment;
  • the assembled vector comprises two overlapping sequences, optionally
  • the inside has an enzyme cleavage site.
  • a screening marker is placed between the cleavage sites of the above assembled vector.
  • the positive control test fragment described above has the same structural characteristics as the DNA fragment.
  • a kit may also be included in the specification, and the composition of the kit of the present invention and the method of use thereof may be described in the specification.
  • the assembly buffer and exonuclease (such as T5 DNA exo-cut)
  • the enzyme, DNA polymerase (such as Phusion DNA polymerase) and DNA ligase (such as Taq DNA ligase) are combined into an assembled master mix.
  • the user can carry out the assembly reaction by simply adding the DNA fragment to be assembled, the corresponding restriction enzyme and the assembly vector as required by the instructions.
  • the assembled vector may include a plasmid having at least one antibiotic resistance such as ampicillin (Amp), kanamycin (Kan), chloramphenicol (Cm), tetracycline (Tet), and spectinomycin (Spc).
  • the positive control test fragment can be a plurality (e.g., 3) of assembled test fragments (e.g., Saccharomyces cerevisiae synthetic sequence fragments) cloned on a vector (e.g., pMD18-T) to verify the effectiveness of the assembly system.
  • assembled test fragments e.g., Saccharomyces cerevisiae synthetic sequence fragments
  • vector e.g., pMD18-T
  • the DNA fragment in vitro assembly method described in the present invention can be used for gene synthesis, has few experimental steps, simple operation, and high success rate.
  • the synthesized single-stranded DNA fragments oligos
  • the synthesized single-stranded DNA fragments can be directly assembled into the target fragment by one-step reaction and simultaneously cloned into the assembled vector;
  • a plurality of circular plasmids carrying small DNA fragments are directly assembled into large DNA fragments by one-step assembly reaction, and simultaneously cloned into an assembly vector.
  • the DNA fragment in vitro assembly method described in the present invention can also be used for PCR product cloning and vector construction, has simple operation and high success rate, and is especially suitable for cloning and vector construction of multi-fragment, large-length PCR products.
  • the PCR product can be cloned into an assembled vector by a one-step assembly reaction by simply introducing an overlapping sequence on the primer.
  • the method of the present invention not only has a higher cloning success rate for smaller PCR products (e.g., below 2.5 kbp), but also for larger PCR products (e.g., 2.5 kbp-20 kbp).
  • the DNA fragment in vitro assembly method described in the present invention can also be used for metabolic pathway optimization. Since the ribosome binding site (RBS) and some promoter sequences are short (several to several tens of bp), conventional methods are difficult to manipulate, so the method of the present invention is particularly suitable for RBS optimization and simultaneous optimization of promoter and RBS. Simply add the corresponding overlapping sequence to both ends of the RBS or promoter to be optimized, and then synthesize it in the form of single-stranded DNA and mix it into the assembly reaction, and insert it into the metabolic pathway accurately by one-step assembly.
  • RBS ribosome binding site
  • the test DNA fragments were designated as TF101, TF102, TF103, TF104, TF105 and TF106, respectively.
  • the test fragments contained 20-40 bp homologous regions, and the upstream of TF101 and the downstream of TF106 each had a 40 bp overlapping sequence, and the restriction sites were BssHII.
  • the six fragments are the synthetic yeast chromosome 2 sequence, and the synthetic yeast chromosome 2 sequence information can be found in the patent application 201510008356.4.
  • the assembled vector carries the corresponding 40 bp overlapping sequence and the red fluorescent protein (mRFP1) screening marker, and the cleavage site is I-SceI.
  • the test fragment TF101 was amplified using the primers TF101-F and TF101-R listed in Table 1, and the test fragment TF102 was amplified using primers TF102-F and TF102-R, and the test fragment TF103 was amplified using primers TF103-F and TF103-R.
  • the test fragment TF104 was amplified using primers TF104-F, TF104-R, the test fragment TF105 was amplified using primers TF105-F, TF105-R, and the test fragment TF106 was amplified using primers TF106-F, TF106-R.
  • the TF101-TF105 template was a chunkC4 plasmid (sequence is SEQ ID NO: 1), and the TF106 template was chunkD4 (sequence is SEQ ID NO: 2).
  • the PCR reaction system was: 0.2 ⁇ L of ExTaq, 2 ⁇ L of 10 ⁇ ExTaq buffer, 1.6 ⁇ L of dNTPs, 1 ⁇ L of template DNA (2 ng/ ⁇ L), 1 ⁇ L of each of the positive and negative primers, and 13.2 ⁇ L of ddH 2 O.
  • the PCR reaction procedure was: 94 ° C for 5 min; 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 1.5 min, 30 cycles; 72 ° C for 5 min.
  • the PCR product was purified using a PCR product purification kit.
  • PCR product was cloned into the pMD18-T vector using the TA cloning kit (TAKARA). Enzyme digestion was performed using the designed cleavage site. TF101, TF102, TF103, TF104, TF105 and TF106 plasmids were obtained, respectively.
  • TAKARA TA cloning kit
  • the red fluorescent protein gene was amplified using the primers RFP-F, RFP-R listed in Table 1, and the amplification template was pSB2K3: J04450 (from iGEM, www.igem.org, sequence is SEQ ID NO: 17).
  • the PCR reaction system was: 0.2 ⁇ L of Phusion DNA polymerase, 4 ⁇ L of 5 ⁇ HF buffer, 1.6 ⁇ L of dNTPs, 0.6 ⁇ L of MgCl 2 , 1 ⁇ L of template DNA (2 ng/ ⁇ L), 1 ⁇ L of positive and negative primers, and ddH 2 O 10.6 ⁇ L. .
  • the PCR reaction procedure was: 98 ° C 30 sec; 98 ° C 10 sec, 55 ° C 30 sec, 72 ° C 1 min, 35 cycles; 72 ° C 5 min.
  • the PCR product was purified using a PCR product purification kit.
  • the red fluorescent protein gene fragment and the pSBGAK vector were ligated by Gibson assembly to construct a group
  • the carrier is named pSBGAK-RFP.
  • the pSBGAK vector information and the Gibson assembly experimental method are described in Chinese Patent Application No. 201310085313.7.
  • the reaction system was prepared in a 1.5 mL centrifuge tube: 2 ⁇ L of assembly buffer, 0.25 ⁇ L of Phusion DNA polymerase, 1 ⁇ L of Taq DNA ligase, 0.8 ⁇ L of 1/100 T5 exonuclease, 1 ⁇ L of BssHII, and 0.5 ⁇ L of I-SceI. 280 ng of each of TF101, TF102, TF103, TF104, TF105, and TF106 plasmids, 28 ng of pSBGAK-RFP plasmid, and 20 ⁇ L of ddH 2 O. After reacting at 37 ° C for 30 min, the reaction was carried out at 50 ° C for 1 h.
  • E. coli DH5 ⁇ competent cells 50 ⁇ L of E. coli DH5 ⁇ competent cells were transformed by heat shock method using 10 ⁇ L of the assembly reaction solution, and plated with LB medium (containing kanamycin 50 ⁇ g/mL), and cultured at 37 ° C overnight.
  • Red and white clones were grown on the assembled transformation plates (Fig. 3), and 6 white clones (red clones were empty) were picked and inoculated into 3 mL of LB liquid medium (containing kanamycin 50 ⁇ g/mL). , shake at 37 ° C 200 rpm overnight.
  • plasmid small kit Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • electrophoresis detection 2 ⁇ L of plasmid DNA plus 1 ⁇ L of 10 ⁇ loading buffer, mix and spot; 3 ⁇ L ⁇ -HindIII DNA Marker (TAKARA); 1.2% agarose gel, electrophoresis at 120 V for 40 min.
  • the plasmids of the six clones (lanes 1, 2, 3, 4, 5, 6) all met the expected size, and the results are shown in Fig. 4A.
  • the restriction endonucleases MluI (TAKARA) and XhoI (TAKARA) were used to identify the assembled plasmids.
  • the total length of the assembled plasmids was 11111 bp, and two bands were generated after digestion, and the sizes were 7679 bp and 3432 bp, respectively.
  • the digestion system was: MluI 0.3 ⁇ L, XhoI 0.3 ⁇ L, 10 ⁇ L buffer 1 ⁇ L, plasmid DNA 4 ⁇ L, and ddH 2 O 4.5 ⁇ L.
  • the enzyme was digested at 37 ° C for 1 h.
  • the results of electrophoresis detection were as follows: 1.1 ⁇ L of 10 ⁇ loading buffer was added to the digested product, 6 ⁇ L of the spot was taken, 3 ⁇ L of ⁇ -HindIII DNA Marker (TAKARA); 1.2% agarose gel, and electrophoresis at 120 V for 40 min.
  • the digestion bands of the six plasmids (lanes 1, 2, 3, 4, 5, 6) were all in line with the expected size, and the results are shown in Figure 4B.
  • the results of digestion of all white clones showed that the assembly accuracy of the six fragments was above 80%.
  • the green fluorescent protein (sfGFP) expression cassette assembly was taken as an example to demonstrate the feasibility of the method of the present invention for assembly of mixed-type DNA fragments.
  • the promoter and terminator are cloned on the pMD18-T vector,
  • the chain form exists, the restriction site is BssHII;
  • the RBS sequence exists as single-stranded linear DNA (oligo);
  • the green fluorescent protein gene fragment is a PCR product and exists as double-stranded linear DNA.
  • the cleavage site in the assembled vector is a high-density multiple cloning site, including SfiI, BsiWI, NgoMIV, PspOMI, EagI and other cleavage sites;
  • the screening marker is lacZ gene.
  • the promoters were amplified using the primers Promoter-F and Promoter-R listed in Table 2, and the terminator-F and terminator-R were used to amplify the terminator.
  • the template was pSB2K3: J04450 plasmid.
  • the PCR reaction system and procedure are the same as in Section 1.1.1 of the first embodiment except that the template and the primer are different.
  • the PCR product was purified using a PCR product purification kit.
  • the PCR product was cloned using the TA cloning kit (TAKARA). Promoter and terminator plasmids were obtained, respectively.
  • the RBS sequence is a mixture of four RBSs of different intensities, which are directly synthesized by a DNA synthesizer and purified by PAGE.
  • the RBS sequence is from iGEM (www.igem.org).
  • the four types of RBS information are shown in Table 3.
  • the green fluorescent protein was amplified using the primers GFP-F and GFP-R listed in Table 4, and the template was pUC19-5_emutS_CBM_3_GFP PCR (sequence is SEQ ID NO: 30).
  • the reaction system and procedure were the same except that the template and the primer were different. Section 1.2.
  • the PCR product was purified using a PCR product purification kit.
  • the lacZ was amplified using the primers lacZ-F, lacZ-R listed in Table 4, and the template was pUC19 (Promega).
  • the PCR reaction system and procedure are the same as in Section 1.2 of the first embodiment except that the template and the primer are different.
  • the lacZ was purified using a PCR product purification kit and ligated into the pSBGAK vector into an assembled vector designated pSBGAK-lacZ.
  • the assembly method is the same as the 1.2 in the first embodiment.
  • the reaction system was prepared in a 1.5 mL centrifuge tube: 10 ⁇ L of assembly buffer, 0.25 ⁇ L of Phusion DNA polymerase, 1 ⁇ L of Taq DNA ligase, 0.8 ⁇ L of 1/100 T5 exonuclease, 1 ⁇ L of BssHII, 0.5 ⁇ L of SfiI, promoter
  • the terminator plasmid was 150 ng each, the RBS fragment was 6.25 nM, the green fluorescent protein fragment was 35 ng, the assembled vector was 28 ng, and ddH 2 O was added to make up 20 ⁇ L.
  • the reaction was carried out at 50 ° C for 1 h.
  • E. coli DH5 ⁇ competent cells 50 ⁇ L of E. coli DH5 ⁇ competent cells were transformed by heat shock method using 10 ⁇ L of the assembly reaction solution, and plated with LB medium (containing kanamycin 50 ⁇ g/mL), and cultured at 37 ° C overnight.
  • plasmid small kit Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • electrophoresis detection 2 ⁇ L of plasmid DNA plus 1 ⁇ L of 10 ⁇ loading buffer, mix and spot; 3 ⁇ L ⁇ -HindIII DNA Marker (TAKARA); 1.5% agarose gel, electrophoresis at 180V for 30 min.
  • the plasmids of the 19 clones all met the expected size, and the results are shown in Fig. 6A.
  • Two assembled plasmids were selected and identified by restriction endonuclease XhoI (TAKARA).
  • the total length of the assembled plasmid was 3245 bp, and three bands were generated after digestion, and the sizes were 1456 bp, 1026 bp and 763 bp, respectively.
  • the digestion system was: XhoI 0.5 ⁇ L, 1 ⁇ L of 10 ⁇ H buffer, 4 ⁇ L of plasmid DNA, and 4.5 ⁇ L of ddH 2 O.
  • the enzyme was digested at 37 ° C for 1 h.
  • Electrophoresis detection results Add 1.1 ⁇ L of 10 ⁇ loading buffer to the digested product, take 6 ⁇ L spot, 3 ⁇ L D2000 DNA Marker (Tiangen Biochemical Technology (Beijing) Co., Ltd.); 1.5% agarose gel, 180V The voltage was electrophoresed for 30 min. The enzymatic cleavage bands of the two plasmids all conformed to the expected size, and the results are shown in Fig. 6B.
  • RBS number The number of occurrences Frequency of occurrence BBa_B0034 2 10.5% BBa_B0030 6 31.6% BBa_B0032 7 36.8% BBa_B0031 4 21.1%
  • the assembly buffer of the present invention can be combined with an exonuclease (such as T5 exonuclease), a DNA polymerase (such as Phusion DNA polymerase), and a DNA ligase (such as Taq DNA ligase) into an assembled premix.
  • concentration of the assembly buffer may be N times (eg, 2, 3, 5, 8, or 10 times) of the working solution.
  • the preparation of the 3 ⁇ assembly buffer and the assembled premix in this example was as follows.
  • the plasmid DNA of the assembled vector and the test fragment was extracted using a plasmid extraction kit.
  • the assembled vector DNA concentration was adjusted to 28 ng/ ⁇ L; the test fragment DNA concentration was adjusted to 93 ng/ ⁇ L and stored at -20 °C.
  • Reaction component Amount added Assembly of premix 8.5 ⁇ L Endonuclease 1 ⁇ L Assembling DNA fragments * Assembly carrier 15ng
  • the formulation of 1 ⁇ assembly buffer was as follows: Tris-acetic acid (pH 7.5) 100 mmol/L, magnesium acetate 10 mmol/L, potassium acetate 20 mmol/L, dNTPs 0.2 mmol/L, DTT 10 mmol/L, NAD + 1 mmol/L, BSA 100 ⁇ g/mL, PEG 8000 5% (mass/volume).
  • each component may be varied within a range of 10% above and below the above value, and the pH is in the range of 7.2 to 7.8; or the content of each component is an equal ratio multiple of the upper and lower 10% of the above value, and the pH is Within the range of 7.2 to 7.8.

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Abstract

一种DNA片段体外组装方法和试剂盒，能够将多个DNA片段和组装载体按照设定的顺序组装起来，其中第一个片段上游和最后一个片段下游分别带有与组装载体相同的重叠序列，其它片段上游带有与上一个片段下游相同的重叠序列，下游带有与下一个片段上游相同的重叠序列；组装载体的重叠序列的内侧任选地分别带有酶切位点，任选地在酶切位点之间带有筛选标记；该方法使多个DNA片段和组装载体与DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶进行反应，实现组装。

Description

一种DNA片段体外组装方法和试剂盒 技术领域
本发明涉及DNA组装技术领域，尤其涉及一种DNA片段体外组装方法和试剂盒。
背景技术
DNA化学合成方法虽然可以从头合成一定长度的DNA序列，但是更大片段的DNA合成则必须要通过DNA组装来完成，所以DNA组装技术就成了合成生物学和代谢工程等领域最重要的基础实验技术之一。最早的DNA组装采用的是酶切、连接的方法。酶切、连接的方法经过了几十年的发展，不断得到扩展和完善，是基因工程领域最为经典的基础实验方法之一。如BioBrick方法，只需重复使用4种标准化的限制性内切酶就可以将各种基因元件依次连接到标准载体上，是酶切、连接方法的重要分支之一。酶切、连接方法的另外一个分支——Golden Gate，可以通过酶切、连接一步同时反应将一个或多个线型或环状双链DNA片段组装并连接到组装载体上，也是比较常用的组装方法之一。但是酶切、连接的方法仍然有一些无法克服的缺陷，如酶切位点的限制，连接的片段数量、长度的限制等。
随着合成生物学的发展，尤其是随着基因组人工合成领域的发展，人们对于DNA大片段的需求量极速增加，常规酶切、连接的方法就显得力不从心了。因此近年来又逐渐发展出了很多新的DNA组装技术，其中以利用同源序列进行组装的方法最为常见，形成了一系列体内、体外不同的组装方法，都可以有效的对多个任意的DNA片段进行组装，摆脱了酶切位点的限制。同源区体外组装方法如CPEC、USER、SLIC、Gibson组装等，这些方法都可以利用片段之间的一段同源序列，将多段双链DNA片段有效地连接起来，只是产生同源粘性末端的方式各不相同。其中尤其以Gibson组装方法最为方便，适用范围广，可以通过一步组装反应将多个带有同源区的双链线性DNA片段组装成一个双链线性DNA大片段。Gibson组装还可以实现多个单链DNA片段的组装和克隆。但Gibson组装一步拼接法合成费用高，拼接效率低。另外，利用同源单链DNA桥实现连接的组装方法LCR、利用酵母同源重组***进行组装的酵母体内组装方 法等也是DNA多片段组装的有效方法，且这两种方法是目前组装效率最高的方法。
但是现有方法仍然不够完美，更高的组装效率、更低的错误率、更简单快速的操作流程仍然是人们在DNA组装技术开发上不断追求的目标。
发明内容
本发明提供一种依赖于重叠序列的DNA片段体外组装方法，可完成多个DNA片段的组装和克隆。相应地，本发明还提供用于DNA片段体外组装的试剂盒。
根据本发明的第一方面，本发明提供一种DNA片段体外组装方法，包括将多个DNA片段和组装载体组装起来的步骤；其中上述多个DNA片段按照设定的片段顺序，第一个片段上游和最后一个片段下游分别带有与上述组装载体相同的重叠序列，其它片段上游带有与上一个片段下游相同的重叠序列，并且其它片段下游带有与下一个片段上游相同的重叠序列；上述组装载体包括两个重叠序列，任选地其内侧分别带有酶切位点，任选地在酶切位点之间带有筛选标记；上述方法使上述多个DNA片段和上述组装载体与识别上述酶切位点的DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶接触进行反应，将上述多个DNA片段按照设定的组装顺序组装到上述组装载体上。
根据本发明的第二方面，本发明提供一种用于DNA片段体外组装的试剂盒，包括组装缓冲液和组装载体；上述试剂盒用于将多个DNA片段和组装载体组装起来，其中上述多个DNA片段按照设定的片段顺序，第一个片段上游和最后一个片段下游分别带有与上述组装载体相同的重叠序列，其它片段上游带有与上一个片段下游相同的重叠序列，并且其它片段下游带有与下一个片段上游相同的重叠序列；上述组装载体包括两个重叠序列，任选地其内侧分别带有酶切位点，任选地在酶切位点之间带有筛选标记；任选地，上述试剂盒还包括阳性对照测试片段、DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶的至少一种。
附图说明
图1为本发明的一个实施方案的双链环状DNA片段组装原理示意图；
图2为本发明的一个实施方案的混合类型DNA片段组装原理示意图；
图3为本发明的一个实施方案的双链环状DNA片段组装测试平板图；
图4为本发明的一个实施方案的双链环状DNA片段组装质粒及酶切电泳图；
图5为本发明的一个实施方案的混合类型DNA片段组装测试平板图；
图6为本发明的一个实施方案的混合类型DNA片段组装质粒及酶切电泳图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
本发明的实施方案描述了一种依赖于重叠序列的DNA片段体外组装方法，组装反应可以在同一反应体系中进行，可完成多个DNA片段的组装和克隆。
如图1所示，本发明的一个实施方案，对于多个克隆在载体上的双链DNA片段(即片段1、片段2和片段3，在其它实施方案中片段数量可以不限于3个)按照设定的片段顺序(即依次是片段1、片段2和片段3的顺序)，第一个片段(片段1)上游(01)和最后一个片段(片段3)下游(02)分别带有与组装载体相同的重叠序列(01和02)，其它片段(片段2)上游(03)带有与上一个片段(片段1)下游(03)相同的重叠序列，并且其它片段(片段2)下游(04)带有与下一个片段(片段3)上游(04)相同的重叠序列，每个片段(片段1、片段2和片段3)的重叠序列(01、03、04、02)的外侧分别带有酶切位点(E)；组装载体具有两个重叠序列(01和02)，其内侧分别带有酶切位点(E)，在酶切位点之间带有筛选标记(图1A)。
需要说明的是，图1示出的片段和载体都是完整的双链环状DNA结构，因此带有酶切位点。如果片段和载体是线性DNA片段，可以不带有酶切位点，因此酶切位点是“任选地”，即视具体情况可以带有也可以不带有酶切位点。类似地，载体上可以有也可以没有筛选标记，优选有筛选标记，以提高筛选效率。
需要说明的是，在上述实施方案中，虽然说明组装载体具有两个重叠序列(01和02)，但是应当理解，本发明的方法并不限定组装载体只能包括两个重叠序列，而是至少两个(比如三个、四个或更多)重叠序列均可，其它重叠序列比如可以位于与筛选标记相邻的位置。
在反应体系中加入上述多个DNA片段和组装载体，并同时加入识别酶切位点(E)的DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶。首先DNA内切酶起作用，将待组装的DNA片段和组装载体骨架从质粒上切下来(图1B)，紧接着DNA外切酶起作用，将片段和组装载体的末端重叠区切割，露出互补的粘性末端，同时粘性末端互补配对，待组装片段和组装载体依次连接成环状(图1C)；然后DNA聚合酶切除突出的酶切位点残留序列，并补齐外切酶留下的缺口，最后DNA连接酶起作用，将补平的开环质粒连接成双链闭合环状质粒(图1D)，组装完成。组装反应在组装缓冲液中进行，组装缓冲液要求可以同时兼容DNA外切酶、DNA聚合酶、DNA连接酶以及多种DNA内切酶。反应产物可直接用于大肠杆菌等细菌转化，在组装载体抗性培养基上长出单克隆，通过组装载体上的筛选标记去除空载体，挑取阳性克隆，提质粒即得到大量组装成功的DNA片段。
需要说明的是，虽然本发明的方法优选在同一反应体系的组装缓冲液中，使多个DNA片段和组装载体同时与识别酶切位点的DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶接触进行反应，也就是优选一步反应；然而，基于本发明的精神，也可以分步进行，即将上述DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶依次加入反应体系中分别进行反应。
如图2所示，本发明的方法不仅可以用于(a)均是克隆在载体上的双链DNA片段(即双链环状DNA片段)的组装，这种情况下，双链DNA片段两侧的重叠序列的外侧分别带有能被上述DNA内切酶识别的酶切位点；同时也可用于(b)均是双链线性DNA片段组装，(c)均是单链线性DNA片段组装，这种情况下，单链线性DNA片段的数量为偶数，以及(d)克隆在载体上的双链DNA片段、双链线性DNA片段和单链线性DNA片段中任意2种或3种类型DNA片段的混合组装，在存在克隆在载体上的双链DNA片段的情况下，双链DNA片段两侧的重叠序列的外侧分别带有能被上述DNA内切酶识别的酶切位点；在存在单链线性DNA片段的情况下，单链线性DNA片段的数量为偶数。片段之间只需带有如上所述及如图1所示的重叠序列即可。组装原理与双链环状DNA片段组装类似。
本发明的一个实施方案中，重叠序列长度大于15bp即可实现有效的组装，优选20～40bp。
本发明的一个实施方案中，组装载体中的筛选标记可以是任何在细菌中可用的标记物，如抗生素抗性基因、荧光蛋白基因、致死基因、lacZ基因等，或它们之间的任意组合，优选红色荧光蛋白基因或lacZ基因与红色荧光蛋白组基因的组合。
本发明的一个实施方案中，酶切位点可以是任意限制性内切酶识别位点或多克隆酶切位点(MCS)，优选归位内切酶(如I-SceI)或高密度多克隆位点(如GGCGCGCCGGCGCC、GTCGACCGGACCGCGGGTC、GGGCCCGCCGGCGCGCC、CGCCGGCGCGCCGCGGCCGC、GACGACGTCGACCGCGGGTC、ACCGGACCGCGGGTCACC、GCGCGCCGGCGCC等)。
需要说明的是，组装片段和组装载体的酶切位点要求：所有组装片段和组装载体上均不存在该酶切位点，酶切位点可相同也可不同。由于是在同一个体系中进行反应，并要求这些酶切位点都必须在所有组装片段和组装载体上都不存在，否则就会有片段或载体被切断，因此优选相同的酶切位点，以减少组装片段和组装载体被切断的风险，并且也简单易行，易于操作，对于反应体系的要求也最简单。
需要说明的是，所有限制性内切酶均可作为本发明的DNA内切酶，优选酶切位点出现概率低的罕见限制性内切酶，如I-SceI、FseI、AscI、NotI、SfiI、AsiSI、SbfI、RsrII、PmeI、SgrAI、TspMI、XmaI、EagI、BssHII、SacII、PacI、NaeI、NgoMIV、MluI、PspXI、AvrII、NruI、PspOMI、PvuI、BsiWI、ApaLI、PmlI、BbvCI、PshAI、AhdI、KasI、NarI、SfoI、BmtI、NheI、SalI、NmeAIII、FspI、AatII、ZraI、BglI、BlpI、AgeI、BsrBI、AleI、DraIII、Bsu36I等。
此外，所有双链DNA外切酶均可用于本发明，优选T5DNA外切酶；所有DNA聚合酶均可用于本发明，优选具有3’-外切活性的DNA聚合酶，如phusion DNA聚合酶等；所有DNA连接酶均可用于本发明，优选Taq DNA连接酶。
在本发明的上述实施方案所描述的方法中，组装载体只需带有重叠序列和酶切位点即可，优选本发明的上述实施方案中描述的组装载体。如上所述，如果组装载体是线性DNA片段，可以不带有酶切位点。
本发明可用于任意数量、任意长度DNA片段的组装。对于双链环状DNA片段，优选用于组装1-20个大于200bp的DNA片段，片段总长度小于20kbp； 对于双链线性DNA片段，优选用于组装1-10个大于200bp的DNA片段，片段总长度小于20kbp；对于单链线性DNA片段，优选用于组装2-20个(偶数)大于40nt的DNA片段，总长度小于1.5kbp。
本发明的上述实施方案所描述的方法，优选的反应条件为50℃反应1h。如遇到有些限制性内切酶在50℃条件下酶切活性较低时，可先在其最适温度(如37℃)下反应0.5-1h，再在50℃反应1h即可。
根据本发明中的DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶的反应需要，本发明的一个实施方案中的组装缓冲液含有Tris-醋酸、镁离子、钾离子、dNTPs、BSA(牛血清白蛋白)和PEG8000；优选上述组装缓冲液还含有DTT(二硫苏糖醇)、NAD+(烟酰胺腺嘌呤二核苷酸，辅酶I)；其中各种成分的含量可以在一定范围内变化。在本发明的一个进一步优选的实施例中，组装缓冲液含有Tris-醋酸100mmol/L，醋酸镁10mmol/L，醋酸钾20mmol/L，dNTPs 0.2mmol/L，DTT 10mmol/L，NAD+1mmol/L，BSA 100μg/mL，PEG8000 5％(质量/体积)，pH为7.5。发明人已经证实，上述各组分的含量在上述数值的上下10％范围内变动，pH在7.2～7.8的范围内，均可作为本发明的组装缓冲液。此外，按照上述各组分的含量是上述数值的上下10％范围的等比例倍数关系(例如2、3、5、6或8倍)，pH在7.2～7.8的范围内，可以配置成作为母液的组装缓冲液，在使用时按照相应倍数稀释，即可得到作为工作液的组装缓冲液。
本发明的一个实施方案中，用于DNA片段体外组装的试剂盒，包括组装缓冲液和组装载体，任选地还包括阳性对照测试片段、DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶的至少一种；该试剂盒用于将多个DNA片段和组装载体组装起来，其中多个DNA片段按照设定的片段顺序，第一个片段上游和最后一个片段下游分别带有与组装载体相同的重叠序列，其它片段上游带有与上一个片段下游相同的重叠序列，并且其它片段下游带有与下一个片段上游相同的重叠序列；组装载体包括两个重叠序列，任选地其内侧分别带有酶切位点。任选地，上述组装载体的酶切位点之间带有筛选标记。任选地，上述阳性对照测试片段具有与DNA片段相同的结构特点。任选地，试剂盒中还可以包括说明书，说明书中可以记载本发明试剂盒的组成以及使用方法。
本发明的一个优选实施例中，组装缓冲液与DNA外切酶(如T5 DNA外切 酶)、DNA聚合酶(如Phusion DNA聚合酶)和DNA连接酶(如Taq DNA连接酶)组合成组装预混液。使用者只需按说明书要求加入待组装的DNA片段、相应的限制性内切酶和组装载体即可进行组装反应。组装载体可以包括带有氨苄青霉素(Amp)、卡那霉素(Kan)、氯霉素(Cm)、四环素(Tet)和壮观霉素(Spc)等至少一种抗生素抗性的质粒。阳性对照测试片段可以是多个(如3个)克隆在载体(如pMD18-T)上的组装测试片段(如酿酒酵母合成序列片段)，用以验证组装体系的有效性。
本发明中所描述的DNA片段体外组装方法可用于基因合成，实验流程步骤少，操作简单，且成功率高。对于较小的DNA片段，可以通过一步反应直接将合成的多个单链DNA片段(oligos)直接组装成目标片段，并同时克隆到组装载体上；对于较大的DNA片段或代谢通路，可以再通过一步组装反应，将多个带有DNA小片段的环状质粒直接组装成DNA大片段，同时克隆到组装载体上。需要说明的是，目前所有基于同源区重叠序列的组装方法均不能通过一步反应完成对克隆在载体上的DNA片段的组装(包括混合有克隆在载体上的DNA片段的多种类型片段的组装)，而本发明能够实现这一点。
本发明中所描述的DNA片段体外组装方法还可用于PCR产物克隆和载体构建，操作简单，成功率高，尤其适用于多片段、大片段PCR产物的克隆和载体构建。对于线性的PCR产物，只需在引物上引入重叠序列，即可通过一步组装反应将PCR产物克隆到组装载体上。而且本发明的方法不仅对较小的PCR产物(如2.5kbp以下)有较高的克隆成功率，对较大的PCR产物(如2.5kbp-20kbp)仍然可以进行。
高效克隆对于多片段PCR产物(若片段太大扩增效果不好，可进行分段PCR扩增)或多片段载体构建，只需在引物上互相引入片段同源区或重叠序列，仍仅需一步组装反应即可将多个PCR产物或载体片段组装并克隆到组装载体上，从而实现多片段的高效克隆，这是常规TA克隆或酶切连接无法完成的。
本发明中所描述的DNA片段体外组装方法还可用于代谢通路优化。由于核糖体结合位点(RBS)和一些启动子序列较短(几至几十个bp)，常规方法很难操作，所以本发明的方法尤其适用于RBS优化以及启动子和RBS同时优化。只需在待优化的RBS或启动子两端加上相应的重叠序列，再以单链DNA的形式合成后混合加入组装反应中，通过一步组装即可准确地***到代谢通路中。
以下通过具体实施例详细描述本发明。实施例中所描述的实验流程仅用于证明专利方法的可行性，发明的方法的应用并不仅限于此。实施例中所提及的实验操作，如无特殊说明，均为常规实验方法；所提及的试剂耗材，如无特殊说明，均为常规试剂耗材。
实施例一：多个环状DNA片段组装测试
以6个1.5kbp左右的DNA片段为例，证明本发明的方法对于多个双链环状DNA片段组装的可行性。测试DNA片段分别命名为TF101、TF102、TF103、TF104、TF105和TF106。测试片段之间分别带有20～40bp同源区，TF101上游、TF106下游分别带有40bp的重叠序列，酶切位点均为BssHII。6个片段均为合成酵母2号染色体序列，合成酵母2号染色体序列信息见专利申请201510008356.4。组装载体带有相应的40bp重叠序列和红色荧光蛋白(mRFP1)筛选标记，酶切位点为I-SceI。
1.1测试DNA片段制备
1.1.1PCR扩增
使用表1所列的引物TF101-F、TF101-R扩增测试片段TF101，使用引物TF102-F、TF102-R扩增测试片段TF102，使用引物TF103-F、TF103-R扩增测试片段TF103，使用引物TF104-F、TF104-R扩增测试片段TF104，使用引物TF105-F、TF105-R扩增测试片段TF105，使用引物TF106-F、TF106-R扩增测试片段TF106。TF101-TF105模版为chunkC4质粒(序列为SEQ ID NO：1)，TF106模版为chunkD4(序列为SEQ ID NO：2)。PCR反应体系为：ExTaq 0.2μL，10×ExTaq buffer 2μL，dNTPs 1.6μL，模版DNA(2ng/μL)1μL，正、反向引物各1μL，ddH2O 13.2μL。PCR反应程序为：94℃ 5min；94℃ 30sec，55℃ 30sec，72℃ 1.5min，30个循环；72℃ 5min。使用PCR产物纯化试剂盒纯化PCR产物。
表1
Figure PCTCN2015092438-appb-000001
Figure PCTCN2015092438-appb-000002
1.1.2TA克隆
使用TA克隆试剂盒(TAKARA)将PCR产物克隆至pMD18-T载体上。使用设计的酶切位点进行酶切鉴定。分别得到TF101、TF102、TF103、TF104、TF105和TF106质粒。
1.2组装载体制备
使用表1所列的引物RFP-F、RFP-R扩增红色荧光蛋白基因，扩增模版为pSB2K3：J04450(来自iGEM，www.igem.org，序列为SEQ ID NO：17)。PCR反应体系为：Phusion DNA聚合酶0.2μL，5×HF缓冲液4μL，dNTPs 1.6μL，MgCl20.6μL，模版DNA(2ng/μL)1μL，正、反向引物各1μL，ddH2O 10.6μL。PCR反应程序为：98℃ 30sec；98℃ 10sec，55℃ 30sec，72℃ 1min，35个循环；72℃ 5min。使用PCR产物纯化试剂盒纯化PCR产物。
通过Gibson组装将红色荧光蛋白基因片段和pSBGAK载体连接，构建成组 装载体，命名为pSBGAK-RFP。pSBGAK载体信息和Gibson组装实验方法见申请号为201310085313.7的中国发明专利申请。
1.3组装测试
1.3.1组装反应
在1.5mL离心管中配制反应体系：2×组装缓冲液10μL，Phusion DNA聚合酶0.25μL，Taq DNA连接酶1μL，1/100 T5 DNA外切酶0.8μL，BssHII 1μL，I-SceI 0.5μL，TF101、TF102、TF103、TF104、TF105、TF106质粒各280ng，pSBGAK-RFP质粒28ng，加ddH2O补足20μL。37℃反应30min后50℃反应1h。
取10μL组装反应液热激法转化50μL大肠杆菌DH5α感受态细胞，涂布LB培养基(含卡那霉素50μg/mL)平板，37℃培养过夜。
1.3.2组装结果鉴定
组装转化平板上长出有红色和白色克隆(如图3)，挑6个白色克隆(红色克隆为空载体)，分别各接种至3mL LB液体培养基(含卡那霉素50μg/mL)中，37℃ 200rpm摇培过夜。
各取1.5mL菌液，使用质粒小提试剂盒(天根生化科技(北京)有限公司)提质粒，电泳检测：2μL质粒DNA加1μL 10×上样缓冲液，混匀点样；3μLλ-HindIII DNA Marker(TAKARA)；1.2％琼脂糖凝胶，120V电压电泳40min。6个克隆(泳道1、2、3、4、5、6)的质粒均符合预期大小，结果如图4A。
选取限制性内切酶MluI(TAKARA)和XhoI(TAKARA)对组装质粒进行酶切鉴定，组装成功的质粒总长度为11111bp，酶切后产生2条带，大小分别为7679bp和3432bp。酶切体系为：MluI 0.3μL，XhoI 0.3μL，10×H缓冲液1μL，质粒DNA 4μL，ddH2O 4.5μL。37℃酶切1h。
电泳检测酶切结果：在酶切产物中加1.1μL 10×上样缓冲液，取6μL点样，3μLλ-HindIII DNA Marker(TAKARA)；1.2％琼脂糖凝胶，120V电压电泳40min。6个质粒(泳道1、2、3、4、5、6)的酶切带型均符合预期大小，结果如图4B。所有白色克隆的酶切结果显示，6个片段的组装正确率都在80％以上。
实施例二：三种类型DNA片段混合组装测试
以绿色荧光蛋白(sfGFP)表达框组装为例，证明本发明的方法对混合类型DNA片段组装的可行性。其中启动子、终止子克隆在pMD18-T载体上，以双 链环状形式存在，酶切位点为BssHII；RBS序列以单链线性DNA(oligo)形式存在；绿色荧光蛋白基因片段为PCR产物，以双链线性DNA形式存在。组装载体中酶切位点为高密度多克隆位点，包含SfiI、BsiWI、NgoMIV、PspOMI、EagI等酶切位点；筛选标记为lacZ基因。
2.1DNA片段及组装载体制备
使用表2所列的引物promoter-F、promoter-R扩增启动子，使用引物terminator-F、terminator-R扩增终止子。模版为pSB2K3：J04450质粒。PCR反应体系和程序除模板和引物不同外，其它同实施例一中的1.1.1节。使用PCR产物纯化试剂盒纯化PCR产物。使用TA克隆试剂盒(TAKARA)克隆PCR产物。分别得到启动子、终止子质粒。
表2
Figure PCTCN2015092438-appb-000003
RBS序列为不同强度的4种RBS混合而成，直接由DNA合成仪合成，PAGE纯化。RBS序列来自iGEM(www.igem.org)。4种RBS信息分别为表3所示。
表3
Figure PCTCN2015092438-appb-000004
Figure PCTCN2015092438-appb-000005
使用表4所列的引物GFP-F、GFP-R扩增绿色荧光蛋白，模版为pUC19-5_emutS_CBM_3_GFPPCR(序列为SEQ ID NO：30)反应体系和程序除模板和引物不同外，其它同实施例一中的1.2节。使用PCR产物纯化试剂盒纯化PCR产物。
使用表4所列的引物lacZ-F、lacZ-R扩增lacZ，模版为pUC19(Promega)。PCR反应体系和程序除模板和引物不同外，其它同实施例一中的1.2节。使用PCR产物纯化试剂盒纯化lacZ，与pSBGAK载体连接成组装载体，命名为pSBGAK-lacZ。组装方法同实施例一中的1.2节。
表4
Figure PCTCN2015092438-appb-000006
Figure PCTCN2015092438-appb-000007
2.2组装测试
在1.5mL离心管中配制反应体系：2×组装缓冲液10μL，Phusion DNA聚合酶0.25μL，Taq DNA连接酶1μL，1/100 T5 DNA外切酶0.8μL，BssHII 1μL，SfiI 0.5μL，启动子、终止子质粒各150ng，RBS片段各6.25nM，绿色荧光蛋白片段35ng，组装载体28ng，加ddH2O补足20μL。50℃反应1h。
取10μL组装反应液热激法转化50μL大肠杆菌DH5α感受态细胞，涂布LB培养基(含卡那霉素50μg/mL)平板，37℃培养过夜。
2.3组装结果鉴定
2.3.1酶切鉴定
在蓝光下可以看出组装转化平板上长出的绿色克隆(如图5，圆圈中为不同荧光强度的绿色克隆)，挑19个克隆分别各接种至3mL LB液体培养基(含卡那霉素50μg/mL)中，37℃ 200rpm摇培过夜。
各取1.5mL菌液，使用质粒小提试剂盒(天根生化科技(北京)有限公司)提质粒，电泳检测：2μL质粒DNA加1μL 10×上样缓冲液，混匀点样；3μLλ-HindIII DNA Marker(TAKARA)；1.5％琼脂糖凝胶，180V电压电泳30min。19个克隆的质粒均符合预期大小，结果如图6A。
选取2个组装质粒，使用限制性内切酶XhoI(TAKARA)对其进行酶切鉴定。组装成功的质粒总长度为3245bp，酶切后产生3条带，大小分别为1456bp、1026bp和763bp。酶切体系为：XhoI 0.5μL，10×H缓冲液1μL，质粒DNA 4μL，ddH2O 4.5μL。37℃酶切1h。
电泳检测酶切结果：在酶切产物中加1.1μL 10×上样缓冲液，取6μL点样，3μL D2000 DNA Marker(天根生化科技(北京)有限公司)；1.5％琼脂糖凝胶，180V电压电泳30min。2个质粒的酶切条带均符合预期大小，结果如图6B。
2.3.2测序分析
对19个组装质粒，使用引物RBSSeq-1F(序列为SEQ ID NO：35)进行sanger 测序，分析4种RBS在组装中的***情况。19个质粒测序均发现预期的RBS序列且具有较好的随机性，RBS分布情况如表5所示。
表5
RBS编号 出现次数 出现频率
BBa_B0034 2 10.5％
BBa_B0030 6 31.6％
BBa_B0032 7 36.8％
BBa_B0031 4 21.1％
实施例三：组装试剂盒制备
3.1组装预混液配制
本发明中的组装缓冲液可以与DNA外切酶(如T5 DNA外切酶)、DNA聚合酶(如Phusion DNA聚合酶)和DNA连接酶(如Taq DNA连接酶)组合成组装预混液。其中组装缓冲液的浓度可以是工作液的N倍(如2、3、5、8或10倍)。本实施例中的3×组装缓冲液和组装预混液的配制如下。
配制3×组装缓冲液；取1mol/L的Tris-醋酸(pH 7.5)300μL，1 mol/L的醋酸镁30μL，1 mol/L的醋酸钾60μL，10 mmol/L的dNTPs 60μL，1 mol/L的DTT30μL，50 mmol/L的NAD+60μL，10 mg/mL的BSA 30μL，PEG8000 0.15g，加ddH2O定容至1 mL，混匀，-20℃保存。
配制组装预混液：3×组装缓冲液333μL，Phusion DNA聚合酶25μL，T5 DNA外切酶0.4μL，Taq DNA连接酶50μL，ddH2O 16.6μL，混匀，分装到1.5 mL离心管，8.5μL/管，-20℃保存。
3.2组装载体及测试片段制备
使用质粒提取试剂盒提取组装载体及测试片段的质粒DNA。组装载体DNA浓度调至28 ng/μL；测试片段DNA浓度调至93 ng/μL，-20℃保存。
3.3反应体系及条件
反应成份 加入量
组装预混液 8.5μL
内切酶 1μL
组装DNA片段 *
组装载体 15ng
ddH2O 补足20μL
50℃反应1h**。
*：组装DNA片段的摩尔浓度为组装载体的5倍。
**：如内切酶在50℃条件下活性较差，可先在37℃反应30min，再在50℃反应1h。
试剂配方
1×组装缓冲液的配方如下：Tris-醋酸(pH7.5)100mmol/L，醋酸镁10mmol/L，醋酸钾20mmol/L，dNTPs 0.2mmol/L，DTT 10mmol/L，NAD+1mmol/L，BSA 100μg/mL，PEG8000 5％(质量/体积)。
上述各组分的含量可在上述数值的上下10％范围内变动，pH在7.2～7.8的范围内；或者上述各组分的含量为上述数值的上下10％范围的等比例倍数关系，pH在7.2～7.8的范围内。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种DNA片段体外组装方法，其特征在于，包括将多个DNA片段和组装载体组装起来的步骤；其中所述多个DNA片段按照设定的片段顺序，第一个片段上游和最后一个片段下游分别带有与所述组装载体相同的重叠序列，其它片段上游带有与上一个片段下游相同的重叠序列，并且其它片段下游带有与下一个片段上游相同的重叠序列；所述组装载体包括两个重叠序列，任选地其内侧分别带有酶切位点，任选地在酶切位点之间带有筛选标记；所述方法使所述多个DNA片段和所述组装载体与识别所述酶切位点的DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶接触进行反应，将所述多个DNA片段按照设定的组装顺序组装到所述组装载体上。
  2. 根据权利要求1所述的方法，其特征在于，所述方法在同一反应体系的组装缓冲液中，使所述多个DNA片段和所述组装载体同时与识别所述酶切位点的DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶接触进行反应。
  3. 根据权利要求1所述的方法，其特征在于，所述多个DNA片段选自如下情况中的任意一种：
    (a)均是克隆在载体上的双链DNA片段，所述双链DNA片段两侧的重叠序列的外侧分别带有能被所述DNA内切酶识别的酶切位点；
    (b)均是双链线性DNA片段；
    (c)均是单链线性DNA片段，且所述单链线性DNA片段的数量为偶数；或
    (d)包括克隆在载体上的双链DNA片段、双链线性DNA片段和单链线性DNA片段中的任意2种或3种；在存在克隆在载体上的双链DNA片段的情况下，所述双链DNA片段两侧的重叠序列的外侧分别带有能被所述DNA内切酶识别的酶切位点；在存在单链线性DNA片段的情况下，所述单链线性DNA片段的数量为偶数。
  4. 根据权利要求1所述的方法，其特征在于，所述筛选标记选自抗生素抗性基因、荧光蛋白基因、致死基因、lacZ基因或它们之间的任意组合；
    任选地，所述酶切位点选自限制性内切酶识别位点或多克隆酶切位点；
    任选地，所述DNA内切酶为限制性内切酶；
    任选地，所述DNA外切酶为T5DNA外切酶；
    任选地，所述DNA聚合酶为phusion DNA聚合酶；
    任选地，所述DNA连接酶为Taq DNA连接酶。
  5. 根据权利要求1所述的方法，其特征在于，所述重叠序列的长度大于15bp。
  6. 根据权利要求2所述的方法，其特征在于，所述组装缓冲液含有Tris-醋酸、镁离子、钾离子、dNTPs、BSA和PEG8000；
    任选地，所述组装缓冲液还含有DTT和NAD+。
  7. 根据权利要求6所述的方法，其特征在于，所述组装缓冲液含有Tris-醋酸100mmol/L，醋酸镁10mmol/L，醋酸钾20mmol/L，dNTPs 0.2mmol/L，DTT10mmol/L，NAD+1mmol/L，BSA 100μg/mL，PEG80005％(质量/体积)，pH为7.5；或者上述各组分的含量在上述数值的上下10％范围内变动，pH在7.2～7.8的范围内；或者上述各组分的含量为上述数值的上下10％范围的等比例倍数关系，pH在7.2～7.8的范围内。
  8. 一种用于DNA片段体外组装的试剂盒，其特征在于，包括组装缓冲液和组装载体；
    所述试剂盒用于将多个DNA片段和组装载体组装起来，其中所述多个DNA片段按照设定的片段顺序，第一个片段上游和最后一个片段下游分别带有与所述组装载体相同的重叠序列，其它片段上游带有与上一个片段下游相同的重叠序列，并且其它片段下游带有与下一个片段上游相同的重叠序列；所述组装载体包括两个重叠序列，任选地其内侧分别带有酶切位点，任选地在酶切位点之间带有筛选标记；
    任选地，所述试剂盒还包括阳性对照测试片段、DNA内切酶、DNA外切酶、DNA聚合酶和DNA连接酶的至少一种。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述DNA内切酶为限制性内切酶，所述DNA外切酶为T5DNA外切酶，所述DNA聚合酶为phusion DNA聚合酶，所述DNA连接酶为Taq DNA连接酶；
    任选地，所述组装缓冲液与所述DNA外切酶、DNA聚合酶和DNA连接酶组合成组装预混液。
  10. 根据权利要求8-9任一项所述的试剂盒，其特征在于，所述组装缓冲液含有Tris-醋酸、镁离子、钾离子、dNTPs、BSA和PEG8000；
    任选地，所述组装缓冲液还含有DTT和NAD+；
    优选地，所述组装缓冲液含有Tris-醋酸100mmol/L，醋酸镁10mmol/L，醋酸钾20mmol/L，dNTPs 0.2mmol/L，DTT 10mmol/L，NAD+1mmol/L，BSA100μg/mL，PEG80005％(质量/体积)，pH为7.5；或者上述各组分的含量在上述数值的上下10％范围内变动，pH在7.2～7.8的范围内；或者上述各组分的含量为上述数值的上下10％范围的等比例倍数关系，pH在7.2～7.8的范围内。
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110951759A (zh) * 2019-12-27 2020-04-03 苏州泓迅生物科技股份有限公司 一种超大质粒的体外组装方法及其应用
CN110964738A (zh) * 2019-12-27 2020-04-07 苏州泓迅生物科技股份有限公司 一种多片段dna组装试剂盒、多片段dna组装方法及其应用
CN110982834A (zh) * 2019-12-27 2020-04-10 苏州泓迅生物科技股份有限公司 一种质粒构建试剂盒、质粒构建方法及其应用
CN111004813A (zh) * 2019-12-27 2020-04-14 苏州泓迅生物科技股份有限公司 一种超大质粒构建试剂盒、超大质粒构建方法及其应用
CN112175939A (zh) * 2019-07-03 2021-01-05 华大青兰生物科技(无锡)有限公司 一种基于同源序列的核酸拼接方法及其应用
WO2021214258A1 (en) 2020-04-22 2021-10-28 Fabmid Methods for circularizing linear double stranded nucleic acids
CN114250242A (zh) * 2021-12-29 2022-03-29 上海英基生物科技有限公司 一种一步法BbsI酶切连接片段组装方法、组装试剂盒及应用
WO2023072958A1 (en) 2021-10-25 2023-05-04 Fabmid Methods for circularizing linear double stranded nucleic acids and the products thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112011471B (zh) * 2019-05-31 2022-07-22 深圳华大生命科学研究院 酿制柠檬风味啤酒的酵母菌株、其制备方法及啤酒酿制方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101445807A (zh) * 2008-12-24 2009-06-03 宜春学院 一种新型高效的复杂载体构建的方法
CN102016070A (zh) * 2008-02-15 2011-04-13 合成基因组公司 体外连接和组合装配核酸分子的方法
US20120245100A1 (en) * 2011-03-22 2012-09-27 Brennan Winkler Plasmid for expression of transgenes in plants
CN104593354A (zh) * 2015-01-14 2015-05-06 江南大学 一种基于体外组合组装快速定向进化dna的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102016070A (zh) * 2008-02-15 2011-04-13 合成基因组公司 体外连接和组合装配核酸分子的方法
CN101445807A (zh) * 2008-12-24 2009-06-03 宜春学院 一种新型高效的复杂载体构建的方法
US20120245100A1 (en) * 2011-03-22 2012-09-27 Brennan Winkler Plasmid for expression of transgenes in plants
CN104593354A (zh) * 2015-01-14 2015-05-06 江南大学 一种基于体外组合组装快速定向进化dna的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JI, ZHICHENG ET AL.: "Application of Gibson Assembly Method for Plant Expression Vector Construction", JOURNAL OF SOUTH CHINA AGRICULTURAL UNIVERSITY, vol. 35, no. 5, 17 July 2014 (2014-07-17), pages 112 - 116, ISSN: 1001-411X *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112175939A (zh) * 2019-07-03 2021-01-05 华大青兰生物科技(无锡)有限公司 一种基于同源序列的核酸拼接方法及其应用
CN110951759A (zh) * 2019-12-27 2020-04-03 苏州泓迅生物科技股份有限公司 一种超大质粒的体外组装方法及其应用
CN110964738A (zh) * 2019-12-27 2020-04-07 苏州泓迅生物科技股份有限公司 一种多片段dna组装试剂盒、多片段dna组装方法及其应用
CN110982834A (zh) * 2019-12-27 2020-04-10 苏州泓迅生物科技股份有限公司 一种质粒构建试剂盒、质粒构建方法及其应用
CN111004813A (zh) * 2019-12-27 2020-04-14 苏州泓迅生物科技股份有限公司 一种超大质粒构建试剂盒、超大质粒构建方法及其应用
WO2021214258A1 (en) 2020-04-22 2021-10-28 Fabmid Methods for circularizing linear double stranded nucleic acids
WO2023072958A1 (en) 2021-10-25 2023-05-04 Fabmid Methods for circularizing linear double stranded nucleic acids and the products thereof
CN114250242A (zh) * 2021-12-29 2022-03-29 上海英基生物科技有限公司 一种一步法BbsI酶切连接片段组装方法、组装试剂盒及应用

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