WO2015103741A1 - Procédé efficace de clonage de gène et ses utilisations - Google Patents

Procédé efficace de clonage de gène et ses utilisations Download PDF

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WO2015103741A1
WO2015103741A1 PCT/CN2014/070296 CN2014070296W WO2015103741A1 WO 2015103741 A1 WO2015103741 A1 WO 2015103741A1 CN 2014070296 W CN2014070296 W CN 2014070296W WO 2015103741 A1 WO2015103741 A1 WO 2015103741A1
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vector
cloning
spacer
dna fragment
dna fragments
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PCT/CN2014/070296
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English (en)
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Junhua Liu
Guanfan MAO
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Pioneer Overseas Corporation
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Priority to CN201480071699.9A priority Critical patent/CN106103712B/zh
Priority to PCT/CN2014/070296 priority patent/WO2015103741A1/fr
Publication of WO2015103741A1 publication Critical patent/WO2015103741A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—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
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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

Definitions

  • the field is in the area of molecular biology, particularly with regards to gene cloning.
  • PCR Polymerase chain reaction
  • a method for gene cloning in which DNA fragments with sticky ends are obtained; the DNA fragments are ligated with a cloning vector that has sticky ends complementary to the sticky ends of the DNA fragments; and competent cells are transformed with the ligation products.
  • the DNA fragments with sticky ends can be obtained byamplifying a target DNA fragment using a pair of primers wherein at least one primer has additional bases at its 5' end and a number of bases that are complementary to an end of the target DNA fragment.
  • the additional bases may comprise a nicking endonuclease recognition site or at least one protection base at its 5' end followed by a nicking endonuclease recognition site.
  • the amplified DNA fragment is digested with the nicking endonuclease that corresponds to the nicking endonuclease recognition site in said primers; andthe digested material can be removed by denaturation.
  • Zeroto ten protection bases can be added to the primer at its 5' end, and the protection bases can be in any arrangement of A, G, C, or T.
  • the DNA fragment may have one sticky end and one blunt end, or may have two sticky ends.
  • the primers have the same endonuclease recognition site, or have different endonuclease recognition sites.
  • the nicking endonuclease can be Nb.BbvCI, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.BbvCI.Nt.Alwl, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, or Nt.CviPII.
  • the nicking endonuclease is Nb.BbvCI.
  • the cloning vector is linearized and has one or two sticky ends.
  • the linearized cloning vector can be obtained by digesting a starting vector or can be prepared using the following steps:
  • the spacer is incorporated into the cloning site of a starting vector to obtain a pre-cloning vector
  • the pre-cloning vector is digested with the type II restriction endonuclease(s) corresponding to the recognition site(s) in the spacer.
  • the spacer is at least 300 bp in length.
  • the spacer can have one type II restriction endonuclease recognition sequence at one end and another type II restriction endonuclease recognition sequence at the other end or the same type II restriction endonuclease recognition sequence at each end.
  • the spacer can be a selection marker, such as ccdB gene.
  • the starting vector can be a PBR322 vector, a PUC vector, a PGEM vector, a pBluescript vector, a pMD19-T vector, a pMD18-T vector, a pMD19-T Simple vector, or a pMD18-T Simple vector.
  • the cloning vector is pMD19GW-Adv.BstX-BstXI.
  • the method for gene cloning can be used to clone DNA fragments up to 12 Kb in length.
  • a method for one-step cloning of multiple DNA fragments into a vector in a designed orientation is presented herein.
  • multiple DNA fragments are obtained in which the DNA fragments have designed variable sticky ends.
  • the DNA fragments with variable sticky ends are obtained by amplifying target DNA fragments using a pair of primers specific for each target DNA fragment; digesting the amplified DNA fragment with the nicking endonuclease that corresponds to the nicking endonuclease recognition site in said primers; and removing the digested materialtoobtain the DNA fragments with designed sticky ends. For each primer set directed towards amplification of a specific target, each primer in the pair has
  • variable sticky ends are produced by altering the nicking endonuclease recognition site and/or altering the number and arrangement of protection bases added at the 5' end of each primer.
  • a ligation step is then performed in which the DNA fragments with sticky ends ligate with each other and with the cloning vector in a designed orientation due to complementary base pairing of the sticky ends. Competent cells are then transformed with the ligation products.
  • the nicking endonuclease can be Nb.BbvCI, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.BbvCI, Nt.Alwl, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, or Nt.CviPII.
  • the nicking endonuclease isNb.BbvCI.
  • the cloning vector is linearized and has sticky end(s).
  • the linearized cloning vector can be obtained by digesting a starting vector, or can be prepared using the following steps:
  • the spacer is incorporated into the cloning site of a starting vector to obtain a pre-cloning vector
  • the pre-cloning vector is digested with the type II restriction endonucleases corresponding to the recognition sites in the spacer.
  • the spacer is at least 300 bp in length.
  • the spacer can have one type II restriction endonuclease recognition sequence at one end and another type II restriction endonuclease recognition sequence at the other end or the same type II restriction endonuclease recognition sequence at each end.
  • the spacer can be a selection marker, such as ccdB gene.
  • the starting vector can be a PBR322 vector, a PUC vector, a PGEM vector, a pBluescript vector, a pMD19-T vector, a pMD18-T vector, a pMD19-T Simple vector, or a pMD18-T Simple vector.
  • the cloning vector is pMD19GW-Adv.BstX-BstXI.
  • the method for one-step cloning of multiple DNA fragments in a designed orientation can be used to construct a vector comprising an RNAi construct.
  • sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1 .821 1 .825.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the lUPAC IUBMB standards described in Nucleic Acids Res. 13:3021 3030 (1985) and in the Biochemical J. 219 (2):345 373 (1984) which are herein incorporated by reference.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1 .822.
  • SEQ ID NO:1 is the nucleotide sequence of the spacer,which encodes ccdB protein.
  • SEQ ID NO:2 is the nucleotide sequence of the forward primer used to amplify the spacer sequence.
  • SEQ ID NO:3 is the nucleotide sequence of the reverse primer used to amplify the spacer sequence.
  • SEQ ID NO:4 is the nucleotide sequence of a genomic sequence comprising the EF1 a gene at LOC_Os03g08010.1 of chromosome 3 in the Zhonghua11 genome.
  • SEQ ID NO:5 is the nucleotide sequence of primer NK-3.
  • SEQ ID NO:6 is the nucleotide sequence of primer NK-4.
  • SEQ ID NO:7 is the nucleotide sequence of primer NK-5.
  • SEQ ID NO:8 is the nucleotide sequence of primer NK-6.
  • SEQ ID NO:9 is the nucleotide sequence of primer NK-7.
  • SEQ ID NO:10 is the nucleotide sequence of primer NK-8.
  • SEQ ID NO:1 1 is the nucleotide sequence of primer NK-9.
  • SEQ ID NO:12 is the nucleotide sequence of primer NK-10.
  • SEQ ID NO:13 is the nucleotide sequence of primer NK-1 1 .
  • SEQ ID NO:14 is the nucleotide sequence of primer NK-12.
  • SEQ ID NO:15 is the nucleotide sequence ofprimer NK-k.
  • SEQ ID NO:16 is the nucleotide sequence of primer check9/1 1 .
  • SEQ ID NO:17 is the nucleotide sequence of primer check5/7.
  • SEQ ID NO:18 is the nucleotide sequence of primer check3.
  • SEQ ID NO:19 is the nucleotide sequence of primer ADVANCED M13F.
  • SEQ ID NO:20 is the nucleotide sequence of primer Nb.DsRed-1 .
  • SEQ ID NO:21 is the nucleotide sequence ofprimer Nb.DsRed-2.
  • SEQ ID NO:22 is the nucleotide sequence of primer Nb.HYG -1 .
  • SEQ ID NO:23 is the nucleotide sequence of primer Nb.HYG-2.
  • SEQ ID NO:24 is the nucleotide sequence of primer Nb.GUS-1 .
  • SEQ ID NO:25 is the nucleotide sequence ofprimer Nb.GUS-2.
  • SEQ ID NO: 26 is the nucleotide sequence ofprimerDsRed-reverse SEQ ID NO: 27 is the nucleotide sequence ofprimerDsRed-forward SEQ ID NO: 28 is the nucleotide sequence ofprimerGUS-reverse SEQ ID NO: 29 is the nucleotide sequence ofprimerGUS-forward SEQ ID NO: 30 is the nucleotide sequence ofprimerHYG-reverse SEQ ID NO: 31 is the nucleotide sequence ofprimerHYG-forward SEQ ID NO: 32 is the nucleotide sequence ofprimerM13R
  • SEQ ID NO: 33 is the nucleotide sequence ofprimerFRiGUS-1
  • SEQ ID NO: 34 is the nucleotide sequence ofprimerFRiGUS-2
  • SEQ ID NO: 35 is the nucleotide sequence ofprimerRRiGUS-1
  • SEQ ID NO: 36 is the nucleotide sequence ofprimerRRiGUS-2
  • SEQ ID NO: 37 is the nucleotide sequence ofprimerlntron-F
  • SEQ ID NO: 38 is the nucleotide sequence ofprimerlntron-R
  • SEQ ID NO: 39 is the nucleotide sequence ofprimerReverse Intron
  • SEQ ID NO: 40 is the nucleotide sequence ofprimerForward Intron
  • Figure l is the structure of the pMD19GW-Adv.BstX vector.
  • ColE1 ori is replication origin of E Coli
  • Amp r is ampicillinresistance gene.
  • Figure 2 shows the structures of the sticky ends of amplified PCR products DNA Fragment 1 , DNA Fragment 2, and DNA Fragment 3 for the experiment shown in EXAMPLE 6 and EXAMPLE 7.
  • Figure 3 shows how the multiple DNA fragments are positioned in a designed orientation relative to the cloning vector as a result of
  • the method involves generating DNA fragments with sticky ends and ligating the DNA fragments with a cloning vector having complementary sticky ends, thereby allowing the DNA fragments to ligate in a correct orientation with the cloning vector.
  • This method offers many advantages including but not limited to:(1 )high efficiency ligation of PCR products with the cloning vector;(2) cloning of DNA fragments up to 12 Kb;(3) a one-step ligation when cloning multiple DNA fragments;(4) no need to consider the nicking endonuclease recognition sequence inside the DNA fragment(s); and (5) a reduction in costs and increases inoverall efficiency as compared to other gene cloning methods available to one of ordinary skill in the art.
  • nucleic acids are written left to right in 5' to 3' orientation.
  • Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the current disclosure pertains. In the current disclosure, the following terminology will be used in accordance with the definitions set out below. The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.
  • Base and “Nucleotide” are used as interchangeably herein to refer to nucleotide by their single-letter designation as follows: “A” for adenylate or deoxyadenylate, “C” for cytidylate or deoxycytidylate, and “G” for guanylate or deoxyguanylate for RNA or DNA, respectively; and “T” for deoxythymidylate.
  • “Denaturation” also called DNA melting, is the process by which double- stranded DNA unwinds and separates into single-stranded strands through the breaking of hydrogen bond(s).
  • a "DNA fragment” is a piece of DNA.
  • a “designed orientation” refers to the desired placement of DNA fragments relative to one another and to the cloning vector.
  • the orientation can be designed by altering the number and arrangement of protection basesand/or nicking endonuclease recognition sequence in the primers specific for each DNA fragment, thus producing overhang of nucleotides at the terminal ends of the DNA fragments.
  • the overhang regions are complementary to one another and pair via hydrogen bonding, ensuring that multiple fragments are connected in a designed orientation.
  • Plasmid is an extrachromosomal genetic unit capable of autonomous replication.
  • the term “plasmid” includes the extrachromosomal DNA molecule in organelles of eukaryote cells and bacterial cells.
  • a "plasmid vector” is an artificial plasmid that generally contains a replication origin, a resistance selection marker, and multiple cloning sites.
  • Existing plasmid vectors can be used as "starting vectors" herein and can be modified by the methods described herein to obtaincloning vectors with sticky ends.
  • the startingvectors can be selected from PBR322 vectors, PUC series vectors, PGEM series vectors, pBluescript vectors, pMD19-T vector, pMD18-T vector, pMD19-T Simple vector, pMD18-T Simple vector, or any other known plasmid vector known to one of ordinary skill in the art.
  • Primer pairs consist of an upstream primer and a downstream primer and are capable of amplifying a target DNA fragment.
  • a “protectionbase” refers to a base that is added to the 5' end of a primer and can be followed by a nicking endonucleaserecognitionsequence.lt can be A, T, C, or G. A protection base(s) added to the 5' ends of the primers will be a part of the digested sticky ends of the DNA fragments.
  • Recognizing sequence "Recognizing site”, “Recognition sequence”, “Recognition site”, and “Cutting site” are used as interchangeably herein to refer to nucleotide sequences recognized by a restriction enzyme.
  • Typell restriction endonuclease is the most common restriction endonuclease and it recognizes and cleaves 4-8 bases in a nucleotide sequence with no methylation. Most recognizing sequences have a palindrome structure. There are three cleaving mechanisms of type II restriction endonucleases: (1 ) some can cleave to produce 5' sticky ends; (2) some can cleave to produce 3' sticky ends; and (3) some cleave to produce blunting ends. Any typellrestriction endonuclease can be used for vector construction in the methods described herein.
  • selection marker is any marker, which when expressed at a sufficient level, confers resistance to a selective agent. Selection markers and their corresponding selective agents include, but are not limited to, herbicide resistance genes and herbicides; antibiotic resistance genes and antibiotics; and other chemical resistance genes with their corresponding chemical agents.
  • Resistance may be conferred to herbicides from several groups, including amino acid synthesis inhibitors, photosynthesis inhibitors, lipid inhibitors, growth regulators, cell membrane disrupters, pigment inhibitors, seedling growth inhibitors, including but not limited to imidazolinones, sulfonylureas, triazolopyrimidines, glyphosate, sethoxydim, fenoxaprop, glufosinate, phosphinothricin, triazines, bromoxynil, and the like. See, for example, Holt (1993) Ann Rev Plant Physiol Plant Mo/S/ ' o/44:203-229; and Miki et al. (2004) J Biotechnol 107:193-232.
  • Selection markers include sequences that confer resistance to such herbicides and include, but are not limited to, the bar gene, which encodes phosphinothricin acetyl transferase (PAT) which confers resistance to glufosinate (Thompson et al. (1987) EMBO J 6:2519-2523); glyphosate oxidoreductase (GOX), glyphosate N-acetyltransferase (GAT), and 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) each of which confers resistance to glyphosate (Barry et al. (1992) in Biosynthesis and Molecular Regulation of Amino Acids in Plants, B.K.
  • PAT phosphinothricin acetyl transferase
  • GOX glyphosate oxidoreductase
  • GAT glyphosate N-acetyltransferase
  • sulfonylureas such as imazethapyr and/or chlorsulfuron (see, e.g., Zu et al. (2000) Nat Biotechnol 18:555-558; U.S. Patents 6,444,875, and 6,660,910; Sathasivan et al. (1991 ) Plant Physiol 97:1044-1050; Ott et al. (1996) J
  • Bacterial drug resistance genes include, but are not limited to, neomycin phosphotransferase II (nptll) which confers resistance to kanamycin, paromycin, neomycin, and G418, and hygromycinphosphotransferase (hph) which confers resistance to hygromycin B. See also, Bowen (1993) Markers for Plant Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization; Everett et al. (1987) Bio/Technology 5:1201 -1204; Bidney et al. (1992) Plant ⁇ / ⁇ / ⁇ /18:301 -313; and WO97/05829.
  • nptll neomycin phosphotransferase II
  • hph hygromycinphosphotransferase
  • chemical resistance genes further include tryptophan decarboxylase which confers resistance to 4-methyl tryptophan (4-mT)
  • the selection marker may comprise cyanamidehydratase (Cah), see, for example, Greiner et al. (1991 )
  • Cyanamidehydratase enzyme converts cyanamide into urea, thereby conferring resistance to cyanamide.
  • Any form or derivative of cyanamide can be used as a selection agent including, but not limited to, calcium cyanamide (PERLKA® (SKW, Trotberg Germany) and hydrogen cyanamide (DORMEX® (SKW)). See also, U.S. Patents 6,096,947, and 6,268,547. Variants of cyanamidehydratase polynucleotides and/or
  • polypeptides will retain cyanamidehydratase activity.
  • a biologically active variant of cyanamidehydratase will retain the ability to convert cyanamide to urea.
  • Methods to assay for such activity include assaying for the resistance of plants expressing the cyanamidehydratase to cyanamide. Additional assays include the cyanamidehydratase colorimetric assay (see, e.g., Weeks et al. (2000) Crop Sci 40:1749-1754; and U.S. Patent 6,268,547).
  • the selection marker can also be the ccdB gene (Bernard, P. 1995. Gene 162:159-160; Bernard, P. et al. 1994. Gene 148:71 -74; Marcil, R.A., et al. 1996. NIH Res. 8:62).
  • ccdB is alethal gene that targets DNA gyrase.
  • the ccdB positive selection marker acts by killing the background of cells with no cloned DNA; only cells containing a recombinant DNA will give rise to viable clones.
  • the "spacer” is a DNA fragment.
  • sticky ends refers to the overhang region of nucleotides that are free to base pair with the sticky ends of other elements (i.e. other DNA fragments, cloning vector) via standard base pairing.
  • a "target DNA fragment” is a DNA fragment of interest that is to be amplified, extracted, and ligated into a cloning vector.
  • Nicking endonucleases are restriction endonucleases( NEW ENGLAND BioLabs Inc. (NEB)) that can hydrolyze only one strand of the double-strand DNA to produce DNA molecules that are "nicked", rather than cleaved. These conventional nicks (3 ' -hydroxyl, 5 ' -phosphate) can serve as initiation points for a variety of further enzymatic reactions such as replacement DNA synthesis, strand-displacement amplification, exonucleolytic degradation, or the creation of small gaps.
  • Nicking endonucleases include but are not limited to: Nb.BbvCI, Nt.BbvCI, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.BbvCI, Nt.Alwl, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII, all of which can be used for the methods described herein.
  • Nb.BbvCI expressed in an E. coli strain contains an altered form of the BbvCI restriction genes [Ra+:Rb(E177G)] from Bacillus brevis (L. Ge).
  • the recognition sequence is as follows with the arrowhead pointing at the cutting position:
  • Nt. BbvCI expressed in anE. coli strain contains an altered form of the BbvCI restriction genes [Ra (K169E):Rb+] from Bacillus brevis (L. Ge).
  • the recognition sequence is as follows with the arrowhead pointing at the cutting position:
  • a primer design method based on nicking endonuclease is disclosed herein.
  • primers are designed in accordance with principles of complementary base pairing and additional sequence is then added to the 5' ends of both the upstream and downstream primers.
  • the additional sequence (in the 5' to 3' direction) comprises a number of protection bases, a nicking endonuclease recognition sequence, and a region of bases specific to the DNA fragment to be amplified.
  • Zero to ten protection bases can be used, and the protection bases can be any arrangement of A, C, G, or T.
  • a method of producing DNA fragments with sticky ends includes: (1 ) designing primers according to the primer design method; (2) obtaining target DNA fragments by PCR amplification; (3) digesting the DNA fragments obtained in step (2) with the corresponding nicking endonuclease(s) of the nicking endonuclease recognition sequences used in step (1 ), and removing the digested materialtoobtain DNA fragments with designed sticky ends.
  • the digested material can be removed by denaturing the DNA. Denaturation can occur at 65°C for 5 minutes.
  • the nicking endonuclease recognition sequences can be ignored in practical application because nicking endonucleases only cut one strand of DNA.
  • the protection bases added as part of the primer design method will be a part of the digested sticky ends.
  • a cloning vector as described herein has sticky ends at each end that are complementary to the sticky ends of the DNA fragments obtained using the primer design method.
  • a linearized cloning vector can be obtained by digesting a starting vector, or it can be prepared using the following steps: 1 ) obtaining a spacer which has a type II restriction endonuclease recognition sites at each end; 2) incorporating the spacer into the cloning site of a starting vector to obtain a pre-cloning vector; and 3) digesting the pre-cloning vector with the type II restriction endonuclease(s) corresponding to the type II restriction endonuclease recognition sites in the spacer to obtain the cloning vector.
  • the spacer is used herein for convenient separation of the restriction enzyme digested cloning vector from the starting vector.
  • a spacer can be of any size as long as it does not form abnormal secondary structure.
  • Thespacer may beat least 100bp, at least 200 bp, or at least 300 bp in length.
  • the spacer is most preferably at least 300 bp in length.
  • the spacer may be but is not limited to the ccdB gene (Bernard, P. 1995. Gene 162:159-160; Bernard, P. et al. 1994. Gene 148:71 -74; Marcil, R.A., et al. 1996. NIH Res. 8:62) or DSRed (Matz, M.V. et al. 1999. Nat Biotechnol 17:969-973).
  • the starting vectors that can be used for constructing cloning vectors can be selected from a PBR322 vector, the PUC series vectors, the PGEM series vectors, the pBluescript vectors, a pMD19-T vector, a pMD18-T vector, a pMD19-T Simple vector, a pMD18-T Simple vector, or any other commonly used cloning vector known to one of ordinary skill in the art.
  • restriction endonuclease(s) that can be used to construct the cloning vector can be a regular type II restriction endonuclease ora nicking endonuclease. Any scenario can be envisioned in which the spacer contains: the same restriction endonuclease site on each end or a different restriction endonuclease site on each end.
  • thecloning vector is pMD19GW-Adv.BstX-BstXI.
  • the construction of this cloning vector is shown in EXAMPLES 2 through 4.
  • An efficient gene cloning method as disclosed herein has the following steps: (1 ) designing primers according to the primer design method; (2) obtaining DNA fragments with sticky ends using the methods described herein; (3) ligating the DNA fragments with sticky ends obtained in step (2) with a cloning vector, wherein the cloning vector has two sticky ends that are complementary to the sticky ends of the DNA fragments; and (4) transforming competent cells with the ligation products.
  • the method for gene cloning is highly efficient and can be used to construct a vector comprising an RNAi construct and to clone DNA fragments up to 12 Kb in length.
  • multiple DNA fragments can be designed to ligate in a certain orientation ("a designed orientation") by changing the numbers and arrangements of protection bases and/or nicking endonuclease recognition sequences in the primers specific for each DNA fragment.
  • a designed orientation After PCR and digestion with nicking endonuclease(s), the DNA fragments have different sticky ends which are complementary with each other, allowing oriented ligation of multiple DNA fragments relative to one another and to the cloning vector (as shown in FIG. 3).
  • This procedure offers the additional advantage in that multiple fragments can be ligated with the cloning vector in a simple one-step process.
  • Elements of the gene cloning method disclosed herein can also be assembled into a gene cloning kit, which can be used for efficient cloning of DNA fragments up to 12 Kb and for one-step ligation of multiple DNA fragments in a designed orientation.
  • the gene cloning kit can comprise the linearized pMD19GW- Adv.BstX-BstXIvector as described in Example 4; nicking endonuclease reaction buffer; nicking endonuclease Nb.BbvCI; and ligase.
  • Primers were designed to amplify a fragment of DNA via the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • additional sequences were added to the 5'end of the gene-specific portion of the primer. For example two protection bases (boxed) and the recognition sequence for nicking
  • Nb.BbvCI (underlined)were added to the gene-specific portion of each primer (as shown below).
  • PCR amplified products were extracted using the E.Z.N. A.® Cycle- Pure Kit from Omega Bio-tek and digested with nicking endonuclease
  • Nb.BbvCI The digested portion was removed by denaturation at 65 ° C for 5 min,thereby producing sticky ends as shown below.
  • Primers were designed to a spacer sequence (SEQ ID NO:1 )which encodes ccdB protein and is found in the pMD19GW-delete vector, a pMD19GW-delete vector without a ccdA gene.
  • the primers are as follows (with the BstXI recognition sequence underlined):
  • the pMD19GW-delete vector was used as a template to amplify the spacer sequence (SEQ ID NO:1 ) using the PCR reaction mix and cycle conditions shown in Tables 1 and 2, respectively.
  • PCR amplified products were isolated by 1 % agarose gel electrophoresis and then extracted using a column kit.
  • Preparation of pre-cloninq vector pMD19GW-Adv.BstX The spacer sequence obtained in Example 2 was ligated with T-vector pMD19T( simple) by mixing 0.5 LT 4 DNA ligase (NEB), 1 T 4 DNA ligation buffer, -300 ng spacer and 0.5 ⁇ pMD19T (40 ng/ L), and adding distilled water to 10 ⁇ . Ligation occurred at 16° C for 3 h. The ligation products were transformed into competent E.Coli cells using the heat shock transformation method. After plating on LB medium containing Amp, the transformed E.Coli cells were cultured at 37 0 C overnight. Three colonies were randomly selected and then validated by colony PCR. The positive colonies were sequenced, and the plasmids in the positive colonies were extracted to obtain the pre-cloning vector pMD19GW-Adv.BstX(FIG. 1 ). EXAMPLE 4
  • the pre-cloning vector pMD19GW-Adv.BstX-BsfXI has restriction endonucleaseSsfXI recognition sequences, which are located at two sides of cloning sites (a SsfXI recognition sequence in the spacer sequence).
  • SsfXI recognition sequence in the spacer sequence.
  • Elongation factor 1a (EF1 a) gene is usually used as an internal reference in real-time PCR, and this gene is located at LOC_Os03g08010.1 of chromosome 3 in the ZH11 genome.
  • a genomic sequence (SEQ ID NO: 4) comprising the EF1 a gene was selected for testing.
  • KOD-FX (TOYOBO) Pfu polymerase was used in PCR amplification (See Table 4 for PCR reaction mix; Tables 5 and 6 show PCR cycling conditions for fragments greater than or equal to 12Kb and for fragments less than 12 Kb, respectively), and the primers are shown in Table 3. (recognition sequence of nicking endonuclease Nb.BbvCI is underlined and the protection bases are boxed).
  • NK-4 CCGCTGAGGCCTATTAATATGGGCAGAAATC 6
  • NK-6 [CCJGCTGAGGGCACACAACTAGAGTCATGC 8
  • NK-7 IC IG TGAGG AACATATTACTTG G GTTACACTAAG G 9 NK-8 CClGCTG AG G AATACAG GTG G ACAGCAACATC 10
  • the primer combinations and lengths of amplified fragments are displayed in Table 7.
  • the GC% of the amplified fragments are among
  • the PCR amplified products were recovered using a column kit and then digested with nicking endonuclease Nb.BbvCI.
  • the enzyme digestion was as follows: 1 pLNb.BbvCI, 5 ⁇ _ NEB Buffer 2, 20 ⁇ _ PCR amplified products, and distilled water to 50 ⁇ _. After digestion at 37 0 C overnight, the digested products were isolated on 1 % agarose gel electrophoresis to obtain the target DNA fragments.
  • the PCR amplified fragments were ligated with the cloning vector constructed in Example 4 using 0.5 ⁇ _ T 4 DNA ligase (NEB), 1 ⁇ T 4 DNA ligation buffer, -300 ng digested PCR products, 0.5 ⁇ _ pMD19GW-Adv.BstX- SsfXI cloning vector (40 ng/ L), and distilled water to 20 ⁇ _. Ligation occurred at 16 ° C for 3 hours. E.Coli DH5aTM competent cells were
  • the E.Coli DH5aTMcells were plated on Amp- containing LB medium and cultured at 37 0 C overnight. Hundreds of bacterial colonies grew on the selection LB plates.
  • Colony PCR was carried out to validate ten randomly selected positive colonies on each plate.
  • the primer pairs used for colony PCR validation are shown in Table 8.
  • the expected lengths of the amplicons produced by each primer pair with respect to each plasmid are shown in Table 9.
  • restriction enzyme analysis was carried out to validate the positive transformants.
  • Two positive colonies validated by colony PCR on each plate were cultured to extract plasmids, and the plasmids were digested by restriction endonuclease in accordance with the manufacturer's standard protocol of the particular restriction endonuclease utilized.
  • the restriction endonucleases were selected such that restriction sites for the specific restriction endonuclease were present on both the vector backbone and the amplified fragment.
  • the selected endonucleases used in the enzyme digestion analysis are displayed in Table 10.
  • digested plasmid Selected Length of digested nucleotide restriction fragment (bp)
  • NK11/12 Gel electrophoresis demonstrated thatdigestion of plasmids extracted from positive colonies on each plate produceddesired-size DNA fragments. The results further indicated that amplified DNA fragments correctly ligated with the cloning vector 100% of the time. Gene cloning methods using the primer design method and the constructed cloning vectorare highly efficient, even when the PCR fragment is 12Kb.
  • PCR was performed using the primers provided in Table 11 , and the products were recovered using a column kit and then digested with Nb.BbvCI. Structures of the sticky ends of amplified PCR productsDNA Fragment 1 , DNA Fragment 2, and DNA Fragment 3 are shown in FIG. 2. The amplified DNA Fragment 1 , DNA Fragment 2, and DNA Fragment 3 were then ligated with cloning vector of pMD19GW-Adv.BstX-SsfXI (FIG. 3).
  • the experiments utilized plasmid pCAMBIA 1301 -DsRed which contains the DsRed gene expression cassette [DNA Fragment 1 ], the GUS gene expression cassette [DNA Fragment 2], and the HYG resistance gene expression cassette [DNA Fragment 3].
  • the primers in Table 12 were used to amplify DNA Fragment 1 (1921 bp), DNA Fragment 2 (1834 bp), and DNA Fragment3 (1739 bp).
  • the Nb.BbvCI restriction sequences are underlined, and the protection bases are boxed.
  • Nb.HYG -1 GCCTGCTGAGGATGGTGGAGCACGACACTCTC 22
  • PCR was perfornned using the reaction mixture and cycling conditions shown in Table 13.
  • the PCR amplified products were recovered using a column kit, and then digested with nicking endonuclease Nb.BbvCI.
  • the enzyme digestion was performed as follows: 1 LNb.BbvCI, 5 ⁇ _ NEB Buffer 2, 20 ⁇ _ PCR amplified products, and distilled water to 50 ⁇ _ and then incubated at 37° C overnight.
  • the digested products were isolated using 1 % agarose gel electrophoresis to obtain the target DNA fragments.
  • the PCR amplified fragments were ligated with the cloning vector constructed in Example 4 using a ligation system including 0.5 ⁇ _ T DNA ligase (NEB), 1 ⁇ T 4 DNA ligation buffer, about 300 ng of each digested PCR product, 0.5 ⁇ _ pMD19GW-Adv.BstX-SsfXI cloning vector (40 ng/ ⁇ ), and residual distilled water, at 16 0 C for 3 hours. Competent cells of
  • E.ColiDH5aTM were transformed with the ligation product by the heat-shock transformation method, and the E.ColiDH5aTM cells were plated on Amp- containing LB medium, and cultured at 37 0 Covernight. Hundreds of bacterial colonies grew on the selection LB plate. PCR was used to validate the colonies that grew on the Amp-containing medium, and the primer
  • the lengths of the expected amplified fragments are: 261 bp for primers ADVANCED M13F and DsRed-reverse, 278 bp for primers DsRed-forward and GUS- reverse, 616 bp for primers GUS-forward and HYG- reverse, and 441 bp for HYG-forward and M13R.
  • Ten randomly selected colonies yielded the desired-size amplified fragments with each primer pair,as shown through colony PCR validation and gel electrophoresis, demonstrating that the three amplified PCR fragments are connected in designed orientation with high efficiency and that they are connected with the constructed cloning vector of pM D 19GW-Ad v. BstX-SsfXI .
  • restriction enzyme analysis was carried out to validate the positive transformants.
  • Two positive colonies validated by PCR on each plate were cultured to extract plasmids, and the plasmids were digested with a restriction endonuclease in accordance with its description in the NEB products guidelines. Restriction endonucleases were selected such that at least one restriction site was present on the vector backbone and the amplified fragments. The selected endonucleases used for enzyme digestion are displayed in Table 15.
  • multiple fragments can be connected in designed orientation in one step by changing the lengths and arrangement of the protection bases added at the primers which are used to produce different sticky ends.
  • ten colonies randomly selected from hundreds of colonies on the selection plate were PCR validated as positive; and two were chosen for further validation by enzyme analysis and were also validated as positive.
  • the method proved to be highly efficient in terms of ligation efficiency, and the designed orientation of multiple DNA fragments was achieved 100% of the time.
  • RNAi vector construction method based on nicking endonuclease
  • RNAi vector to interferethe GUS gene was constructed.
  • the amplified templates were plasmids pUCCRNAi and pCAMBIA 1301 .
  • the target amplifying region of pUCCRNAi was the second intron (2 nd Intron) of the gibberellin 20-oxidase gene from SolanumLycopericum.
  • the target amplifying region of pCAMBIA 1301 was a region of the second exon of the GUS gene.
  • Primers shown in Table 16 were designed similarly to what has been described previously (Nb.BbvCI restriction sequences are underlined and the protection bases are boxed).
  • the sense chain of F-GUS was DNA Fragment 1
  • the 2 nd Intron of gibberellin 20-oxidase gene was DNA Fragment 2
  • the antisense chain of GUS was DNA Fragment 3.
  • the expected lengths of the amplified fragments are 250 bp, 215 bp, and 250 bp, respectively.
  • FRiGUS-1 ICTiGCTGAGGGACGCTCACACCGATACCAT 33
  • FRiGUS-2 GCTTIGCTGAGGACATATCCAGCCATGCACAC 34
  • PCR was performed as shown in Table 17.
  • the PCR amplified products were recovered with a column kit, and then digested with nicking
  • Enzyme digestion system occurred as follows: 1 ⁇ _ ⁇ , 5 ⁇ _ NEB Buffer 2, 20 ⁇ _ PCR amplified products, and distilled water to 50 ⁇ _. Digestion occurred at 37° C overnight. The digested products were isolated on 1 % agarose gel electrophoresis to obtain the target DNA fragments.
  • the PCR amplified fragments were ligated with the cloning vector constructed in Example 4 using 0.5 ⁇ _ T 4 DNA ligase (NEB), 1 ⁇ T 4 DNA ligation buffer, about 300 ng of each digested PCR product, 0.5
  • PCR was used to validate the grown colonies on Amp-containing medium, and the primer combinations used for validation consisted of the downstream primer of the prior fragment and the upstream primer of the next connected fragment.
  • the expected lengths of the amplified fragments were: 499 bp for primers ADVANCED M13F and Reverse Intron, and 659bp for primers
  • restriction enzyme analysis was carried out to validate the positive transformants.
  • Two positive colonies validated by PCR on theplate were cultured to extract plasmids, and the plasmids were digested by restriction endonuclease in accordance with the appropriate protocol described in the NEB catalog.
  • a restriction endonuclease was selected if the restriction site was present on both the vector backbone and an amplified fragment. The selected endonucleases used in enzyme digestion analysis aredisplayed in Table 19.
  • RNAi construct can be constructed in a designed orientation using the primer design and gene cloning methods disclosed herein.
  • the elements of the gene cloning method disclosed herein can also be assembled into a gene cloning kit.
  • the gene cloning kit can be used for efficient cloning of DNA fragments up to 12 Kb and for one-step ligation of multiple DNA fragments in a designed orientation.
  • the gene cloning kit can comprise the linearized pMD19GW-Adv.BstX- BstXI as described in Example 4; nicking endonuclease reaction buffer; nicking endonuclease Nb.BbvCI; and ligase.

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Abstract

La présente invention concerne des procédés et compositions destinés au clonage moléculaire d'ADN. Les procédés incluent la préparation de fragments d'ADN ayant des extrémités collantes en utilisant un procédé de conception d'amorce basé sur des endonucléases de coupure et la ligature de fragments d'ADN avec des vecteurs de clonage qui ont été techniquement modifiés pour posséder des extrémités collantes qui sont complémentaires à celles des fragments d'ADN. Les procédés sont hautement efficaces et peuvent être utilisés pour cloner des fragments d'ADN ayant jusqu'à 12 Kb. Les procédés permettent également une ligature en une étape de multiples fragments d'ADN dans une orientation désignée. L'invention concerne également des kits de clonage de gènes commerciaux.
PCT/CN2014/070296 2014-01-08 2014-01-08 Procédé efficace de clonage de gène et ses utilisations WO2015103741A1 (fr)

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WO2018039599A1 (fr) * 2016-08-26 2018-03-01 Life Technologies Corporation Commandes d'extraction et d'amplification d'acides nucléiques et procédés d'utilisation associés

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WO2007135354A1 (fr) * 2006-05-19 2007-11-29 Plant Bioscience Limited Clonage moléculaire amélioré à base d'excision de l'uracile
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WO2018039599A1 (fr) * 2016-08-26 2018-03-01 Life Technologies Corporation Commandes d'extraction et d'amplification d'acides nucléiques et procédés d'utilisation associés
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