WO2015103741A1

WO2015103741A1 - Efficient gene cloning method and uses thereof

Info

Publication number: WO2015103741A1
Application number: PCT/CN2014/070296
Authority: WO
Inventors: Junhua Liu; Guanfan MAO
Original assignee: Pioneer Overseas Corp
Current assignee: Pioneer Overseas Corp
Priority date: 2014-01-08
Filing date: 2014-01-08
Publication date: 2015-07-16
Anticipated expiration: 2016-07-08
Also published as: CN106103712B; CN106103712A

Abstract

Provided are methods and compositions for molecular cloning of DNA. The methods comprise preparing DNA fragments with sticky ends using a primer design method based on nicking endonucleases and ligating the DNA fragments with cloning vectors that have been engineered to have sticky ends that are complementary to those of the DNA fragments. The methods are highly efficient and can be used to clone fragments of DNA up to 12 Kb. The methods also allow one-step ligation of multiple DNA fragments in a designed orientation. Also provided are commercial gene cloning kits.

Description

EFFICIENT GENE CLONING METHOD AND USES THEREOF

FIELD

The field is in the area of molecular biology, particularly with regards to gene cloning.

BACKGROUND

Polymerase chain reaction (PCR) is the most common method for amplifying DNA in-vitro. Products obtained from PCR can be cloned into a vector for the purposes of sequencing, further vector construction, in vitro transcription and translation, and gene expression and functional analysis in living organisms. While there are many methods for cloning DNA fragments, the most common method is TA cloning (Holton, TA. et al. 1991 . Nucleic Acids Research.^ 9(5)^{^}.^ ^ 56). In this method, an overhang deoxy-thymidine (T) is made at 3' terminus of the DNA vector, and forms complementary base pairing with one "A" base of the inserted PCR amplification product. The TA cloning is then completed by ligation of T4 DNA Iigase.

However, there are some defects in the traditional TA cloning. One, the overhang of "T" or "A" is unstable and can be lost during extraction or repeated freezing and thawing. Two, all kinds of T vectors connect with PCR amplified products through a "TA" dihydrogen bond, and there is only one base at the sticky end, so the bond energy of "TA" ligation is weak. Three, the connection of DNA fragments is a molecular collision process dependent on probability events, which makes efficiency less than optimum. Four, the ligation efficiency is low when T vector connects PCR products more than 5 Kb (Szatytowicz and Sadlej-Sosnowska, 2010. J. Chem. Inf. Model. 50(12, 2151-61). Finally, for oriented connection of PCR product with the TA cloning vector, further identificationneeds to be carried out to select clones having the plasmid in the correct orientation (Peijun et al. 2010. Curr Issues MolBiol. 12, 1 1 -16).

Other methods for cloning DNA fragments exist (Champoux.2001 . AnnuRev Biochem.70, 369-413; Cheo et al. 2004. Genome Res. 14, 2111 -20; van den Ent andl_o^"we.2006. JBiochemBiophys Methods. 67, 67-74.

However, these methods are also plagued by problems such as low ligation efficiency, complex process steps, incorrect orientation of the DNA fragment, etc. There is a need for an efficient cloning method that can overcome these problems.

SUMMARY

Methods for gene cloning are provided herein.

In one embodiment, a method for gene cloning is presented 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. In one aspect, 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:

1 ) a spacer bounded by a type II restriction endonuclease recognition sequence at each end is obtained;

2) the spacer is incorporated into the cloning site of a starting vector to obtain a pre-cloning vector; and.

3) the pre-cloning vector is digested with the type II restriction endonuclease(s) corresponding to the recognition site(s) in the spacer. In some aspects, 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. In one aspect, 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.

In another embodiment, a method for one-step cloning of multiple DNA fragments into a vector in a designed orientation is presented herein. In the method, 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

additional bases at 5' end and a number of bases that are complementary to an end of the target DNA fragment, wherein the additional bases comprise a nicking endonuclease recognition site or at least one protection basefollowed by a nicking endonuclease recognition site. The 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.

To create the sticky ends, zeroto ten protection bases can be added at the 5' end, and the protection bases can be in any arrangement of A, G, C, or T. 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. In one aspect, 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:

2) the spacer is incorporated into the cloning site of a starting vector to obtain a pre-cloning vector; and

3) the pre-cloning vector is digested with the type II restriction endonucleases corresponding to the recognition sites in the spacer. In some aspects, 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. In one aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The current disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.

The 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:4is 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, and 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

complementary base pairing of the sticky ends.

DETAILED DESCRIPTION

An efficient gene cloning method is presented herein. 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.

It is to be understood that the current disclosure is not limited to particular embodiments, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular

embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, terms in the singular and the singular forms "a", "an" and "the", for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "plant", "the plant" or "a plant" also includes a plurality of plants; also, depending on the context, use of the term "plant" can also include genetically similar or identical progeny of that plant; use of the term "a nucleic acid" optionally includes, as a practical matter, many copies of that nucleic acid molecule.

Unless otherwise indicated, 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. Unless defined otherwise, 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.

The following definitions are provided as an aid to understand thecurrent disclosure.

"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.

A "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.

"Restriction enzymes" and "Restriction endonuclease" are

interchangeably used herein refer to a nucleic acid hydrolase capable of recognizing and cleaving specific nucleotide sequence of the double-strand DNA molecule. 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.

A "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. Singh et al. (Eds) pp.139- 145; Kishore et al. (1992) Weed Tech 6:626-634; Castle (2004) Science 304:1 151 -1 154; Zhou et al. (1995) Plant Cell Rep 15:159-163; WO97/04103; WO02/36782; and WO03/092360). Other selection markers include dihydrofolate reductase (DHFR), which confers resistance to methotrexate (see, e.g., Dhir et al. (1994) Improvements of Cereal Quality by Genetic Engineering, R.J. Henry (ed), Plenum Press, New York; and Hauptmann et al. (1988) Plant Physiol 86:602- 606). Acetohydroxy acid synthase (AHAS or ALS) mutant sequences lead to resistance to imidiazolinones and/or

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

MolBiol 263:359-368; and Fang et al. (1992) Plant MolBiol 18:1 185-1 187).

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 Μο/β/Ό/18:301 -313; and WO97/05829.

In addition, chemical resistance genes further include tryptophan decarboxylase which confers resistance to 4-methyl tryptophan (4-mT)

(Goodijn et al. (1993) Plant MolBiol 22:907-912); and bromoxynilnitrilase which confers resistance to bromoxynil. The selection marker may comprise cyanamidehydratase (Cah), see, for example, Greiner et al. (1991 )

ProcNatlAcadSci USA 88:4260-4264; and Weeks et al. (2000) Crop Sci 40:1749-1754. 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.

The term "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.

Methods using nicking endonucleases and a primer design method to clone DNA

Nicking endonucleases

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.

For example, 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:

, * , ? Λ L # , , ,

In yet another example, 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:

.. .. ψ

Primer design method

A primer design method based on nicking endonuclease is disclosed herein. In this method, 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. By altering the number and arrangement of protection bases and/or nicking endonuclease recognition site in the additional sequence, DNA fragments can be designed to ligate in a certain orientation. This feature is particularly useful for one-step ligation of multiple DNA fragments in a designed orientation, as discussed below.

Producing DNA fragments with sticky ends

A method of producing DNA fragments with sticky ends is provided. The method 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 materialcan 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.

Preparation of a cloning vector

Methods for preparing a linearized cloning vector for use in gene cloning as described herein are also provided. 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.

The 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.

In one aspect, thecloning vector is pMD19GW-Adv.BstX-BstXI. The construction of this cloning vector is shown in EXAMPLES 2 through 4.

Gene cloning methods and uses thereof

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.

As shown herein, 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.

One-step ligation of multiple DNA fragments with a cloning vector

Using the method described above, 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. 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.

Kits

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.

EXAMPLES

The following examples are offered to illustrate, but not to limit, the current disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the scope of the appended claims.

EXAMPLE 1

PCR primer design based on the recognition sequence of nicking

endonuclease Nb.BbvCI and the production of sticky ends

Primers were designed to amplify a fragment of DNA via the polymerase chain reaction (PCR). In designing the primers, 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

endonuclease Nb.BbvCI (underlined)were added to the gene-specific portion of each primer (as shown below).

F-Primer: 5' ^••TcT CTGAGG+NNNN-

R-Primer: 5 ' --jcflGCTGAGG+NNNN-

Standard PCR protocols were then employed to amplify a target DNA fragment. 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 portionwas removed by denaturation at 65 ° C for 5 min,thereby producing sticky ends as shown below.

5 ' - TGAGGNNNNNNNNNNNNNNNNNNNNCCTCAGCGG - 3' 3 ' - GACGACTCCNNNNNNNNNNNNNNNNNNNNGGAGT - 5 '

EXAMPLE 2

Preparation of spacer

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):

Spacer-F:

CCAGCCGCTTGGAGATCCGGCTTACTAAAAGCCAGATAACAGT

(SEQ ID NO:2)

Spacer-R:

CCATCTGCTTGGCTCGACGGAGCCTGACATTTATATTCCCCAGAACATCA

GGTTA

(SEQ ID NO:3)

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.

Table 1 : PCR reaction mixture

Reaction mix 50 uL

Template 5 μΙ_

TaKaRa LA-Taq (5 ΙΙ/μΟ 0.5 μΙ_

I Q LA TaqBufferll ( Mg²⁺ Plus) 5 μΙ_ dNTP Mixture (2.5 mM each ) 8 μΙ_

Spacer-F/R ( 10 μΜ ) 2 μΙ_ each ddH₂O 27.5 μΙ_ Table 2: PCR cycle conditions

94 ° C 3 min

72° C 5 min

PCR amplified products were isolated by 1 % agarose gel electrophoresis and then extracted using a column kit.

EXAMPLE 3

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 LT₄ DNA ligase (NEB), 1 T₄ DNA ligation buffer, -300 ng spacer and 0.5 μί pMD19T (40 ng/ L), and adding distilled water to 10 μί. 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

Preparation of cloning vector pMD19GW-Adv.BstX-BsfXI As shown in FIG. 1 , the pre-cloning vector pMD19GW-Adv.BstX has restriction endonucleaseSsfXI recognition sequences, which are located at two sides of cloning sites (a SsfXI recognition sequence in the spacer sequence). After digesting the pre-cloning vector pMD19GW-Adv.BstX with SsfXI, three DNA fragments with lengths of 131 bp, 324 bp, and 2923 bp were obtained. The largest DNA fragment was extracted using a column kit to obtainthe cloning vector.

EXAMPLE 5

Cloning efficiency analysis

To investigate the ligation efficiency of the cloning vector and an amplified PCR product, DNA fragments with different lengths were amplified using Zhonghua11 (ZH11 ) rice genomic DNA as template. 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).

Table 3: Primers for PCR amplification

Primer Sequence SEQ ID NO:

NK-3 ICTIGCTGAGGTCGACCTCATAAAGCAAGTG 5

NK-4 CCGCTGAGGCCTATTAATATGGGCAGAAATC 6

NK-5 ICTIGCTG AG G AACATATTACTTG G GTTACACTAAG 7

NK-6 [CCJGCTGAGGGCACACAACTAGAGTCATGC 8

NK-7 IC IG TGAGG AACATATTACTTG G GTTACACTAAG G 9 NK-8 CClGCTG AG G AATACAG GTG G ACAGCAACATC 10

NK-9 CTiGCTGAGGCTTGGAGTGAAGCAGATGATCT 11

NK-10 CCGCTGAGGGACCGTCGACAAAACTTAACG 12

NK-11 CTiGCTG AG G CTTG GAGTG AAGCAG ATG ATCT 13

NK-12 CClGCTGAG G AATACAG GTG G ACAGCAACATC 14

NK-k CCIGCTGAGGCCTTGGCTTTGTCTTCATGTCGG 15

Table 4. PCR reaction mixture

Reaction mix 50 uL

ZH11 genomic DNA (80 ng/ L) 1 μΙ_

TOYOBO KOD-FX (1.0 ΙΙ/μΙ_) 1 μΙ_

2xPCR buffer for KOD-FX 25 μΙ_

2 mMdNTPs (0.4 mM each) 10μΙ_

Primer-F/R (10 μΜ) 2 μΙ_ each ddH₂O 9 μΙ_

Table 5. PCR cycle conditions for fragments of 12 Kb

94 °C 3 min

98 °C 10s Ί I.

^■ 5

74 °C 12 min J

98 °C 10s \5

72 °C 12 min J

70 °C 12 min

98 °C 10s 1 L x20

68 °C 12 min J

68 °C 5 min

Table 6. PCR cycle conditions for fragments less than 12 Kb 94 ° C 3 min

68 ° C 5 min

The primer combinations and lengths of amplified fragments are displayed in Table 7. The GC% of the amplified fragments are among

60%~80%.

Table 7. Primer combination, position of amplified fragments in SEQ ID NO:4, and length of amplified fragments

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 μΙ_ NEB Buffer 2, 20 μΙ_ PCR amplified products, and distilled water to 50 μΙ_. 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 μΙ_ T₄ DNA ligase (NEB), 1 χ T₄ DNA ligation buffer, -300 ng digested PCR products, 0.5 μΙ_ pMD19GW-Adv.BstX- SsfXI cloning vector (40 ng/ L), and distilled water to 20 μΙ_. Ligation occurred at 16 ° C for 3 hours. E.Coli DH5a™ competent cells were

transformed with the ligation product using a standard heat-shock

transformation method. The E.Coli DH5a™cells 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.

Table 8: Primer pairs used for colony PCR validation

Primer Sequence SEQ ID NO: check9/1 1 ATTCACAGAAGTTGCTCATCA 16 check5/7 GACTAGTGCACGCAGGAT 17 check3 GACAACTCGGCTTCCATCTC 18

ADVANCED M13F GTAAAACGACGGCCAGTGAATTC 19

Table 9. Expected lengths of amplicons for each primer pair

Length of amplified fragment

Plasmid name Primer combination (bp)

ADVANCED

GW-Adv.B-NK-5/NK-k M13F&check5/7 596

ADVANCED

GW-Adv.B-NK-3/NK-4 M13F&check3 465

ADVANCED

GW-Adv.B-NK-5/NK-6 M13F&check5/7 596

ADVANCED

GW-Adv.B-NK-7/NK-8 M13F&check5/7 596

ADVANCED

GW-Adv.B-NK-9/NK-10 M13F&check9/1 1 416

GW-Adv.B-NK-11/NK-12 ADVANCED 416 I M13F&check9/1 1 I

Agarose gel electrophoresisshowedthat PCR with the ten randomly selected colonies on each plate produceddesired-sizeamplified fragments (not shown),thereby demonstrating that PCR fragments amplified using primers containing the nicking endonuclease sequence can be ligated with the constructed cloning vector with high efficiency.

To further confirm the correct ligation of the PCR amplified products with cloning vector pMD19GW-Adv.BstX-SsfXI, 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.

Table 10. Selected enzymes and length of digested fragments in the

restriction endonuclease analysis

digested plasmid Selected Length of digested nucleotide restriction fragment (bp)

endonuclease

GW-Adv.B-NK5/K EcoRI 432 4008 10267

GW-Adv.B-NK3/4 Pst\ 261 3528 5131

GW-Adv.B-NK5/6 Pst\ 261 3232 4231

GW-Adv.B-NK7/8 Pst\ 261 1432 4231

GW-Adv.B- Xcm\&Pst\ 359 4265

NK9/10

GW-Adv.B- EcoR\&Pst\ 359 4265

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.

EXAMPLE 6

Multiple DNA fraqments ligation method with high efficiency based on nicking endonuclease Nb.BbvCI

Experiments were performed to demonstrate that 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. In these experiments, primers were designed with additional sequence at the 5' end as shown in Table 11 to amplify three target DNA fragments. In Table 11 , Nb.BbvCI restriction sequences are underlined; the protection bases are boxed; "NNNN " refers to the gene specific sequence; and L/R represent upstream primer and downstream primer, respectively. The overhang connecting terminals of connected DNA fragments are complementary to ensure that multiple fragments are connected in designed orientation.

Table 11 : Primers designed to amplify target DNA fragments for a ligation involving multiple DNA fragments

Primer Sequence

Genel L 55''...ICTiGCTGAGG+NNNN...

GeneI R 55'' .. JGCTTIGCTGAGG+ NNNN.

Gene2L 55''.. JGCAA!GCTGAGG+NNNN..

Gene2R 55''... GCAGGCTGAGG+NNNN ...

Gene3L 55'' .. JGCCT1GCTGAGG+ NNNN...

Gene3R 55''...lCClGCTGAGG+NNNN.

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). In Table 12, the Nb.BbvCI restriction sequences are underlined, and the protection bases are boxed.

Table 12: Primers designed to DsRed, GUS, and HYG in pCAMBIA 1301 -

DsRed

Primer Sequence SEQ ID NO:

Nb.DsRed-1 CT CTGAGGCGAAGCTGGCCGCTCTAGA20

Nb.DsRed-2lGCTTiGCTGAGGGGCCGCATTCGCAAAACAC 21

N b . G US- 11GCAAGCTGAGGAGGACCTAACAGAACTCGCCGTA 24

Nb.GUS-1 GCAGGCTGAGGCGCGCGCGATAATTTATCCTAG 25

Nb.HYG -1 GCCTGCTGAGGATGGTGGAGCACGACACTCTC 22

Nb.HYG-2lCClGCTGAGGTAATTCGGGGGATCTGGATTTT 23

Table 13:PCR reaction mixture and procedure:

Reaction mix 50 uL pCAMBIA 1301 (5 ng/pL) 1 μΙ_

TOYOBO KOD-FX (1.0 U/μί) 1 μΙ_

2xPCR buffer for KOD-FX 25μΙ_ 2 mMdNTPs(0.4 mM each) 10 μΙ_

Primer-F/R (10 μΜ) 2 μΙ_ each

ddH₂O 9 μΙ_ PCR cycle:

94 ° C 3 min

98 ° C 10 s

68 ° C(1 Kb/min) min -J

68 ° C 5 min

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 μΙ_ NEB Buffer 2, 20 μΙ_ PCR amplified products, and distilled water to 50 μΙ_ 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 μΙ_ T DNA ligase (NEB), 1 χ T₄ DNA ligation buffer, about 300 ng of each digested PCR product, 0.5 μΙ_ pMD19GW-Adv.BstX-SsfXI cloning vector (40 ng/μί), and residual distilled water, at 16 ⁰ C for 3 hours. Competent cells of

E.ColiDH5a™ were transformed with the ligation product by the heat-shock transformation method, and the E.ColiDH5a™ 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

combinations used contained a downstream primer of the prior fragment and an upstream primer of the next connected fragment. The primers are shown in Table 14.

Table 14: Primers for colony validation

Primer Name Sequence SEQ ID NO:

DsRed-reverse GCCACGGTTATTCTCACGA 26

DsRed-forward TGGGCATCAAAGTTGTGTGT 27

GUS-reverse CCAGTCTTTACGGCGAGTTC 28 GUS-forward GAATCCTGTTGCCGGTCTT 29

HYG-reverse TTCAGCGTGTCCTCTCCAA 30

HYG-forward TCCGAGGGCAAAGAAATAGA 31

M13R AGCGGATAACAATTTCACACAGGA 32

ADVANCED M13F GTAAAACGACGGCCAGTGAATTC 19

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 .

To further confirm the correct ligation of the PCR products with cloning vector pMD19GW-Adv.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.

Table 15. Restriction endonuclease analysis of multiple fragments ligation

endonuclease

GW-Adv.B- Xho\&Bgl\\&Pst\ 326 1094 1243 2761 DsRed^*GUS^*HYG 4023 Gel electrophoresis demonstrated that the resulting fragment sizes were obtained, further demonstrating that the three DNA fragments connected with the cloning vector according to the designed connection order.

Using the method and vector described herein, 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. Moreover, 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.

EXAMPLE 7

One-step RNAi vector construction method based on nicking endonuclease

Nb.BbvCI

An 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, and 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.

Table 16: Primers designed to F-GUS, the 2^nd intron of gibberellin 20-oxidase and the antisense chain of GUS

Primer Sequence SEQ ID NO:

FRiGUS-1 ICTiGCTGAGGGACGCTCACACCGATACCAT 33 FRiGUS-2 GCTTIGCTGAGGACATATCCAGCCATGCACAC 34

RRiGUS-1 CCIGCTGAGGGACGCTCACACCGATACCAT 35

RRiGUS-2 GCCTGCTGAGGACATATCCAGCCATGCACAC 36

Intron-F GCAA!GCTGAGGGTACGGACCGTACTACTCTATT 37

Intron-R GCAGIGCTGAGGCTATATAATTTAAGTGGAAAAAAAGG 38

Table 17: PCR reaction mixture and procedure:

Reaction mix 50 uL

pCAMBIA 1301 /pUCCRNAi(5 ng/pL) 1 μΙ_

TOYOBO KOD-FX (1 .0 ΙΙ/μΙ_)1 μΙ_

2xPCR buffer for KOD-FX 25 μΙ_

2 mMdNTPs(0.4 mM each)10 μΙ_

Primer-F/R (10 μΜ) 2 μΙ_ each

ddH₂O 9 μΙ_

PCR cycle:

94 ° C 3 min

98 ° C 10 s

χ30

68 ° C 20 s

68 ° C 5 min

PCR was performed as shown in Table 17. The PCR amplified products were recovered with a column kit, and then digested with nicking

endonuclease Nb.BbvCI. Enzyme digestion system occurred as follows: 1 μΙ_Ι\^^νΟΙ, 5 μΙ_ NEB Buffer 2, 20 μΙ_ PCR amplified products, and distilled water to 50 μΙ_. 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 μΙ_ T₄ DNA ligase (NEB), 1 ^χ T₄ DNA ligation buffer, about 300 ng of each digested PCR product, 0.5

μίρΜϋΙ

cloning vector (40 ng/μΐ.), and distilled water, and the ligation mix was placed at 16 ⁰ C for 3 hours. Competent cells of E.Coli DH5a™ were transformed with the ligation product using the heat- shock transformation method; the E.Coli DH5a™ cells were plated on Amp- containing LB medium, and then cultured at 37 ⁰ C overnight. Hundreds of colonies grew on the selection plate. Colony PCR was carried out to validate the positive colonies on theplate. Ten colonies were randomly selected on theplate. The primers of M13F and Reverse Intron, M13R and Forward Intron were used to validate positive colonies (shown in Table 18).

Table 18: Primers for colony PCR validation

Primer Sequence SEQ ID NO:

Reverse Intron TTGAAACGAATAGAGTAGTA 39

Forward Intron GGACCGTACTACTCTATTCGTTTC 40

M13R AGCGGATAACAATTTCACACAGGA 32

ADVANCED M13F GTAAAACGACGGCCAGTGAATTC 19

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

Forward Intron and M13R. Gel electrophoresis confirmed that eight of ten colonies produced expected amplified fragments of 499 bp, and all ten colonies produced amplified fragments of 659bp. Thus, at least 80%of validated colonies hadgenes in the desired order.

To further confirm the correct ligation of the PCR products with the cloning vector of pMD19GW-Adv.BstX-SsfXI, 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.

Table 19. Restriction endonuclease analysis of RNAi vector

Digestion of the RNAi plasmid GW-RiGUSwith restriction

endonucleasesEcoRI andPsflresulted in the expected fragment sizes shown in Table 19, thereby demonstrating that the three DNA fragments connected with the cloning vector in the designed connection order.

Eight of ten colonies randomly selected from hundreds of colonies on the selection plate were PCR validated as positive; and two colonies chosen for validation by enzyme analysis were positive. The method proved to be highly efficient in terms of ligation efficiency and the frequency with which multiple DNA fragments aligned correctly with one another and with the linearized cloning vector was at least 80%. The results demonstrated that an RNAi construct can be constructed in a designed orientation using the primer design and gene cloning methods disclosed herein.

EXAMPLE 8

Gene cloning kit

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.

Claims

CLAIMS What is claimed is:

1 . A method for gene cloning, said method comprising:

a. obtaining DNA fragments with sticky ends, wherein said DNA fragments are obtained by:

I. amplifying a target DNA fragment using a pair of primers wherein at least one primer has additional bases at its 5'endand a region of bases specific to the target DNA fragment, wherein theadditional bases comprise a nicking endonuclease recognition site or at least one protection base followed by a nicking endonuclease recognition site;

II. digesting the DNA fragment amplified in step I. with the nicking endonuclease that corresponds to the nicking endonuclease recognition site in the saidprimer(s); and

III. denaturing to remove digested material;

b. ligating the DNA fragment with a cloning vector that has sticky ends that are complementary to the sticky ends of the DNA fragments; and

c. transforming competent cells with the product of step (b).

2. The method of claim 1 , wherein zeroto ten protection bases are added to the primer at its 5' end.

3. The method of claim 1 , wherein the protection bases are in any

arrangement of A, G, C, or T.

4. The method of claim 1 , wherein the said DNA fragment has one sticky end and one blunt end.

5. The method of claim 1 , wherein the said DNA fragment has two sticky ends.

6. The method of claim 1 , wherein the primers have the same

endonuclease recognition site.

7. The method of claim 1 , wherein the primers have different

endonudease recognition sites.

8. The method of claim 1 , wherein said nicking endonudease is Nb.BbvCI, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.BbvCI, Nt.Alwl, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, or Nt.CviPII.

9. The method of claim 1 , wherein said nicking endonudease is Nb.BbvCI.

10. The method of claim 1 , wherein the denaturing step comprises incubating at 65 ° C for 5 min.

1 1 . The method of claim 1 , wherein said cloning vector is obtained by digesting a starting vector.

12. The method of claim 1 , wherein said cloning vector is obtained using a method comprising:

a. obtaining a spacer that is bounded by a type II restriction

endonudease recognition sequence at each end;

b. incorporating the spacer into the cloning site of a starting vector to obtain a pre-cloning vector; and

c. digesting the pre-cloning vector with the type II restriction

endonudease corresponding to the recognition sites in the spacer.

13. The method of claim 12, wherein said spacer is at least 300 bp in

length.

14. The method of claim 12, wherein said spacer is a selection marker.

15. The method of claim 14, wherein said selection marker is ccdB.

16. The method of claim 12, wherein said spacer has one type II restriction endonudease recognition sequence at one end and another type II restriction endonudease recognition sequence at the other end.

17. The method of claim 12, wherein said spacer has the same type II restriction endonudease recognition sequence at each end.

18. The method of claim 11 or 12, wherein the starting vector is 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.

19. The method of claim 1 , wherein said cloning vector is pMD19GW- Adv.BstX-SsfXI.

20. The method of claim 1 , wherein said DNA fragment is up to 12 Kb in length.

21 .A method for one-step cloning of multiple DNA fragments into a vector in a designed orientation, said method comprising:

a. obtaining DNA fragments with variable sticky ends, wherein said DNA fragments are obtained by:

I. amplifying target DNA fragments using a pair of primers specific for each target DNA fragment, wherein each primer hasadditional bases at its 5' endand a number of bases that are complementary to an end of the target DNA fragment, wherein the additional bases comprise a nicking endonuclease recognition site or at least one protection base followed by a nicking endonuclease recognition site;

II. digesting the amplified DNA fragment with the nicking endonuclease that corresponds to the nicking

endonuclease recognition site in eachprimer; and

III. denaturing to removed igested material;

wherein the variable sticky ends are produced by altering the number and arrangement of protection bases added at the 5' end of each primerand/or by altering the nicking endonuclease recognition site;

b. performing a ligation such that the DNA fragments ligate with each other and with the cloning vector in a designed orientation due to complementary base pairing of the sticky ends; and c. transforming competent cells with the ligation products.

22. The method of claim 21 , wherein the at least one protection base can be from one to ten.

23. The method of claim 21 , wherein the protection bases are in any

arrangement of A, G, C, or T.

24. The method of claim 21 , wherein the primers specific for each target DNA fragment have the same endonuclease recognition site.

25. The method of claim 21 , wherein the primers specific for each target DNA fragment have different endonuclease recognition sites.

26. The method of claim 21 , wherein said nicking endonuclease is

Nb.BbvCI, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.BbvCI,Nt.Alwl, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, or Nt.CviPII.

27. The method of claim 21 , wherein said nicking endonuclease is Nb.BbvCI.

28. The method of claim 21 , wherein said denaturing step comprises

incubating at 65 ° C for 5 min.

29. The method of claim 21 , wherein said cloning vector is obtained by digesting a starting vector.

30. The method of claim 21 , wherein said cloning vector is obtained using a method comprising:

a. obtaining a spacer that is bounded by a type II restriction

endonuclease recognition sequence at each end;

c. digesting the pre-cloning vector with the type II restriction

endonuclease corresponding to the recognition sites in the spacer.

31 .The method of claim 30, wherein said spacer is at least 300 bp in

length.

32. The method of claim 30, wherein said spacer isa selection marker.

33. The method of claim 32, wherein said selection marker is ccdB.

34. The method of claim 30, wherein said spacer has one type II restriction endonuclease recognition sequence at one end and another type II restriction endonuclease recognition sequence at the other end.

35. The method of claim 30, wherein said spacer has the same type II restriction endonuclease recognition sequence at each end.

36. The method of claim 29 or 30, wherein the starting vector is 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.

37. The method of claim 21 , wherein said cloning vector is pMD19GW- Adv.BstX-SsfXI.

38. The method of claim 21 , wherein said method is used to construct a vector comprising an RNAi construct.

39. A gene cloning kit comprising the linearized pMD19GW-Adv.BstX- BstXIcloning vector; nicking endonuclease reaction buffer; nicking endonuclease Nb.BbvCI; and ligase.