| Method for ligating nucleic acids and molecular cloning -> Monitor Keywords |
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Method for ligating nucleic acids and molecular cloningRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal CellMethod for ligating nucleic acids and molecular cloning description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070072297, Method for ligating nucleic acids and molecular cloning. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 10/057,050, filed Jan. 25, 2002, which will issue as U.S. Pat. No. 7,109,178 on Sep. 19, 2006, and which is a Continuation of U.S. application Ser. No. 09/513,710, filed Feb. 25, 2000, the entireties of each of which are incorporated herein by reference. TECHNICAL FIELD [0002] The invention relates to methods of covalently joining nucleic acid molecules and methods of molecular cloning of nucleic acid molecules. BACKGROUND OF THE INVENTION [0003] Construction of recombinant nucleic acid molecules requires two enzymatic steps. First, site-specific restriction endonuclease digestion or PCR amplification are used to generate linear nucleic acid molecules with defined termini. Second, the linear molecules are covalently joined at their termini in the presence of a ligase enzyme. Methods of covalently joining and cloning nucleic acid molecules that require only one step or that eliminate the use of restriction endonucleases or ligases would be advantageous over the traditional method of constructing recombinant nucleic acid molecules. SUMMARY OF THE INVENTION [0004] It is an object of the invention to provide methods of covalently joining nucleic acid molecules. It is a further object of the invention to provide methods of cloning nucleic acid molecules. This and other objects of the invention are provided by one or more of the embodiments described below. [0005] One embodiment of the invention provides a method of covalently joining a nucleic acid insert molecule to first and second nucleic acid flanking molecules to form a ligated molecule. The method comprises incubating the insert molecule and the flanking molecules under conditions which permit their covalent joining to form a ligated molecule wherein an insert molecule is positioned between the first and the second flanking molecule. Each end of the insert molecule comprises a 5'-hydroxyl group. One end only of each of the first and second flanking molecules comprises a covalently bound topoisomerase polypeptide. [0006] Another embodiment of the invention provides a method of covalently joining a nucleic acid insert molecule to first and second nucleic acid flanking molecules to form a ligated molecule. The method comprises incubating an insert molecule, wherein one end of the insert molecule comprises a 5'-hydroxyl group and the other end comprises a 5'-phosphate group, with the first flanking molecule, wherein one end only of the first flanking molecule comprises a covalently bound topoisomerase polypeptide. The incubation is done under conditions which permit their covalent joining to form a ligated nucleic acid wherein the insert molecule is positioned adjacent to the first flanking molecule. This ligated nucleic acid is incubated with phosphatase under conditions which permit removal of a 5'-phosphate group from the ligated nucleic acid. The ligated nucleic acid is incubated with the second flanking molecule. One end only of the second flanking molecule comprises a covalently bound topoisomerase polypeptide. The incubation is done under conditions which permit covalent joining to form a ligated molecule where the insert molecule is positioned between the first and the second flanking molecule. [0007] In still another embodiment of the invention a method of covalently joining a nucleic acid insert molecule to first and second nucleic acid flanking molecules to form a ligated molecule is provided. The method comprises incubating an insert molecule and flanking molecules under conditions which permit their covalent joining to form a ligated molecule wherein an insert molecule is positioned between the first and the second flanking molecule. One end of the insert molecule comprises a 5'-hydroxyl group and the other end comprises a 5'-phosphate group. One end only of the first flanking molecule comprises a covalently bound topoisomerase polypeptide and one end of the second flanking molecule comprises a ligase substrate site. [0008] Any of the first and second nucleic acid flanking molecules can together comprise a pair of left and right vector arms. Further, the ends of the vector arms not covalently joined to the insert can be covalently or non-covalently joined to each other by a method selected from the group consisting of nucleic acid ligase mediated ligation, complementary sequence annealing, topoisomerase mediated ligation, in vitro site-specific recombination, in vivo site-specific recombination, and in vivo homologous recombination. [0009] In still another embodiment of the invention a method of molecular cloning is provided. The method comprises incubating a nucleic acid insert molecule comprising a 5'-hydroxyl group at one end and a 5'-phosphate at the other end, and a linear cloning vector. The linear cloning vector comprises a covalently bound topoisomerase polypeptide at one end only and a ligation substrate site at the other end. The incubation is done under conditions sufficient for their covalent joining to form a ligated circular vector. The ligated circular vector is transformed into a host cell. [0010] Another embodiment of the invention provides a method for molecular cloning. The method comprises incubating a nucleic acid insert molecule where each end of the insert molecule comprises a 5'-hydroxyl group with a first and a second linear arm where one end only of each of the first and second linear arms comprises a covalently bound topoisomerase and the other end comprises a cloning substrate site. The incubation is done under conditions sufficient for their covalent joining to form a ligated insert molecule where the insert molecule is positioned between the first and the second linear arm. The ligated insert molecule is transformed into a host cell. [0011] Even another embodiment of the invention provides a method for molecular cloning. A nucleic acid insert molecule, wherein one end of the insert molecule comprises a 5'-hydroxyl group and the other end comprises a 5'-phosphate group, and a first linear arm, wherein one end only of the first linear arm comprises a covalently bound topoisomerase polypeptide and the other end comprises a cloning substrate site are incubated together. The incubation is done under conditions which permit their covalent joining to form a ligated nucleic acid wherein the insert molecule is positioned adjacent to the first linear arm. The ligated nucleic acid is incubated with phosphatase under conditions which permit removal of a 5'-phosphate group from the ligated nucleic acid. The ligated nucleic acid is incubated with a second linear vector arm, wherein one end only of the second linear vector arm comprises a covalently bound topoisomerase polypeptide and the other end comprises a cloning substrate site. The incubation is done under conditions which permit covalent joining to form a ligated insert molecule wherein the insert molecule is positioned between the first and the second linear vector arm. The ligated insert molecule is transformed into a host cell. [0012] In yet another embodiment of the invention a method for molecular cloning is provided comprising incubating a nucleic acid insert molecule, wherein one end of the insert molecule comprises a 5'-hydroxyl group and the other end comprises a 5'-phosphate group; a first linear arm, wherein one end only of the first linear arm comprises a covalently bound topoisomerase polypeptide and the other end comprises a cloning substrate site; and a second linear arm, wherein one end of the second linear arm comprises a ligase substrate site and the other end comprises a cloning substrate site. The incubation is done under conditions which permit their covalent joining to form a ligated insert molecule wherein the insert molecule is positioned between the first and the second linear arm. The ligated insert molecule is transformed into a host cell. [0013] The cloning substrate site can be selected from the group consisting of a cos site, a LIC site, and a loxP site. [0014] Where the cloning substrate site is loxP, the method can further comprise incubating in vitro the ligated insert molecule with a Cre recombinase and a circular plasmid comprising a loxP site. The incubation is done under conditions sufficient for site-specific recombination to form a circular plasmid comprising the ligated insert molecule. The circular plasmid comprising the ligated insert molecule is transformed into a host cell. [0015] Where the cloning substrate site is loxP the method can further comprise transforming the ligated insert molecule into a host cell comprising a circular plasmid comprising a loxP site, wherein the cell expresses Cre recombinase. The transformation is done under conditions sufficient for site-specific recombination to form a circular plasmid comprising the ligated insert molecule within the cell. [0016] Where the cloning substrate site is a site for homologous recombination with a circular plasmid vector the transformation step further comprises transforming the ligated insert molecule into a host cell comprising a circular plasmid vector. The circular plasmid vector comprises a site for homologous recombination with the ligated insert molecule, and the host cell is recA+. The transformation is done under conditions sufficient for homologous recombination to form a circular plasmid comprising the ligated insert molecule within the host cell. [0017] The first linear arm can comprise a left lambda arm comprising at one end only a covalently bound topoisomerase. The second linear arm can comprise a right lambda arm comprising at one end only a covalently bound topoisomerase. [0018] As used herein, the term "join" or "joining" refers to both covalent and noncovalent attachment of one nucleic acid to another, or one end of a nucleic acid to another end of a nucleic acid. "Covalent" joining refers to the attachment of one end of a nucleic acid strand to another end of a nucleic acid strand via a phosphate bond or to attachment of one end of a double-stranded nucleic acid to another double-stranded end via phosphate bonding on one or both strands. "Non-covalent" joining refers to attachment of one end of a nucleic acid to another end via annealing of a single-stranded regions to each other; that is, no phosphate bond is generated in this embodiment. [0019] "Ligate" or "ligated" refers to the covalent joining of two ends of one or more nucleic acid molecules. [0020] "Complementary annealing" refers to annealing, or the pairing of bases, of complementary regions of one or more nucleic acids, and thus to the formation of hydrogen bonds and other non-covalent interactions between pairs of bases. Continue reading about Method for ligating nucleic acids and molecular cloning... Full patent description for Method for ligating nucleic acids and molecular cloning Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for ligating nucleic acids and molecular cloning patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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