Recombinational cloning using nucleic acids having recombination sites

Abstract

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

1 . A method for cloning or subcloning one or more desired nucleic acid molecules comprising
 (a) combining in vitro or in vivo
 (i) one or more Insert Donor molecules comprising one or more desired nucleic acid segments flanked by at least two recombination sites, wherein said recombination sites do not substantially recombine with each other; 
 (ii) one or more Vector Donor molecules comprising at least two recombination sites, wherein said recombination sites do not substantially recombine with each other; and 
 (iii) one or more site-specific recombination proteins; 
   (b) incubating said combination under conditions sufficient to transfer one or more of said desired segments into one or more of said Vector Donor molecules, thereby producing one or more desired Product nucleic acid molecules;   (c) combining in vitro or in vivo
 (i) one or more of said Product molecules comprising said desired segments flanked by two or more recombination sites, wherein said recombination sites do not substantially recombine with each other; 
 (ii) one or more different Vector Donor molecules comprising two or more recombination sites, wherein said recombination sites do not substantially recombine with each other; and 
 (iii) one or more site-specific recombination proteins; and 
   (d) incubating said combination under conditions sufficient to transfer one or more of said desired segments into one or more different Vector Donor molecules, thereby producing one or more different Product molecules.   
     
     
         2 . The method of  claim 1 , further comprising incubating said different Product molecules with one or more different Vector Donor molecules under conditions sufficient to transfer one or more of said desired segments into said different Vector Donor molecules. 
     
     
         3 . The method of  claim 1 , wherein said Vector Donor molecules comprise at least one Selectable marker. 
     
     
         4 . The method of  claim 3 , wherein the Selectable marker comprises at least one DNA segment selected from the group consisting of:
 (a) a DNA segment that encodes a product that provides resistance in a recipient cell against otherwise toxic compounds;   (b) a DNA segment that encodes a product that is otherwise lacking in a recipient cell;   (c) a DNA segment that encodes a product that suppresses the activity of a gene product in a recipient cell;   (d) a DNA segment that encodes a product that can be identified;   (e) a DNA segment that encodes a product that inhibits a cell function in a recipient cell; (f) a DNA segment that inhibits the activity of any of the DNA segments of (a)-(e) above; (g) a DNA segment that binds a product that modifies a substrate;   (h) a DNA segment that encodes a specific nucleotide recognition sequence which can be recognized by a protein, an RNA, DNA or chemical.   (i) a DNA segment that, when deleted, directly or indirectly confers sensitivity to cell killing by particular compounds within a recipient cell;   (j) a DNA segment that encodes a product that is toxic in a recipient cell; and   (k) a DNA segment that can be used to isolate or identify a desired molecule.   
     
     
         5 . The method of  claim 4 , wherein said Selectable marker comprises at least one marker selected from the group consisting of an antibiotic resistance gene, a tRNA gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an antisense oligonucleotide, a restriction endonuclease, a restriction endonuclease cleavage site, an enzyme cleavage site, a protein binding site, and a sequence complementary to a PCR primer sequence. 
     
     
         6 . The method of  claim 1 , wherein said Vector Donor molecules comprise prokaryotic and/or eukaryotic vectors. 
     
     
         7 . The method of  claim 6 , wherein said eukaryotic vectors comprise vectors which propagate and/or replicate in yeast cells, plant cells, fish cells, eukaryotic cells, mammalian cells, and/or insect cells. 
     
     
         8 . The method of  claim 6 , wherein said prokaryotic vectors comprise vectors which propagate and/or replicate in gram negative or gram positive bacteria. 
     
     
         9 . The method of  claim 1 , wherein said recombination sites are selected from the group consisting of loxP, attB, attP, attL, and attR. 
     
     
         10 . The method of  claim 1 , wherein said recombination proteins are selected from the group consisting of Int, Cre, Flp, Res. 
     
     
         11 . A method for preparing a nucleic acid molecule comprising two or more recombination sites or portions thereof comprising
 (a) mixing a nucleic acid template with a polypeptide having polymerase activity and one or more primers comprising one or more recombination sites or portions thereof; and   (b) incubating said mixture under conditions sufficient to synthesize a nucleic acid molecule which is complementary to all or a portion of said template and which comprises one or more recombination sites or portions thereof.   
     
     
         12 . The method of  claim 11 , further comprising incubating said synthesized molecule in the presence of one or more primers comprising one or more recombination sites or portions thereof under conditions sufficient to synthesize a second nucleic acid molecule complementary to all or a portion to said first nucleic acid molecule, thereby producing a double stranded nucleic acid molecule comprising two or more recombination sites or portions thereof. 
     
     
         13 . The method of  claim 12 , wherein said recombination sites or portions thereof are located at or near one or more termini of said synthesized double stranded nucleic acid molecule. 
     
     
         14 . A method for amplifying a nucleic acid molecule comprising
 (a) contacting a first nucleic acid molecule with a first primer which is complementary to a portion of said first nucleic acid molecule, and a second nucleic acid molecule with a second primer which is complementary to a portion of said second nucleic acid molecule with a polypeptide having polymerase activity;   (b) incubating said mixture under conditions sufficient to form a third nucleic acid molecule complementary to all or a portion of said first nucleic acid molecule and a fourth nucleic acid molecule complementary to all or portion of all or said portion of said second nucleic acid molecule;   (c) denaturing said first and third and said second and fourth nucleic acid molecule; and   (d) repeating steps (a) through (c) one or more times, wherein said first primer and/or second primer comprise one or more recombination sites or portions thereof.   
     
     
         15 . A method for synthesizing one or more nucleic acid molecules comprising one or more recombination sites, said method comprising:
 (a) obtaining one or more linear nucleic acid molecules; and   (b) contacting said molecules with one or more adapters which comprise one or more recombination sites or portions thereof under conditions sufficient to add one or more of said adapters to one or more termini of said linear nucleic acid molecule.   
     
     
         16 . A method for adding one or more recombination sites or portions thereof to one or more nucleic acid molecules, said method comprising:
 (a) contacting one or more nucleic acid molecules with one or more integration sequences which comprise one or more recombination sites or portions thereof; and   (b) incubating said mixture under conditions sufficient to incorporate said integration sequences into said nucleic acid molecules.   
     
     
         17 . The method of  claim 16 , wherein said integration sequences are selected from the group consisting of transposons, integrating viruses, integrating elements, integrons and recombination sequences.