Recombinational cloning using nucleic acids having recombination sites
First Claim
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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.
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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).
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Citations
51 Claims
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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. - View Dependent Claims (2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 49, 50)
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3. A method for cloning or subcloning desired nucleic acid molecules comprising
a) combining in vitro or in vivo i) one or more Insert Donor molecules comprising one or more nucleic acid segments flanked by two or more recombination sites, wherein said recombination sites do not substantially recombine with each other; -
ii) two 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
b) incubating said combination under conditions sufficient to transfer one or more of said desired segments into said different Vector Donor molecules, thereby producing two or more different Product molecules.
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26. 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.
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36. A method for amplifing 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.
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37. A method for producing one or more cDNA molecules or a population of cDNA molecules comprising
(a) mixing an RNA template or population of RNA templates with a reverse transcriptase and one or more primers wherein said primers comprise one or more recombination sites or portions thereof and (b) incubating said mixture under conditions sufficient to make a first DNA molecule complementary to all or a portion of said template, thereby forming a first DNA molecule comprising one or more recombination sites or portions thereof.
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41. A method for synthesizing one or more nucleic acid molecules comprising one or more recombination sites, said method comprising:
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(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. - View Dependent Claims (42, 43, 44, 45)
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46. 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. - View Dependent Claims (47, 48, 51)
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Specification