Recombinational cloning using nucleic acids having recombination sites
AssigneeLife Technologies Corporation
InventorsJames HARTLEY, Michael Brasch, Gary Temple, Donna Fox
Life of this patent
Estimating the term…
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).
Claims17
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.
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.
(canceled)
(canceled)
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.
Dependencies are inferred from the claim text.