Recombinational cloning using engineered recombination sites
First Claim
1. A Vector Donor DNA molecule comprising a first DNA segment and a second DNA segment, said first or second DNA segment containing at least one Selectable marker, wherein the first and second segments are separated either by, (i) in a circular Vector Donor, a first and a second recombination site, or (ii) in a linear Vector Donor, at least a first recombination site, wherein each pair of flanking recombination sites are engineered and do not recombine with each other.
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Accused Products
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
34 Claims
- 1. A Vector Donor DNA molecule comprising a first DNA segment and a second DNA segment, said first or second DNA segment containing at least one Selectable marker, wherein the first and second segments are separated either by, (i) in a circular Vector Donor, a first and a second recombination site, or (ii) in a linear Vector Donor, at least a first recombination site, wherein each pair of flanking recombination sites are engineered and do not recombine with each other.
- 5. An Insert Donor DNA molecule, comprising a desired DNA segment flanked by a first recombination site and a second recombination site, wherein the first and second recombination sites are engineered and do not recombine with each other.
- 7. A kit comprising a container being compartmentalized to receive in close confinement therein at one compartment, wherein a first compartment contains a Vector Donor DNA molecule comprising a first DNA segment and a second DNA segment, said first or second DNA segment containing at least one Selectable marker, wherein the first and second segments are flanked either by, (i) in a circular Vector Donor, a first and a second recombination site, or (ii) in a linear Vector Donor, a first recombination site, wherein each pair of flanking recombination sites are engineered and do not recombine with each other.
- 10. A nucleic acid molecule, comprising at least one DNA segment having at least two recombination sites flanking a Selectable marker or a desired DNA segment, wherein at least one of said recombination sites comprises a core region having at least one engineered mutation that enhances recombination in vitro in the formation of a Cointegrate DNA or a Product DNA.
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16. A method for making a nucleic acid molecule, comprising providing a nucleic acid molecule having at least one engineered recombination site comprising at least one DNA sequence having at least 90% homology to at least one of SEQ ID NOS:
- 1-16.
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22. A method of making a Cointegrate DNA molecule, comprising combining in vitro:
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(i) an Insert Donor DNA molecule, comprising a desired DNA segment flanked by a first recombination site and a second recombination site, wherein the first and second recombination sites do not recombine with each other;
(ii) a Vector Donor DNA molecule containing a third recombination site and a fourth recombination site, wherein the third and fourth recombination sites do not recombine with each other; and
(iii) at least one site specific recombination protein capable of recombining said first and third recombinational sites said second and fourth recombinational sites;
thereby allowing recombination to occur, so as to produce a Cointegrate DNA molecule comprising said first and third or said second and fourth recombination sites. - View Dependent Claims (21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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Specification