Cloning method and kit
First Claim
1. A method of cloning a target DNA, wherein(a) said target DNA is amplified by PCR to obtain single-stranded amplified target DNA,(b) the single-stranded, amplified target DNA is contacted with a single-stranded, linear vector DNA having terminal regions which are complementary to respective terminal regions of said amplified target DNA, wherein said complementary terminal regions overlap and hybridize to form a cyclic DNA product comprising single-stranded target DNA, single-stranded vector DNA, and two double-stranded regions formed by hybridization of said overlapping complementary terminal regions of the single stranded vector and the single stranded amplified target DNA, wherein said double-stranded regions are separated from each other by a region of single-stranded target DNA, and wherein said cyclic DNA product is essentially single-stranded apart from said double-stranded complementary regions,(c) said cyclic DNA product is introduced into a host organism, and(d) said cyclic DNA product is cloned by replication of said host organism.
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Abstract
A method of amplifying target DNA is disclosed wherein said DNA is first amplified by PCR, the amplified DNA then being contacted with a single stranded linearised plasmid vector having terminal regions which are complementary to terminal regions of the PCR amplified DNA, whereby a cyclic product is formed comprising single stranded sequences from said target DNA and said vector and two double stranded regions from the overlapping terminal regions of the vector and the PCR amplified DNA; the cyclic product then being introduced into a host organism. Two-stage PCR may be performed and site-specific mutagenesis may be effected between PCR amplification and formation of the cyclic product. The single-stranded linearised plasmid vector and/or the target DNA may be immobilised. Kits are disclosed for performing various aspects of the method which can be used in a method of diagnosis wherein the target DNA is characteristic of a physiological condition.
47 Citations
14 Claims
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1. A method of cloning a target DNA, wherein
(a) said target DNA is amplified by PCR to obtain single-stranded amplified target DNA, (b) the single-stranded, amplified target DNA is contacted with a single-stranded, linear vector DNA having terminal regions which are complementary to respective terminal regions of said amplified target DNA, wherein said complementary terminal regions overlap and hybridize to form a cyclic DNA product comprising single-stranded target DNA, single-stranded vector DNA, and two double-stranded regions formed by hybridization of said overlapping complementary terminal regions of the single stranded vector and the single stranded amplified target DNA, wherein said double-stranded regions are separated from each other by a region of single-stranded target DNA, and wherein said cyclic DNA product is essentially single-stranded apart from said double-stranded complementary regions, (c) said cyclic DNA product is introduced into a host organism, and (d) said cyclic DNA product is cloned by replication of said host organism.
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10. A kit for cloning target DNA comprising:
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(a) a linear vector (i) in single stranded form having terminal regions that are complementary to respective terminal regions of a target DNA, whereby said complementary terminal regions overlap and hybridize to form a cyclic DNA product comprising single stranded target DNA, single stranded vector DNA, and two double stranded DNA regions wherein said double stranded regions are separated from each other by a region of single stranded target DNA, and wherein said cyclic DNA product is essentially single stranded apart from said double stranded complementary regions;
or(ii) in double stranded form and immobilized by one end of one strand thereof, wherein the single stranded linear vector is produced by separation of the non-immobilized strand from the immobilized strand; (b) a polymerase; (c) two PCR primers for amplification of the target DNA, wherein said PCR primers correspond to the terminal regions of said vector; (d) nucleoside triphosphates. - View Dependent Claims (11, 12)
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Specification