Methods and compositions for generating recombinant nucleic acid molecules

Abstract

A method of generating a double stranded (ds) recombinant nucleic acid molecule covalently linked in both strands by contacting two or more ds nucleotide sequences with a topoisomerase under conditions such that both termini of at least one end of a first ds nucleotide sequence are covalently linked by the topoisomerase to both termini of at least one end of a second ds nucleotide sequence is provided. Also provided is a method for generating a ds recombinant nucleic acid molecule covalently linked in one strand, by contacting two or more ds nucleotide sequences with a type IA topoisomerase under conditions such that one strand, but not both strands, of one or both ends of a first ds nucleotide sequence are covalently linked by the topoisomerase. Compositions for performing such methods, and compositions generated from such methods also are provided, as are kits containing components useful for conveniently practicing the methods.

Description

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Ser. No. 60 / 520,946, filed Nov. 17, 2003; and is a Continuation-in-Part of U.S. Ser. No. 10 / 014,128, filed Dec. 7, 2001; which claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 60 / 326,092, filed Sep. 28, 2001, and U.S. Ser. No. 60 / 254,510, filed Dec. 8, 2000.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention relates generally to compositions and methods for facilitating the construction of recombinant nucleic acid molecules, and more specifically to compositions for using one or more topoisomerases to generate covalently closed recombinant nucleic acid molecules and to methods of making such recombinant nucleic acid molecules. [0004] 2. Background Information [0005] The advent of recombinant DNA technology has allowed the cloning and identification of genes from many different organisms, and the determination of the complete genomes of an ever-...

AI Technical Summary

Benefits of technology

[0059] A method of the invention is additionally exemplified by contacting 1) a first ds nucleotide sequence having a first end and a second end, wherein, at the first end, second end, or both ends, the first ds nucleotide sequence has a first topoisomerase covalently bound to the 5′ terminus and a second topoisomerase covalently bound to the 3′ terminus of the first end, the second end, or both ends (i.e., one or both ends contain a topoisomerase-charged 5′ terminus and a topoisomerase-charged 3′ terminus); and 2) at least a second ds nucleotide sequence, which, preferably, has, or can be made to have, hydroxyl groups at the 5′ terminus and 3′ terminus of an end to be covalently linked to an end of the first ds nucleotide sequence containing the topoisomerases. The method also can be performed wherein either the 5′ terminus or 3′ terminus of the end containing a topoisomerase-charged 3′ terminus or topoisomerase-charged 5′ terminus, respectively, contains a topoisomerase recognition site, wherein the method further includes contacting the components with a topoisomerase that can effect its activity with respect to the topoisomerase recognition site. Such a method of the invention can be performed using only a first ds nucleotide sequence and a second ds nucleotide sequence, or can include a third, fourth or more ds nucleotide sequence as desired, wherein the ds nucleotide sequences are as defined for the first ds nucleotide sequence, the second ds nucleotide sequence, or a combination thereof. A first or second (or other) ds nucleotide sequence independently can, but need not, have one or more topoisomerases covalently bound to a 5′ terminus, 3′ terminus, or both 5′ and 3′ termini of the second end (i.e., the undefined end). Unless indicated otherwise, the first and second (or other) ds nucleotide sequences can be the same or can be different.

[0060] The present invention further relates to a method of generating a ds recombinant nucleic acid molecule covalently linked in both strands by 1) amplifying a portion of a first ds nucleotide sequence using a PCR primer pair, wherein at least one primer of the primer pair encodes a complement of a topoisomerase recognition site, thereby producing an amplified first ds nucleotide sequence having a first end and a second end, wherein the first end or second end or both has a topoisomerase recognition site at or near the 3′ terminus; and 2) contacting a) the amplified first ds nucleotide sequence; b) at least a second ds nucleotide sequence having a first end and a second end, wherein the first end or second end or both has a topoisomerase recognition site, or cleavage product thereof, at or near the 3′ terminus and has, or can be made to have, a hydroxyl group at the 5′ terminus of the same end; and c) a site specific topoisomerase, under conditions such that the topoisomerase can cleave the end of the amplified first ds nucleotide sequence having a topoisomerase recognition site and the end (or ends) of the at least second ds nucleotide sequence having a topoisomerase recognition site, and can effect its ligating activity. The PCR primer that encodes a complement of a topoisomerase recognition site can have a hydroxyl group at its 5′ terminus, or the amplified first ds nucleotide sequence generated using the primer can be contacted with a phosphatase to generate a hydroxyl group at its 5′ terminus. The PCR primer encoding the complement of a topoisomerase recognition site also can comprise a nucleotide sequence at its 5′ terminus such that, upon cleavage by a site specific topoisomerase of a first ds nucleotide sequence amplified using the primer, the ds nucleotide sequence contains a 5′ overhanging sequence, which is complementary to a 5′ overhang of a second (or other) ds nucleotide sequence to which the first ds nucleotide sequence is to be covalently linked according to a method of the i...

Problems solved by technology

A significant bottleneck in recombinant DNA methodology is the requirement that each nucleic acid sequence that is to be used to prepare a construct must be cloned into a vector, the vector must be introduced into and amplified in a host cell (generally a bacterial cell), the amplified vector must be isolated from the host cell, and then must be transformed or transfected into the appropriate cell type for expression.

However, such vectors are limited in that only the most co...

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