US 8,999,673 B2 · Method for specifically producing a joined DNA fragment comprising a sequence derived from a target gene

Abstract

Provided is a method for selectively obtaining, for a given target gene, a “joined DNA fragment” wherein just a target gene fragment is joined with desired other DNA fragments, regardless of whether a DNA fragment containing a target gene sequence has been purified. In the provided method, a double-stranded joining DNA fragment containing a sequence A and/or a sequence B is selectively joined to the ends of a target gene fragment. A mixture of a double-stranded gene fragment, the 3′ end of which is protruding, and the double-stranded joining DNA fragment, which are related in a prescribed manner, undergoes at least two cycles of thermal denaturation, reassociation, and DNA synthesis, resulting in a “joined DNA fragment,” which is a double-stranded DNA fragment including at least one instance of a sequence resulting from joining sequence A, the target gene sequence, and sequence B. A “single-side joined DNA fragment” can also be obtained, by a similar method.

A method for producing a joined DNA fragment in which joining DNA regions have been joined on both ends of a target gene, comprising:

(1) providing a double-stranded gene fragment comprising a target gene sequence as a template;
amplifying the template to produce a 3′ end protruding double-stranded gene fragment containing a target gene having associative regions on the two termini of the target gene, the two associative regions having base sequences that do not mutually associate, the base sequence of one or both of the associative regions being a base sequence contained in the target gene with a protruding sequence of one or more nucleotides being present on the 3′ ends of both of the associative regions joined to a 3′ end protruding double-stranded gene fragment containing a non-target gene;
(2) providing a double-stranded DNA joining fragment, containing a joining region, and having associative regions on the two termini of the joining DNA region;
wherein
(a) one of the associative regions of the 3′ end protruding double-stranded gene fragment is comprised of a base sequence that is homologous with the associative region of one terminus of the double-stranded joining DNA fragment, with the sequence on the terminus to which the 3′ protruding end has been added being the side that connects to the joining DNA region in one of the associative regions of the double-stranded joining DNA fragment;
(b) the protruding sequence from the one associative region of the 3′ end protruding double-stranded gene fragment does not have a strand-elongating function in a DNA synthesis reaction;
(c) the other associative region of the 3′ end protruding double-stranded gene fragment is comprised of a base sequence that is homologous with the associative region of the other terminus of the double-stranded joining DNA fragment, with the sequence on the terminus to which the 3′ protruding end has been added being the side that connects to the joining DNA region in the other of the associative regions of the double-stranded joining DNA fragment;
(d) the protruding sequence from the other associative region of the 3′ end protruding double-stranded gene fragment does not have a strand-elongating function in the DNA synthesis reaction;
and
(3) subjecting the product of step 1 and the double-stranded joining DNA fragment to at least two cycles of a thermal denaturation, re-association, and DNA synthesis reaction to obtain a joined DNA fragment in which joining DNA regions have been joined on both ends of a target gene while suppressing generation of a joined DNA fragment in which joining DNA regions have been joined on both ends of a non-target gene, without a purification step for removal of the 3′ end protruding double-stranded gene fragment containing a non-target gene from the 3′ end protruding double-stranded gene fragment containing a target gene sequence;
wherein the method for producing the joined DNA fragment does not employ chimeric RNA/DNA primers and wherein the method for producing the joined DNA fragment produces a joined DNA fragment in which joining DNA regions are joined to both sides of a target gene, the joined DNA fragment comprising:
(A) a target gene sequence, comprising associative regions on each of the two termini of the target gene sequence, the two associative regions comprising mutually non-associative base sequences, the base sequence of one or both of the regions being a base sequence contained in the target gene sequence, and both of the associative regions having protruding sequences of one or more nucleotides on the 3′ ends thereof;
and
(B) double-stranded joining DNA fragments comprising a joining DNA region, and having associative regions on each terminus of the joining DNA region, respectively.

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Method for specifically producing a joined DNA fragment comprising a sequence derived from a target gene

Life of this patent

Method for specifically producing a joined DNA fragment comprising a sequence derived from a target gene

Life of this patent

Abstract

Claims31