Ligation of double-stranded DNAs
AssigneeAisin Seiki Kabushiki Kaisha
InventorsYasushi Shigemori, Michio Oishi
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Abstract
The present invention relates to a DNA ligation method. Specifically, a DNA complex comprising a three-stranded structure in each of the ends of double-stranded DNAs, one of which has single-stranded regions at the end, and the other of which does not have single-stranded regions at the end, is formed under the presence of a homologous recombinant protein. Moreover, according to needs, the DNA complex is introduced into host cells, and said cells are cultured.
Claims10
A method of ligating a double-stranded end of a first double-stranded DNA and a single-stranded end of second double-stranded DNA, wherein the method comprises:
- a) contacting, in the presence of a homologous recombination protein, the single-stranded end of said second double-stranded DNA and the double-stranded end of said first double-stranded DNA, wherein the nucleotide sequence of one strand of said first double-stranded end is homologous to the nucleotide sequence of said single-stranded end, to form a three-stranded structure comprising said single-stranded end and said double-stranded end, and
- b) completing the ligation by converting the three-stranded structure into a double-stranded structure by inserting a DNA complex comprising the three-stranded structure into cells and replicating it therein;
- wherein the homologous recombination protein is a Rec A protein.
A gene-cloning kit consisting essentially of the following components:
- a) a double-stranded DNA comprising single-stranded regions at both ends, wherein the nucleotide sequences of these single-stranded regions are mutually non-complementary;
- b) an oligonucleotide primer comprising as a part of the 5′ end sequence, a sequence that is complementary to one single-stranded region of the nucleotide sequence of (a), and further comprising a sequence that is complementary to a part of one end of a sequence of a gene to be cloned;
- c) an oligonucleotide primer comprising as a part of the 5′ end sequence, a sequence that is complementary to the other single-stranded region of the nucleotide sequence of (a), and further comprising a sequence that is complementary to a part of the other end of the sequence of the gene to be cloned;
- and
- d) a homologous recombination protein;
- wherein the homologous recombination protein is a Rec A protein.
Dependencies are inferred from the claim text.