Patent ID: 6184000
Filing Date: 2001-02-06
Classification: C12N,C12Y

Abstract:
A method for directionally inserting DNA sequences into a vector comprising:a) providing a recipient DNA vector having a first restriction site therein, wherein said first restriction site contains a degenerate recognition sequence and which generates cohesive ends when digested with its corresponding restriction endonuclease, and digesting said recipient DNA vector with said restriction endonuclease corresponding to said first restriction site, said first restriction site generating first and second cohesive ends on the digested vector;b) providing a first insert DNA segment comprising a first target DNA sequence having second and third restriction sites adjacent to its first and second ends, respectively, wherein said second restriction site is different from said first restriction site of said recipient DNA vector, contains a degenerate recognition sequence and generates a cohesive end when digested with its corresponding restriction endonuclease, wherein the nucleotide sequence of said degenerate recognition sequence in said second restriction site has been selected such that the cohesive end generated from digestion of said second restriction site is complementary to said first cohesive end on the digested vector of (a); and wherein said third restriction site is essentially the same as said first restriction site of said recipient DNA vector, contains a degenerate recognition sequence and generates a cohesive end when digested with its corresponding restriction endonuclease, wherein the nucleotide sequence of said degenerate recognition sequence in said third restriction site has been selected such that the cohesive end generated from digestion of said third restriction site is complementary to said second cohesive end on the digested recipient DNA vector of (a), and when annealed thereto regenerates a restriction site on the second end of said first target DNA sequence which is essentially the same as said third restriction site, and digesting said first insert DNA segment with said restriction endonucleases corresponding to said second restriction site and said third restriction site;c) ligating the digested recipient DNA vector of (a) and the digested first insert DNA segment of (b) to produce a recombinant vector comprising said first target DNA sequence, with a restriction site on the second end of said first target DNA sequence being regenerated within said recombinant vector which is essentially the same as said third restriction site.