US 6,184,000 B2Granted

System for the sequential, directional cloning of multiple DNA sequences

AssigneeThe United States of America as represented by the Secretary of Agriculture

InventorsJames D. Jones, Thomas M. Hohn, Timothy D. Leathers

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Abstract

A method which combines the use of polymerase chain reaction (PCR) or oligonucleotide linkers and restriction enzymes which cleave degenerate or variable recognition site sequences to allow the cloning of multiple DNA sequences into a vector is disclosed. In this invention, a plurality of unrelated DNA sequences may be directionally cloned within a single vector by adding onto the ends of the sequences, restriction sites with specific sequences which are cleaved by corresponding restriction endonucleases which recognize degenerate or variable recognition sites and which generate cohesive ends upon cleavage. The compatibility (or ability to anneal) of the cohesive ends on different DNA sequences is controlled by the choice of the nucleotide sequence within the recognition sequences of the restriction endonucleases, allowing the DNA sequences to be inserted or joined in any desired orientation. These restriction sites may be selectively incorporated onto either or both end of any DNA sequence of interest using oligonucleotidelinkers or using PCR by adding the restriction sites onto the termini of the 5′ and/or 3′ primers.

Claims27

Dependent claims

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.

1 more element

A method for the sequential and directional cloning of multiple DNA sequences into a single vector comprising:

a) providing a vector having one or more restriction sites effective for insertion of DNA therein;
providing a first DNA segment comprising a first DNA sequence of interest having a first restriction site adjacent to a selected end, wherein said first restriction site contains a degenerate recognition sequence and which generates a cohesive end when digested with its corresponding restriction endonuclease, and inserting said first DNA segment into said vector to produce a first recombinant vector comprising said first DNA sequence having said first restriction site adjacent to its selected end;
b) digesting said first recombinant vector with said restriction endonuclease corresponding to said first restriction site, said first restriction site generating first and second cohesive ends on said recombinant vector, said first cohesive end being on the end of the cleaved vector adjacent to said selected end of said first DNA sequence, and said second cohesive end being on the opposite end of the cleaved vector;
c) providing a second DNA segment comprising a second DNA sequence of interest 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 first DNA segment, and also contains a degenerate recognition sequence and which 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 said cohesive end generated from digestion of said second restriction site is complementary to said first cohesive end on the digested vector of (b);
and wherein said third restriction site is essentially the same as said first restriction site of said first DNA segment, and also contains a degenerate recognition sequence and which 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 said cohesive end generated from digestion of said third restriction site is complementary to said second cohesive end on the digested vector of (b), and when annealed thereto regenerates a restriction site adjacent to the second end of said second DNA sequence which is essentially the same as said third restriction site, digesting said second DNA segment with said restriction endonucleases corresponding to said second restriction site and said third restriction site;
d) ligating the digested recombinant vector of (b) and the digested second DNA segment of (c) to produce a second recombinant vector comprising said first DNA sequence and said second DNA sequence, with said first end of said second DNA sequence being adjacent to said selected end of said first DNA sequence and a restriction site adjacent to the second end of said second DNA sequence being regenerated within said second recombinant vector which is essentially the same as said third restriction site.

3 more elements

Dependencies are inferred from the claim text.

Facts

Application: 09/360,083Filed 23 Jul 1999
Granted: 6 Feb 2001
Term ends: Not estimatedThe source records don’t support an estimate.

Classification5 codes

C12N 15/66
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for inserting a…
C12N 15/52
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: genes encoding for enzymes or proenzymes
C12Y 205/01029
Transferring alkyl or aryl groups, other than methyl groups: geranylgeranyl diphosphate synthase
C12N
Microorganisms or enzymes