Vectors for gene-self-assembly

• US 20020025561A1
• Filed: 04/17/2001
• Published: 02/28/2002
• Est. Priority Date: 04/17/2000
• Status: Abandoned Application

• Current Assignee: Clague Pitman Hodgson
• Original Assignee: Clague Pitman Hodgson
• Inventors: Hodgson, Clague Pitman
• Application Number:
  US09/836,737
• Publication Number:
  US 20020025561A1
• Time in Patent Office: Days
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention provides novel vectors and methods for assembling complex DNA molecules starting with a plurality of input gene sequences. The input gene sequences (which overlap with each other by a defined number of bases) are cloned into a vector at a unique restriction site that is flanked on each side by class IIS restriction endonuclease sites. When the clones are digested with the class IIS restriction enzyme, the inserts are released from the vector with a defined number of bases removed from either the 5′ or 3′ termini, corresponding to the overlap sequences. The overlap sequences, which are unique, non-palindromic sequence strings, permit the fragments to self-assemble. When the fragments are ligated, a seamless, unambiguous linear array fragments is created. The invention can be used for assembling synthetic genes, constructs, vectors and chromosomes.

Claims

1. A method for assembling gene constructs from a plurality of DNA fragments, comprising:

(a) preparing a series of overlapping DNA molecules, said DNA molecules having a defined length of overlap, said overlap comprising unique, non-palindromic DNA sequences;

(b) cloning the DNA molecules into a vector, said vector comprising a cloning site that is flanked on both sides by class IIS restriction endonuclease recognition sites, said sites positioned to allow removal by digestion with the class IIS enzyme or enzymes of a defined number of bases from one strand on both ends of the fragment;

(c) validating the insert fragments;

(d) digesting the clones with the appropriate class IIS restriction enzyme or enzymes, releasing the insert DNA fragments, now modified by the removal of the defined number of bases from one strand at each terminus;

(e) purifying the insert fragments away from the vector fragments;

(f) annealing and ligating the insert fragments together; and

(g) characterizing the resulting DNA construct,

Whereby a DNA construct, vector, gene or chromosome with the desired order and orientation of fragments is created.

2. The method of claim 1, wherein the vector is pWB (SEQ ID NO:

1).

3. The method of claim 1, wherein the class IIS enzyme site of choice is SapI.

4. The method of claim 1, wherein the vector provides blue-white colony selection to determine whether inserts are present.

5. The method of claim 1, wherein the insert DNA fragments are joined in order by determining non-palindromic overlap sequences from a table, list, chart, or computer database.

6. The method of claim 1, wherein the insert sequences are characterized by means of sequencing or mapping.

7. The method of claim 1, wherein a computer algorithm is used to determine either the choice of primers, sequences, address labels, or the detection of conflicting internal sites within the construct.

8. The composition of matter comprising the plasmid pWB. (SEQ ID No:

1)

9. The composition of matter comprising a vector, said vector comprising a plasmid or bacterial origin of replication, a selectable gene, and a DNA cloning site, said cloning site comprising class IIS restriction endonuclease recognition sites that are not found elsewhere in the vector, flanking at least one unique cloning site,

whereby digesting the vector containing a DNA insert cloned at the said cloning site with the eponymous class IIS restriction endonuclease results in release of the insert from the vector, with removal of a defined number of bases from one strand at each end of the DNA insert sequence.

10. The composition of claim 9, wherein the insertion of a DNA insert causes a change in phenotype that allows identification of clones containing inserts.

11. The composition of claim 9, comprising the sequence:

5′

-GCTCTTCGCGAAGAGC-3′

(SEQ. ID. NO;

     2).

12. The composition of claim 9, comprising a bacterial origin of replication.

13. The composition of claim 9, wherein said composition is used for the construction of a gene, vector, construct, combinatorial library, or chromosome.

14. The composition of claim 9, comprising two half sites, each half-site comprising the sequence GCTCTTCGCGA (SEQ ID NO:

4), wherein the two half sites are located on opposite strands and are separated by one or more nucleotides, wherein the vector is used for trimming one or more bases from the ends of insert fragments.