Accessible polynucleotide libraries and methods of use thereof

• US 20060281113A1
• Filed: 05/17/2006
• Published: 12/14/2006
• Est. Priority Date: 05/18/2005
• Status: Abandoned Application

• Current Assignee: Codon Devices Incorporated
• Original Assignee: Codon Devices Incorporated
• Inventors: Pitcher, Edmund R., Church, George
• Application Number:
  US11/436,478
• Publication Number:
  US 20060281113A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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

Disclosed are methods of assembling nucleic acid constructs from component parts in a manner that is not dependent on the sequence of the component parts. The methods may be used to assemble large nucleic acid constructs from multiple component parts in one or more reactions. The methods may also be used to assemble two or more nucleic acid constructs in the same reaction mixture. In exemplary embodiments, the methods involve formation of a Holliday junction or bridge structure using a junction oligonucleotide.

Claims

1. A method of directed scarless ligation of a construction polynucleotide to another to produce a larger polynucleotide construct, the method comprising:

a) providing double stranded construction polynucleotides comprising a medial segment comprising a DNA sequence for inclusion within a said larger polynucleotide construct and left and right flanking terminal double stranded sequences of a length sufficient to permit selective hybridization of a complementary DNA thereto;

b) creating a single stranded overhang corresponding to a 3′

flanking region in a first said construction polynucleotide and to a 5′

flanking region in a second said construction polynucleotide while retaining the medial segments of said polynucleotides intact and free of residual flanking sequence bases;

c) contacting the construction polynucleotides under hybridization conditions with a junction oligonucleotide comprising sequence complementary to at least a portion of both said construction polynucleotides to form a complex wherein the two medial segments are aligned end to end; and

d) exposing the complex to ligation conditions, thereby forming a larger polynucleotide construct comprising fused said medial segments.

2. The method of claim 1, wherein step c) is conducted by contacting said first and second construction polynucleotides with a junction oligonucleotide complementary to at least a portion of the medial segments of both said construction polynucleotides.

3. The method of claim 1, wherein step c) is conducted by contacting said first and second construction polynucleotides with a junction oligonucleotide complementary to a 3′

flanking sequence of said first construction polynucleotide and a 5′

flanking sequence of said second construction polynucleotide.

4. The method of claim 3, wherein the construction polynucleotides and the junction oligonucleotide form a Holliday junction, the method further comprising contacting said Holiday junction with a resolvase.

5. The method of claim 1, wherein the left and right flanking terminal double stranded sequences of the construction polynucleotides comprise nested binding sites for two or more primer pairs.

6. The method of claim 5, further comprising providing a desired said construction polynucleotide by amplifying it selectively from a construction polynucleotide mixture using combination of two or more said primer pairs.

7. The method of claim 1, wherein at least five construction polynucleotides are joined together in a single reaction mixture.

8. The method of claim 1, wherein at least two larger polynucleotide construct are formed in a single reaction mixture.

9. The method of claim 1, further comprising amplifying the larger polynucleotide construct after step d) using primers complementary to the terminal flanking regions of said larger polynucleotide.

10. The method of claim 1, wherein a construction polynucleotide is coupled to a solid support.

11. The method of claim 10, wherein the construction polynucleotide is coupled to the solid support by a cleavable linker.

12. The method of claim 10, wherein the construction polynucleotide is coupled to the solid support by hybridization to an oligonucleotide attached to the support.

13. A method of producing a polynucleotide construct by joining together in a preselected order a selected pair of construction polynucleotides, the method comprising:

a) providing a mixture of different candidate construction polynucleotides comprising;

a medial segment for joinder with another, flanked by 5′ and

3′

flanking sequences, wherein the flanking sequences comprise nested binding sites for two or more primer pairs;

b) providing a mixture of junction oligonucleotides comprising (i) a sequence that hybridizes to both a 5′ and

a 3′

flanking sequence of at least one pair of construction polynucleotides, flanked 5′ and

3′

by (ii) junction oligonucleotide flanking sequences comprising binding sites for at least one pair of primers, thereby to enable amplification of a said junction oligonucleotide;

c) providing a plurality of primer pairs;

d) selecting at least a pair of construction polynucleotides from said mixture of candidate construction polynucleotides by amplification thereof with one or more of the primer pairs;

e) selecting a junction oligonucleotide from said mixture of junction oligonucleotides by amplification thereof with one or more of the primer pairs;

f) forming single stranded overhangs on the selected pair of construction polynucleotides thereby to produce a 3′

single stranded overhang corresponding to at least a portion of the 3′

flanking region of a first construction polynucleotide and a 5′

single stranded overhang corresponding to at least a portion of the 5′

flanking region of a second construction polynucleotide;

g) contacting the construction polynucleotide pair with their respective junction oligonucleotide under hybridization conditions to form a complex wherein the junction oligonucleotide is hybridized to the single stranded overhangs of the construction polynucleotides and the medial segments are aligned end to end; and

h) exposing the complex to ligation conditions, thereby to form a larger polynucleotide construct comprising fused said medial segments.

14. The method of claim 13, further comprising removing the 5′

and 3′

flanking regions from the junction oligonucleotide.

15. The method of claim 13, wherein the complex forms a Holliday junction.

16. The method of claim 15, further comprising contacting the complex with a resolvase.

17. A composition comprising a plurality of different junction oligonucleotides, each respective said junction oligonucleotides comprising a nucleotide sequence complementary to a 3′

terminal sequence of one construction polynucleotide and a nucleotide sequence complementary to a 5′

terminal sequence of another construction polynucleotide, said junction oligonucleotides having a sequence error rate less than about one base in 1000 so as to enable simultaneous selective hybridization of plural said junction oligonucleotides with their respective plural complementary construction polynucleotides and the preparation in parallel of multiple fusions between construction polynucleotides.

18. The composition of claim 17, wherein said junction oligonucleotides further comprise common removable primer binding sites on the ends thereof to permit amplification thereof with a pair of common primers.

19. The composition of claim 17, wherein said different junction oligonucleotides are immobilized on a surface, and are adapted for severance therefrom.

20. The composition of claim 17, wherein said junction oligonucleotides have a sequence error rate less than about one base in 1500, 2000, 3000, 5000, or 10,000 bases.