In vitro recombination method

• US 7,723,077 B2
• Filed: 08/11/2006
• Issued: 05/25/2010
• Est. Priority Date: 08/11/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Synthetic Genomics, Inc.
• Inventors: Young, Lei, Smith, Hamilton O., Gibson, Daniel Glenn
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): Woolwine; Samuel C
• Application Number:
  US11/502,746
• Publication Number:
  US 20070037197A1
• Time in Patent Office: 1,383 Days
• Field of Search: None
• US Class Current: 435/91.1
• 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 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/62 involving uric acid
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 301/11003 Exodeoxyribonuclease (lambda-induced) (3.1.11.3)
• CPC Class Codes: C12Y 605/01001 DNA ligase (ATP) (6.5.1.1)

Abstract

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

Claims

1. An in vitro method, using isolated proteins, for joining two double strand (ds) DNA molecules of interest, comprising:

providing a first DNA molecule and a second dsDNA molecule which share a region of sequence identity at a terminal end on each DNA molecule, wherein the region is less than about 150 base pairs in length; and

contacting the two dsDNA molecules with;

(a) a purified 5′

exonuclease;

(b) a purified single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing;

(c) a purified non strand-displacing DNA polymerase; and

(d) a purified ligase,

under conditions whereby;

a 3′

single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme;

the two single-stranded overhangs anneal to form a gapped molecule;

the gaps are filled in by the polymerase; and

nicks are sealed by the ligase, thereby joining the molecules and forming a substantially intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.

2. The method of claim 1, wherein the 3′

single stranded overhangs comprise the region of sequence identity.

3. The method of claim 1, wherein the proteins of (a), (b), (c) and (d) are contacted simultaneously with the DNA molecules.

4. The method of claim 1, wherein the proteins of (b), (c) and (d) are contacted simultaneously or sequentially, in any order, with the DNA molecules, and the protein of (a) is contacted last.

5. The method of claim 1, wherein the proteins are contacted with the DNA molecules sequentially, in the following order:

(b), (d), (c), and (a).

6. The method of claim 1, wherein the exonuclease activity is substantially lower than the activity of the DNA polymerase, such that the gaps are filled in by the polymerase immediately as they are formed.

7. The method of claim 1, wherein

(a) the 5′

exonuclease is the phage T7 gene 6 product, RedA of lambda phage, or RecE of Rac prophage;

(b) the SSB is the phage T7 gene 2.5 product, the E. coli recA protein, RedS of lambda phage, or RecT of Rac prophage;

(c) the DNA polymerase is the phage T7 gene 5 product, phage T4 DNA polymerase, or E coli pol I; and

/or

(d) the ligase is the phage T7 gene 1.3 product, phage T4 DNA ligase, or E coli DNA ligase.

8. The method of claim 1, wherein the four proteins of (a), (b), (c), and (d) function in a concerted manner.

9. The method of claim 1, wherein one or more of the DNA molecules to be joined are generated synthetically.

10. The method of claim 9, wherein:

the DNA molecules to be joined comprise adjacent portions of a gene or genome of interest and are synthesized so as to comprise overlapping regions of sequence identity at their ends; and

the joining reaction joins the DNA molecules to form part or all of a synthetic gene or genome.

11. The method of claim 1, wherein the regions of sequence identity are added to the DNA molecules to be joined by PCR amplification.

12. The method of claim 1, further comprising subjecting the joined DNA molecules to a sizing procedure, isolating DNA molecules of a desired length, and introducing the isolated DNA molecules into a cell of interest.

13. The method of claim 1, which is a method for inserting a DNA fragment of interest into a linearized vector to form a circular molecule, further comprising adding sequences by PCR amplification to each end of the substantially intact double-stranded DNA molecule, which sequences added by PCR amplification are identical to sequences on either end of the linearized vector.

14. The method of claim 1, wherein the region of sequence identity is about 40 base pairs in length.

15. The method of claim 1, wherein the proteins of (a), (b), (c), and (d) are contacted with the DNA molecules in a single reaction vessel.

16. The method of claim 1, wherein the 5′

exonuclease is the phage T7 gene 6 product, RedA of lambda phage, or RecE of Rac prophage.

17. The method of claim 1, wherein the 5′

exonuclease is the T7 gene 6 product.

18. An in vitro method for joining more than two double stranded (ds) DNA molecules in a defined orientation and order, comprising

(a) selecting the more than two DNA molecules such that, for each pair of molecules to be joined, the molecules share a region of sequence identity at terminal ends, wherein each region of sequence identity is unique for each pair of DNA molecules to be joined;

and

(b) contacting the DNA molecules in a reaction mixture in a single reaction vessel with

(i) a purified 5′

exonuclease;

(ii) a purified single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing;

(iii) a purified non strand-displacing DNA polymerase; and

(iv) a purified ligase, under conditions whereby;

a 3′

single-stranded overhang is generated in each molecule by the exonuclease without the use of a restriction enzyme;

the single-stranded overhangs anneal to form a gapped molecule;

the gaps are filled in by the polymerase; and

nicks are sealed by the ligase, thereby joining the plurality of DNA molecules to form a substantially intact duplex DNA molecule in which a copy of each region of sequence identity is retained, wherein none of the enzymatic reactions is actively terminated prior to beginning another of the reactions.

19. The method of claim 18, wherein the more than two dsDNA molecules comprise at least 10 molecules and the region of identity is from about 150 to 300 base pairs in length.