In vitro recombination method

• US 9,534,251 B2
• Filed: 03/30/2010
• Issued: 01/03/2017
• 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): WOOLWINE, SAMUEL C
• Application Number:
  US12/750,571
• Publication Number:
  US 20100184187A1
• Time in Patent Office: 2,471 Days
• Field of Search: None
• US Class Current: 1/1
• 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′ exonuclease; (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. A kit for in vitro joining a plurality of dsDNA molecules, consisting essentially of in a single container,

a mixture of the isolated proteins

(i) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing,

(ii) a non strand-displacing DNA polymerase, and

(iii) a ligase;

(iv) an isolated non-processive 5′

exonuclease; and

(v) glycerol in an amount effective as a preservative;

wherein the ratios of activities of (i), (ii), (iii), and (iv) are effective to achieve in vitro joining of dsDNA molecules, and

wherein the dsDNA molecules are joined via a region of sequence homology to form a DNA molecule in which a single copy of the region of sequence homology is retained.

2. The kit of claim 1, wherein

the mixture contains

(i) as the SSB, the phage T7 gene 2.5 product, the E. coli recA protein, RedB of lambda phage, or RecT of Rae prophage;

(ii) as the DNA polymerase, the T7 gene 5 product, T4 polymerase, or E. coli pol; and

/or

(iii) as the ligase, the phage T7 gene 1.3 product, phage T4 DNA ligase, or E. coli DNA ligase; and

/or

(iv) as the 5′

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

3. The kit of claim 2 wherein:

(i) the phage T7 gene 2.5 product, the E. coli recA protein, RedB of lambda phage, or RecT of Rae prophage are substantially purified;

(ii) the T7 gene 5 product, T4 polymerase, or E. coli pol are substantially purified; and

/or

(iii) the phage T7 gene 1.3 product, phage T4 DNA ligase, or E. coli DNA ligase are substantially purified.

4. The kit of claim 3 wherein the non-processive 5′

exonuclease is substantially purified.

5. The kit of claim 3 further comprising instructions for performing a method for the in vitro joining of a plurality of dsDNA molecules.

6. The kit of claim 1 wherein:

(i) the single stranded DNA binding protein (SSB) is substantially purified;

(ii) the non strand-displacing DNA polymerase is substantially purified, and

(iii) the ligase is substantially purified.

7. The kit of claim 6 wherein the non-processive 5′

exonuclease is substantially purified.

8. The kit of claim 6 further comprising instructions for performing a method for the in vitro joining of a plurality of dsDNA molecules.

9. A composition consisting essentially of:

(a) a substantially purified single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing,

(b) a substantially purified non strand-displacing DNA polymerase,

(c) a substantially purified ligase, and

(d) substantially purified non-processive 5′

exonuclease, and

(e) glycerol in an amount effective as a preservative;

wherein the ratios of activities of (a), (b), (c) and (d) are effective to achieve in vitro joining of dsDNA molecules via a region of homology.