Direct cloning

• US 10,443,051 B2
• Filed: 06/10/2011
• Issued: 10/15/2019
• Est. Priority Date: 06/10/2010
• Status: Active Grant

• Current Assignee: Gene Bridges GmbH
• Original Assignee: Gene Bridges GmbH
• Inventors: Zhang, Youming, Fu, Jun, Stewart, Adrian Francis
• Primary Examiner(s): Weiler, Karen S.
• Application Number:
  US13/806,086
• Publication Number:
  US 20130210681A1
• Time in Patent Office: 3,049 Days
• Field of Search: None
• 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 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/16 Exonucleases active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters (3.16)

Abstract

A method for performing homologous recombination between at least a first nucleic acid molecule and a second nucleic acid molecule which share at least one region of sequence homology. A method for improving the efficiency of homologous recombination.

Claims

1. A method for performing homologous recombination between at least a first linear nucleic acid molecule and a second linear nucleic acid molecule which share at least one region of sequence homology, wherein the method comprises bringing the first nucleic acid molecule into contact with the second nucleic acid molecule in a first step of linear to linear homologous recombination, in the presence of a 5′

to 3′

exonuclease, RecT, and Red gamma;

wherein the 5′

to 3′

exonuclease is an N-terminally extended RecE expressed from heterologous DNA that comprises or consists of amino acids 1-866 of SEQ ID NO;

1, or a variant of this sequence having at least 95% sequence identity to SEQ ID NO;

1 over the entire length of the 866 amino acid sequence,

wherein said first step is carried out in the absence of truncated RecE, Red alpha and Red beta,

and wherein the method further comprises bringing the product of the linear to linear homologous recombination reaction into contact with a further nucleic acid molecule in a second step of linear to circular homologous recombination in the presence of Red alpha, Red beta, and Red gamma,

wherein said second step is carried out in the absence of the N-terminally extended RecE.

2. The method of claim 1, wherein the method is carried out in a host cell which expresses RecE/T and Red alpha/beta under the control of different inducible promoters that may be independently temporally expressed.

3. The method of claim 1, wherein the product of the linear to linear homologous recombination reaction is circular and the second step involves bringing the circular product into contact with a linear nucleic acid molecule.

4. The method of claim 1, wherein the method comprises performing the homologous recombination between the first nucleic acid molecule and the second nucleic acid molecule in vitro.

5. The method of claim 4, wherein the method further comprises transforming the product of the linear to linear homologous recombination reaction into a host cell and carrying out the second step of linear to circular homologous recombination in the presence of Red alpha, Red beta and Red gamma.

6. The method of claim 1, wherein the second nucleic acid is a linear vector with a selectable antibiotic resistance gene and the method comprises performing the homologous recombination between the first nucleic acid molecule and the second nucleic acid molecule in a host containing full length RecE and RecT and selecting for the antibiotic resistance gene;

wherein the second step comprises taking the resistant colonies and electroporating the resistant colonies with the further nucleic acid molecule, wherein the further nucleic acid molecule is a linear DNA molecule encoding a second selectable gene,

and wherein the method further comprises selecting for the second selectable gene and identifying the correct products as the colonies that grow after selection for the second selectable gene.

7. The method of claim 1, wherein the homologous recombination is carried out in a host cell.

8. The method of claim 7, wherein expression of the 5′

to 3′

exonuclease is driven by an inducible promoter.

9. The method of claim 8, wherein the inducible promoter is an arabinose inducible promoter or a rhamnose inducible promoter.

10. The method of claim 1, wherein the second nucleic acid is a linearised cloning vector selected from a linearised BAC, a linearised p15A origin based vector, a linearised pBR322 origin based vector, a linearised fosmid, a linearised pUC origin based vector or a linearised ColE1 origin based vector.

11. The method of claim 1, wherein the first nucleic acid molecule:

a) comprises a sequence of interest of 10 kb or more in length; and

/or

b) comprises a sequence of interest which is a gene cluster encoding a secondary metabolite pathway or a fatty acid synthesis pathway; and

/or

c) is a fragment of genomic DNA; and

/or

d) is a linearised BAC and the method is used to subclone a sequence of interest from the BAC into a cloning vector.

13. The method of claim 1, wherein:

a) the efficiency of linear to linear homologous recombination in step 1 is increased by using the N-terminally extended RecE compared to a truncated RecE consisting of amino acids 602-866 of SEQ. ID. NO;

1; and

b) the efficiency of linear to circular homologous recombination in step 2 is increased by using Red alpha/beta, compared to the N-terminally extended RecE used in step 1.

12. A method for performing homologous recombination between at least a first linear nucleic acid molecule and a second linear nucleic acid molecule which share at least one region of sequence homology, wherein the method comprises bringing the first nucleic acid molecule into contact with the second nucleic acid molecule in a first step of linear to linear homologous recombination, in the presence of a 5′

to 3′

exonuclease, RecT, and Red gamma;

wherein the 5′

to 3′

exonuclease is an N-terminally extended RecE expressed from heterologous DNA that comprises or consists of amino acids 1-866 of SEQ ID NO;

1, or a variant of this sequence having at least 95% sequence identity to SEQ ID NO;

1 over the entire length of the 866 amino acid sequence,

and wherein the method further comprises bringing the product of the linear to linear homologous recombination reaction into contact with a further nucleic acid molecule in a second step of linear to circular homologous recombination in the presence of Red alpha, Red beta, and Red gamma,

wherein the method is carried out in a host cell which expresses the N-terminally extended RecE under the control of a first inducible promoter, and Red alpha/beta under the control of a second inducible promoter that is independently temporally expressed,

wherein only the first inducible promoter is induced immediately prior to the first step of linear to linear homologous recombination such that the N-terminally extended RecE mediates the first step of linear to linear homologous recombination,

and wherein only the second inducible promoter is induced immediately prior to the second step of linear to circular homologous recombination such that Red alpha/beta mediates the second step of linear to circular homologous recombination.

14. The method of claim 12, wherein:

a) the efficiency of linear to linear homologous recombination in step 1 is increased by using the N-terminally extended RecE compared to a truncated RecE consisting of amino acids 602-866 of SEQ. ID. NO;

1; and

b) the efficiency of linear to circular homologous recombination in step 2 is increased by using Red alpha/beta, compared to the N-terminally extended RecE used in step 1.