Compartmentalised self replication method for in vitro evolution of molecular libraries

• US 7,514,210 B2
• Filed: 03/13/2003
• Issued: 04/07/2009
• Est. Priority Date: 09/13/2000
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Ong, Jennifer Lee, Ghadassy, Farid, Holliger, Phillipp
• Primary Examiner(s): Ramirez; Delia M
• Application Number:
  US10/387,387
• Publication Number:
  US 20040005594A1
• Time in Patent Office: 2,217 Days
• Field of Search: 435/91.2, 435/91.31, 435/91.5, 435/91.52, 435/194, 435/440, 536/23.1, 536/23.2
• US Class Current: 435/6.16
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase

Abstract

We describe a method of selecting an enzyme having replicase activity, the method comprising the steps of: (a) providing a pool of nucleic acids comprising members each encoding a replicase or a variant of the replicase; (b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool together with the replicase or variant encoded by the nucleic acid member; (c) allowing nucleic acid replication to occur; and (d) detecting amplification of the nucleic acid member by the replicase. Methods for selecting agents capable of modulating replicase activity, and for selecting interacting polypeptides are also disclosed.

Claims

1. A method of selecting an active nucleic acid replicase, the method comprising the steps of:

(a) providing a pool of nucleic acids comprising nucleic acid members each encoding a nucleic acid replicase;

(b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member and the contents of each compartment are not in contact with the contents of other compartments;

(c) expressing the nucleic acid member in the compartment to form the nucleic acid replicase encoded by said nucleic acid member;

(d) providing conditions within the compartment under which the nucleic acid member may be processed by the nucleic acid replicase encoded by said nucleic acid member; and

(e) detecting processing of the nucleic acid member by the nucleic acid replicase encoded by said nucleic acid member, whereby an active nucleic acid replicase is selected.

2. The method of claim 1, wherein the processing of the nucleic acid member results in one copy of said nucleic acid member.

3. The method of claim 1, wherein the processing of the nucleic acid member comprises either a fill-in reaction of a 5′

overhang appended to said nucleic acid member or an extension of a 3′

end of said nucleic acid member.

4. The method of claim 1 in which the processing of the nucleic acid member results in more than one copy of said nucleic acid member.

5. The method of claim 4, in which the processing of the nucleic acid member is an exponential amplification.

6. The method of claim 4, in which the processing is carried out by a polymerase chain reaction (PCR), a nested PCR, a ligase chain reaction (LCR), a transcription based amplification system (TAS), a self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), a transcription-mediated amplification reaction (TMA), or a strand-displacement amplification (SDA).

7. The method of claim 4, wherein the number of copies of the nucleic acid member is proportional to the activity of the nucleic acid replicase.

8. The method of claim 4, in which the processing is detected by assaying the copy number of the nucleic acid member.

9. The method of claim 4, in which the processing is detected by assaying the presence of a tag on the nucleic acid member.

10. The method of claim 4, in which the processing is detected by determining the nucleic acid replicase activity of the polypeptide encoded by the nucleic acid member.

11. The method of claim 1, in which the conditions in the compartment are selected to permit selection of a active nucleic acid replicase with a particular desired property.

12. The method of claim 4, in which the replicase activity of the nucleic acid replicase is a template-dependent replicase activity selected from a polymerase activity, a reverse transcriptase activity and a ligase activity.

13. The method of claim 1, wherein the step of expressing the nucleic acid member to form the nucleic acid replicase encoded by said nucleic acid member is carried out by in vitro transcription and translation.

14. The method of claim 1, wherein the step of expressing the nucleic acid member to form the nucleic acid replicase encoded by said nucleic acid member is carried out by in vivo transcription and translation in an expression host cell.

15. The method of claim 14 wherein said expression host cell is a bacterial cell.

16. The method of claim 1, in which the compartments comprise aqueous compartments of a water-in-oil emulsion.

17. The method of claim 16, in which the water-in-oil emulsion is produced by emulsifying an aqueous phase with an oil phase and a surfactant comprising Span80, Tween80, and TritonX100.

18. The method of claim 17 wherein said surfactant comprises 4.5% v/v Span80, 0.4% v/v Tween80 and 0.1% v/v TritonX100.

19. The method of claim 1 wherein the active nucleic acid replicase that is selected is a variant of a Taq polymerase which has greater thermostability than said Taq polymerase.

20. The method of claim 1 wherein the active nucleic acid replicase that is selected is a variant of a Taq polymerase which is inhibited to a lesser extent by heparin than is said Taq polymerase.