Compartmentalized self tagging

• US 7,691,576 B2
• Filed: 05/03/2006
• Issued: 04/06/2010
• Est. Priority Date: 11/03/2003
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Oliynyk, Zoryana, Holliger, Philipp
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US11/417,449
• Publication Number:
  US 20070134682A1
• Time in Patent Office: 1,434 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass

Abstract

The present invention relates to the development of a novel method for the selection of nucleic acid processing and other enzymes. In particular the invention relates to a method for the selection of nucleic acid polymerases and other enzymes with desired properties based on the method of compartmentalized self-tagging.

Claims

1. A method for the selection of an enzyme capable of modifying an oligonucleotide wherein the method is not dependent on the complete replication of the oligonucleotide modifying enzyme encoding gene, which method comprises the steps of:

(a) providing one or more parent nucleic acids encoding one or more enzymes of interest, wherein the parent nucleic acid provides the gene sequence of the selected enzyme of interest;

(b) compartmentalizing those parent nucleic acids according to step (a), such that each compartment comprises a parent nucleic acid together with the one or more enzymes encoded by the parent nucleic acid and an oligonucleotide specific for a region on the parent nucleic acid according to step (a);

(c) providing conditions that permit the stable association of the oligonucleotide of step (b) with a region on the parent nucleic acid;

(d) providing conditions that permit the modification of the oligonucleotide of step (b) by the enzyme encoded by the parent nucleic acid, such that the resultant modified oligonucleotide comprises a molecular tag and forms a modified oligonucleotide/parent nucleic acid complex; and

(e) capturing the modified oligonucleotide/parent nucleic acid complex.

2. The method of claim 1 wherein, under the selection conditions, the method selects an enzyme having a property selected from the group consisting of:

low catalytic turnover, low processivity, and the incorporation of modified substrates which generate a nucleic acid-like polymer that cannot be reamplified.

3. The method of claim 1 wherein the enzyme is a nucleic acid processing enzyme.

4. The method of claim 3 which comprises the steps of:

(a) providing one or more parent nucleic acid molecules encoding one or more nucleic acid polymerases, wherein the parent nucleic acid provides the gene sequence of the nucleic acid processing enzyme of interest;

(b) compartmentalizing those parent nucleic acids of step (a) into compartments, such that each compartment comprises a parent nucleic acid together with the one or more nucleic acid polymerases encoded by the parent nucleic acid;

(c) providing an oligonucleotide specific for a sequence on the parent nucleic acid of step (b) under conditions that permit stable association of said oligonucleotide with that sequence of the parent nucleic acid;

(d) extending the oligonucleotide of step (b) using the nucleic acid processing enzyme in the presence of a nucleotide comprising a molecular tag to generate a modified oligonucleotide/parent nucleic acid complex; and

(e) capturing the modified oligonucleotide/parent nucleic acid complex.

5. The method of claim 3 wherein the nucleic acid processing enzyme is selected from the group consisting of:

poly family DNA polymerases, polX family DNA polymerases, low processivity variants of high processivity polymerases;

DNA ligases, RNA ligases and polynucleotide kinases.

6. The method of claim 3 wherein the nucleic acid processing enzyme is a DNA polymerase.

7. The method of claim 6 wherein the method comprises the following steps:

(a) providing one or more parent nucleic acid molecules encoding one or more DNA polymerases, wherein the parent nucleic acid provides the gene sequence of the polymerase of interest;

(b) compartmentalizing the parent nucleic acid molecules of step (a) into compartments, such that each compartment comprises a parent nucleic acid together with one or more DNA polymerases encoded by the parent nucleic acid; and

an oligonucleotide specific for a sequence on the parent nucleic acid of step (a);

(c) providing conditions that permit stable association of the oligonucleotide specific for a sequence on the parent nucleic acid of step (a) with that sequence on the parent nucleic acid;

(d) providing conditions that permit the 3′

end of the oligonucleotide of step (b) to be extended by the DNA polymerase of step (a) in the presence of a nucleotide comprising a molecular tag to generate a modified oligonucleotide/parent nucleic acid complex; and

(e) capturing the 3′

extended modified oligonucleotide/parent nucleic acid complex.

8. The method of claim 5 wherein the nucleic acid processing enzyme is a polynucleotide kinase and the oligonucleotide modification step (d) involves phosphorylation of the oligonucleotides 5′

end, followed by ligation of a further oligonucleotide linked to a molecular tag.

9. The method of claim 1 wherein the method permits the selection of a DNA ligase and each compartment comprises (i) a nucleic acid encoding the ligase of interest, (ii) an oligonucleotide that associates with a nucleic acid encoding the ligase of interest, (iii) the ligase encoded by the parent nucleic acid, and (iv) one or more tagged nucleotides.

10. The method of claim 1 as applied to the selection of enzymes which act directly on a substrate molecule linked to an oligonucleotide.

11. The method of claim 1 wherein the enzyme is one which is indirectly involved in nucleic acid processing.

12. The method of claim 11 wherein each compartment comprises a parent nucleic acid which encodes an enzyme which is indirectly involved in nucleic acid processing, an enzyme encoded by the nucleic acid, a DNA replicase and a modified substrates of the enzyme encoded by the parent nucleic acid.

13. The method of claim 12 wherein the enzyme either participates in the production of nucleic acid replicase substrates from a blocked or inactive substrate of an enzyme or participates in the functional inactivation of nucleic acid replicase inhibitors present within the compartment.

14. The method of claim 13 wherein the enzyme participates in the production of nucleic acid replicase substrates from a blocked or inactive substrate.

15. The method of claim 4 wherein the efficiency of nucleic acid capture is increased using a technique selected from the following:

increasing the Tm of the oligonucleotide/nucleic acid hybrid;

extending the oligonucleotide by more than 3 bases;

including a linker more than 40 atoms long between the molecular tag and the oligonucleotide; and

using an oligonucleotide more than 10 bases long.

16. The method of claim 15 wherein the efficiency of nucleic acid capture is increased by increasing the Tm of the oligonucleotide/nucleic acid hybrid using a base selected from the group consisting of:

LNA bases;

diaminopurine, a G-clamp; and

INA.

17. The method of claim 15 wherein the efficiency of nucleic acid capture is increased by extending the oligonucleotide by one base or more.

18. The method of claim 15 wherein the efficiency of nucleic acid capture is increased by extending the oligonucleotide by 3 bases or more.

19. The method of claim 15 wherein the efficiency of nucleic acid capture is increased by extending the oligonucleotide by 9 bases or more.

20. The method of claim 15 wherein the efficiency of nucleic acid capture is increased by the use of a linker more than 50 atoms long between the molecular tag and the oligonucleotide.

22. The method of claim 1, wherein said one or more parent nucleic acids are plasmids.

21. A method for the selection of an enzyme capable of modifying an oligonucleotide, comprising the steps of:

(a) providing one or more parent nucleic acids encoding one or more enzymes of interest, wherein the parent nucleic acid provides the gene sequence of the selected enzyme of interest;

(b) compartmentalizing those parent nucleic acids according to step (a), such that each compartment comprises a parent nucleic acid together with the one or more enzymes encoded by the parent nucleic acid and an oligonucleotide specific for a region on the parent nucleic acid according to step (a);

(c) providing conditions that permit the stable association of the oligonucleotide of step (b) with a region on the parent nucleic acid;

(d) providing conditions that permit the modification of the oligonucleotide of step (b) by the enzyme encoded by the parent nucleic acid, such that the resultant modified oligonucleotide comprises a molecular tag and associates with the encoding parent nucleic acid to form a modified oligonucleotide/parent nucleic acid complex; and

(e) capturing the modified oligonucleotide/parent nucleic acid complex using a molecular tag binding agent attached to a solid support.