Compartmentalized self tagging
First Claim
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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.
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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.
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Citations
22 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22)
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21. A method for the selection of an enzyme capable of modifying an oligonucleotide, comprising the steps of:
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(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.
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Specification