Compartmentalized self tagging
View Patent ↗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.
1. A method for the selection of an enzyme capable of modifying an oligonucleotide wherein the method is not dependent on the 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 comprises the gene sequence of the selected enzyme(s) of interest;
(b) compartmentalizing said 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), which oligonucleotide bears a capture tag;
(c) providing conditions that permit the specific hybridization of the oligonucleotide of step (b) with a region on the parent nucleic acid;
(d) providing conditions that permit 5′ or 3′ extension of the oligonucleotide of step (b) by the enzyme encoded by the parent nucleic acid to form an oligonucleotide extension product/parent nucleic acid complex by which the parent nucleic acid may be subsequently captured; and
(e) capturing the oligonucleotide extension product/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 said enzyme is selected from the group consisting of: polY family DNA polymerases, polX family DNA polymerases, and low processivity variants of high processivity polymerases.
4. A method for the selection of an enzyme capable of modifying an oligonucleotide wherein the method is not dependent on the 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 comprises the gene sequence of the selected enzyme of interest;
(b) compartmentalizing said 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), which oligonucleotide bears a capture tag;
(c) providing conditions that permit the specific hybridization of the oligonucleotide of step (b) with a region on the parent nucleic acid;
(d) providing conditions that permit 5′ or 3′ extension of the oligonucleotide of step (b) by the enzyme encoded by the parent nucleic acid to form an oligonucleotide extension product/parent nucleic acid complex by which the parent nucleic acid may be subsequently captured; and
(e) capturing the oligonucleotide extension product/parent nucleic acid complex using a capture tag binding agent attached to a solid support.
5. The method according to claim 1 , wherein said one or more parent nucleic acids are plasmids.