US 12,270,026 B1
Directed evolution method for template-independent polymerases
Jared Ellefson, Oakland, CA (US); Daniel Arlow, San Francisco, CA (US); and Sebastian Palluk, Oakland, CA (US)
Assigned to Ansa Biotechnologies, Inc., Emeryville, CA (US)
Filed by Ansa Biotechnologies, Inc., Berkeley, CA (US)
Filed on Mar. 4, 2021, as Appl. No. 17/192,824.
Claims priority of provisional application 62/985,744, filed on Mar. 5, 2020.
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1058 (2013.01) 9 Claims
OG exemplary drawing
 
1. A method of selecting a template-independent polymerase active under desired conditions, comprising:
providing a plurality of host cells each comprising a plasmid comprising a gene expressing a unique template-independent polymerase variant;
subdividing the plurality of host cells into compartments;
exposing said compartments to conditions desirable for template-independent polymerase activity;
contacting the contents of said host cell in each compartment with reagents to generate a nucleic acid extension product when said expressed template-independent polymerase variant is active under said conditions, wherein said nucleic acid extension reaction product is coupled with said plasmid encoding said active template-independent polymerase variant in each compartment, and wherein said coupling comprises synthesis of said nucleic acid extension reaction product at a free 3′ end of said plasmid, wherein said plasmid has been cleaved;
pooling said compartments into a mixture; and
selectively enriching for nucleic acid extension reaction products coupled with said plasmids encoding said active template-independent polymerase variant from said mixture, thereby selecting plasmids comprising genes encoding template-independent polymerase variants active under said conditions.