US 11,993,791 B2
Modification of 3′ terminal ends of nucleic acids by DNA polymerase theta
Richard T. Pomerantz, Brooklyn, NY (US); and Tatiana Kent, Philadelphia, PA (US)
Assigned to Temple University-Of The Commonwealth System of Higher Education, Philadelphia, PA (US)
Filed by Temple University-Of The Commonwealth System of Higher Education, Philadelphia, PA (US)
Filed on Dec. 14, 2020, as Appl. No. 17/120,973.
Application 17/120,973 is a continuation of application No. 15/772,192, granted, now 10,865,396, previously published as PCT/US2016/059419, filed on Oct. 28, 2016.
Claims priority of provisional application 62/338,119, filed on May 18, 2016.
Claims priority of provisional application 62/248,083, filed on Oct. 29, 2015.
Prior Publication US 2021/0171920 A1, Jun. 10, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/12 (2006.01); C07H 21/04 (2006.01); C12P 19/34 (2006.01); C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6844 (2018.01); C12Q 1/6853 (2018.01)
CPC C12N 9/1252 (2013.01) [C12P 19/34 (2013.01); C12Q 1/68 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/6844 (2013.01); C12Q 1/6853 (2013.01); C07H 21/04 (2013.01); C12N 2310/344 (2013.01); C12Q 2521/101 (2013.01); C12Q 2525/101 (2013.01); C12Q 2563/137 (2013.01); C12Y 207/07007 (2013.01)] 27 Claims
 
1. A method of modifying a 3′ terminal end of a nucleic acid with a substrate, the method comprising:
forming an in vitro mixture comprising an A family polymerase consisting essentially of SEQ ID NO 1, a substrate, a nucleic acid comprising a single-stranded 3′ end or overhang, and a reaction solution, wherein the reaction solution comprises at least one divalent metal;
incubating the mixture, wherein the A family polymerase attaches the substrate to the single-stranded 3′ end or overhang of the nucleic acid in vitro by a template-independent terminal transferase activity thereby forming a 3′-terminal end modified nucleic acid; and
isolating a 3′-terminal end modified nucleic acid.