US 12,480,106 B2
Reactions with non-retroviral reverse transcriptase
Heather E. Upton, Berkeley, CA (US); Lucas Ferguson, Berkeley, CA (US); Sidney Pimentel, Berkeley, CA (US); and Kathleen Collins, Berkeley, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Filed by The Regents of the University of California, Oakland, CA (US)
Filed on Apr. 4, 2023, as Appl. No. 18/295,268.
Application 18/295,268 is a continuation of application No. PCT/US2021/054072, filed on Oct. 8, 2021.
Claims priority of provisional application 63/090,116, filed on Oct. 9, 2020.
Prior Publication US 2023/0235302 A1, Jul. 27, 2023
Int. Cl. C12N 9/12 (2006.01); C12N 15/10 (2006.01); C12Q 1/6848 (2018.01)
CPC C12N 9/1276 (2013.01) [C12N 15/1096 (2013.01); C12Q 1/6848 (2013.01); C12Y 207/07049 (2013.01); C12N 2310/321 (2013.01); C12Q 2600/16 (2013.01)] 24 Claims
OG exemplary drawing
 
1. A method of performing 3′ extension of a nucleic acid in an ordered two-template relay (OTTR) reaction with a non-retroviral reverse transcriptase (RT), wherein the method further comprises:
(a) addition of a non-canonical nucleotide to the reaction mixture to suppress excessive non-templated cDNA 3′ extension in RT reaction conditions without inhibiting templated DNA synthesis;
(b) addition of a modified passenger strand of a primer duplex to the reaction mixture;
(c) labeling of input templates with ddATP then ddGTP by sequential terminal transferase (TT); or
(d) suppression of unwanted products by termination of template copying by adding to the reaction mixture a Cy5 5′ extension of the 3′ acceptor template, primer, and/or other oligonucleotides present except those of the input template pool,
wherein said OTTR reaction exhibits improved reaction yield or suppression of unwanted products in the reaction.