US 11,898,204 B2
Generation of single-stranded circular DNA templates for single molecule sequencing
Aruna Arcot, Fremont, CA (US); Daleen Badenhorst, Wellington (ZA); Rui Chen, Fremont, CA (US); Toummy Guettouche, Pleasanton, CA (US); Ashley Hayes, San Francisco, CA (US); Jenny A. Johnson, Castro Valley, CA (US); Severine Margeridon, Castro Valley, CA (US); Martin Ranik, Cape Town (ZA); Persis Wadia, Fremont, CA (US); and Alexandra Hui Wang, Mountain View, CA (US)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and KAPA Biosystems, Inc., Wilmington, MA (US)
Appl. No. 16/977,421
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
PCT Filed Feb. 28, 2019, PCT No. PCT/EP2019/054938
§ 371(c)(1), (2) Date Sep. 1, 2020,
PCT Pub. No. WO2019/166530, PCT Pub. Date Sep. 6, 2019.
Claims priority of provisional application 62/685,817, filed on Jun. 15, 2018.
Claims priority of provisional application 62/638,034, filed on Mar. 2, 2018.
Prior Publication US 2023/0183797 A1, Jun. 15, 2023
Int. Cl. C12Q 1/6869 (2018.01); C12Q 1/6874 (2018.01); C12N 15/10 (2006.01); C12Q 1/6855 (2018.01)
CPC C12Q 1/6874 (2013.01) [C12N 15/1068 (2013.01); C12Q 1/6855 (2013.01)] 15 Claims
 
1. A method of separately sequencing each strand of a target nucleic acid comprising the steps of:
a) in a reaction mixture, joining the ends of a double stranded target nucleic acid to adaptors to form a doubly-adapted target nucleic acid wherein the adaptor is a single strand forming a double stranded stem and a single-stranded loop and the stem comprises at least one strand cleavage site;
b) contacting the reaction mixture with an exonuclease thereby enriching for the doubly-adapted target nucleic acid;
c) contacting the reaction mixture with a cleavage agent to cleave the doubly-adapted target nucleic acid at the cleavage sites forming extendable termini on each strand;
d) extending the extendable termini thereby separately sequencing each strand of the target nucleic acid, wherein extension is terminated at the cleavage site and does not proceed onto the complementary strand.