US 12,110,534 B2
Generation of single-stranded circular DNA templates for single molecule sequencing
Daleen Badenhorst, Wellington (ZA); Michael S. Berry, Woodstock (ZA); Richard Dannebaum, Pleasant Hill, CA (US); Ashley Hayes, San Francisco, CA (US); Severine Margeridon, Castro Valley, CA (US); Martin Ranik, Kenilworth (ZA); and Etienne Slabbert, Stellenbosch (ZA)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
Filed on Jul. 31, 2020, as Appl. No. 16/945,099.
Application 16/945,099 is a continuation of application No. PCT/EP2019/052764, filed on Feb. 5, 2019.
Claims priority of provisional application 62/626,589, filed on Feb. 5, 2018.
Prior Publication US 2021/0115510 A1, Apr. 22, 2021
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6869 (2013.01); C12Q 2521/319 (2013.01); C12Q 2525/155 (2013.01); C12Q 2525/191 (2013.01); C12Q 2525/307 (2013.01); C12Q 2535/122 (2013.01)] 11 Claims
OG exemplary drawing
 
1. A method of separately sequencing each strand of a double-stranded target nucleic acid, wherein the method comprises the following steps:
(a) in a reaction mixture, joining a double-stranded target nucleic acid to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises primer-binding sites, and wherein the adaptor does not consist of a long strand and a short strand;
(b) amplifying the adapted target nucleic acid with a pair of primers, wherein the pair of primers are complementary to the primer-binding sites, thereby forming an amplicon, wherein one primer in the pair of primers comprises a modified nucleotide affecting a rate of digestion by an exonuclease;
(c) contacting the reaction mixture with an exonuclease, thereby eliminating from the reaction mixture the first of the two complementary strands of the amplicon;
(d) circularizing the second of the two complementary strands of the amplicon to form a single stranded circle;
(e) annealing a sequencing primer to the single-stranded circle; and
(f) extending the primer, thereby sequencing one strand of the target nucleic acid.