US 12,012,631 B2
Methods and compositions for isolating asymmetric nucleic acid complexes
Lei Sun, San Jose, CA (US); Sassan Sheikholeslami, San Francisco, CA (US); Natasha Popovich, Belmont, CA (US); and David Christopher Scherer, Palo Alto, CA (US)
Assigned to Pacific Biosciences of California, Inc., Menlo Park, CA (US)
Filed by Pacific Biosciences of California, Inc., Menlo Park, CA (US)
Filed on Jan. 15, 2021, as Appl. No. 17/150,444.
Application 17/150,444 is a continuation of application No. 16/036,511, filed on Jul. 16, 2018, granted, now 10,920,268.
Claims priority of provisional application 62/534,065, filed on Jul. 18, 2017.
Prior Publication US 2021/0207190 A1, Jul. 8, 2021
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6874 (2013.01); C12Q 2525/301 (2013.01); C12Q 2531/119 (2013.01); C12Q 2535/122 (2013.01); C12Q 2537/1376 (2013.01); C12Q 2565/519 (2013.01)] 17 Claims
 
1. A method for isolating nucleic acid templates comprising:
providing nucleic acid templates that comprise a double-stranded insert region with hairpin adapters at both ends, wherein the nucleic acid templates comprise a mixture of symmetrically-primed nucleic acid templates and asymmetrically-primed nucleic acid templates, wherein each symmetrically-primed nucleic acid template comprises capture primers hybridized to both terminal hairpin adapters or synthesis primers hybridized to both terminal hairpin adapters, and wherein each asymmetrically-primed nucleic acid template comprises a capture primer hybridized to the hairpin adapter at one end and a synthesis primer hybridized to the hairpin adapter at the opposite end;
immobilizing the capture-primer hybridized templates to a solid support through the capture primer;
maintaining the immobilized templates and a nucleic acid polymerase having strand displacement activity under conditions that promote nucleic acid synthesis from the synthesis primer, thereby eluting asymmetrically-primed nucleic acid templates from the solid support; and
collecting the templates eluted from the solid support by the strand displacement activity of the nucleic acid polymerase, thereby isolating asymmetrically-primed nucleic acid templates.