US 11,788,121 B2
Methods, systems, and compositions for counting nucleic acid molecules
Matthew Sekedat, Ann Arbor, MI (US); Jeffrey Buis, Ann Arbor, MI (US); Ronald David Beaubien, Jackson, MI (US); Sharat Singh, Rancho Santa Fe, CA (US); and Jeff Perry, Ann Arbor, MI (US)
Assigned to Enumera Molecular, Inc., Ann Arbor, MI (US)
Filed by Enumera Molecular, Inc., Ann Arbor, MI (US)
Filed on Dec. 7, 2021, as Appl. No. 17/544,669.
Application 17/544,669 is a continuation of application No. 16/373,568, filed on Apr. 2, 2019, granted, now 11,230,731.
Claims priority of provisional application 62/660,699, filed on Apr. 20, 2018.
Claims priority of provisional application 62/651,676, filed on Apr. 2, 2018.
Prior Publication US 2022/0090180 A1, Mar. 24, 2022
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6834 (2018.01)
CPC C12Q 1/6834 (2013.01) [C12Q 2565/537 (2013.01)] 22 Claims
OG exemplary drawing
 
1. A method for counting circularized nucleic acids on a solid support, comprising:
a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid, wherein the primer is bound to a surface on a solid support;
b) detecting formation of the at least one complex in a process comprising:
i) extending the primer in the complex in a rolling circle amplification (RCA) reaction to form RCA product, wherein forming the RCA product comprises extending the primer bound to the surface on the solid support on the circularized nucleic acid in a reaction mixture comprising at least 12% PEG, wherein the PEG has an average molecular weight between 400 and 800;
ii) hybridizing a plurality of labeled probes to the RCA product; and
iii) detecting hybridized labeled probe;
wherein hybridized labeled probe at a locus on the surface on the solid support is indicative of formation of a complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid at that locus on the solid support.