US 12,110,550 B2
Methods of amplifying circular polynucleotides in situ
Daan Witters, San Diego, CA (US); Allen Lipson, San Diego, CA (US); and Eli N. Glezer, Del Mar, CA (US)
Assigned to Singular Genomics Systems, Inc., San Diego, CA (US)
Filed by Singular Genomics Systems, Inc., San Diego, CA (US)
Filed on Feb. 27, 2024, as Appl. No. 18/588,765.
Application 18/588,765 is a continuation of application No. 17/934,407, filed on Sep. 22, 2022.
Application 17/934,407 is a continuation of application No. 17/719,214, filed on Apr. 12, 2022, granted, now 11,486,004, issued on Nov. 1, 2022.
Application 17/719,214 is a continuation of application No. PCT/US2021/041139, filed on Jul. 9, 2021.
Claims priority of provisional application 63/051,094, filed on Jul. 13, 2020.
Prior Publication US 2024/0271207 A1, Aug. 15, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6874 (2013.01) 38 Claims
 
1. A method of incorporating nucleotides into a primer in situ, the method comprising:
(A) extending a first immobilized oligonucleotide hybridized to a circular polynucleotide with a strand-displacing polymerase to generate a first immobilized extension product comprising one or more complements of the circular polynucleotide, wherein said first immobilized oligonucleotide is attached to a protein in or on a cell or tissue;
(B) hybridizing a first primer to the first immobilized extension product, and extending the first primer with a polymerase to incorporate one or more modified nucleotides; and
(C) hybridizing the first immobilized extension product to a second immobilized oligonucleotide, wherein the second immobilized oligonucleotide is immobilized to a cellular component or a matrix in or on the cell or tissue.