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US 12,110,550 B2Granted
Methods of amplifying circular polynucleotides in situ
AssigneeSingular Genomics Systems, Inc.
InventorsDaan Witters, Allen Lipson, Eli N. Glezer
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Abstract
Disclosed herein, inter alia, are complexes, kits, and efficient methods of sequencing two strands of a double stranded polynucleotide.
Claims38
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.
1 more element
A method of incorporating a modified nucleotide into a primer in situ, the method comprising:
- extending a first immobilized oligonucleotide hybridized to a circular polynucleotide with a polymerase to generate an extension product comprising one or more complements of the circular polynucleotide, wherein said first immobilized oligonucleotide is attached to a cellular component of a cell;
- hybridizing the extension product to a second oligonucleotide, wherein the second oligonucleotide tethers the extension product to a localized space;
- and
- hybridizing a primer to the immobilized extension product, and extending the primer with a polymerase to incorporate the modified nucleotide.
1 more element
Dependencies are inferred from the claim text.