US 12,188,088 B1
Multiplexed targeted amplification of polynucleotides
Eli N. Glezer, Del Mar, CA (US); Daan Witters, San Diego, CA (US); and Tung Thanh Le, San Diego, CA (US)
Assigned to Singular Genomics Systems, Inc., San Diego, CA (US)
Filed by Singular Genomics Systems, Inc, San Diego, CA (US)
Filed on Jun. 15, 2023, as Appl. No. 18/335,936.
Application 18/335,936 is a continuation of application No. PCT/US2022/078470, filed on Oct. 20, 2022.
Claims priority of provisional application 63/271,837, filed on Oct. 26, 2021.
Claims priority of provisional application 63/311,576, filed on Feb. 18, 2022.
Claims priority of provisional application 63/373,017, filed on Aug. 19, 2022.
Int. Cl. C12P 19/34 (2006.01); B01L 3/00 (2006.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6874 (2013.01) [B01L 3/502761 (2013.01); B01L 2200/0652 (2013.01); B01L 2200/16 (2013.01); B01L 2300/0829 (2013.01); C12Q 2600/16 (2013.01)] 21 Claims
OG exemplary drawing
 
1. A method of sequencing, said method comprising
contacting a solid support with a sample comprising a first target polynucleotide and a second target polynucleotide, wherein said first target polynucleotide is different from said second target polynucleotide, and wherein said solid support comprises
a first oligonucleotide attached to wells of the solid support at its 5′ end, comprising a sequence capable of hybridizing to a first endogenous region of the first target polynucleotide and a sequencing primer binding sequence, and a second oligonucleotide comprising a sequence capable of hybridizing to the complement of a second endogenous region of said first target polynucleotide; and
a third oligonucleotide attached to wells of the solid support at its 5′ end, comprising a sequence capable of hybridizing to a first endogenous region of the second target polynucleotide and the sequencing primer binding sequence, and a fourth oligonucleotide comprising a sequence capable of hybridizing to the complement of a second endogenous region of said second target polynucleotide;
forming a first extension product comprising the sequencing primer binding sequence by hybridizing the first target polynucleotide to the first oligonucleotide and extending the first oligonucleotide; forming a second extension product comprising the sequencing primer binding sequence by a hybridizing the second target polynucleotide to the third oligonucleotide and extending the third oligonucleotide; and
sequencing the first extension product and sequencing the second extension product, wherein said sequencing the first extension product and sequencing the second extension product comprises hybridizing a sequencing primer to the sequencing primer binding sequence of each of the first extension product and sequencing the second extension product, binding one or more labeled nucleotides to the hybridized sequencing primer, and detecting the bound labeled nucleotides,
wherein the solid support comprises 2, 4, 6, 12, 24, 48, 96, 384, or 1536 wells, wherein each of the wells comprises a plurality of nanowells, wherein each of the nanowells is about 0.1 μm to about 2.0 μm in depth, and about 0.1 μm to about 2.0 μm in diameter.