US 11,788,133 B2
Methods and compositions for sequencing complementary polynucleotides
Daan Witters, San Diego, CA (US); Eli N. Glezer, Del Mar, CA (US); and Allen Lipson, 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 Oct. 21, 2022, as Appl. No. 18/48,808.
Application 18/048,808 is a continuation of application No. 17/934,425, filed on Sep. 22, 2022.
Application 17/934,425 is a continuation of application No. 17/666,458, filed on Feb. 7, 2022, granted, now 11,486,001, issued on Nov. 1, 2022.
Claims priority of provisional application 63/183,585, filed on May 3, 2021.
Claims priority of provisional application 63/163,638, filed on Mar. 19, 2021.
Claims priority of provisional application 63/147,167, filed on Feb. 8, 2021.
Prior Publication US 2023/0193377 A1, Jun. 22, 2023
Int. Cl. C12Q 1/6869 (2018.01); C12Q 1/6834 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12Q 1/6834 (2013.01)] 30 Claims
 
1. A method of identifying a detectable nucleotide analogue, said method comprising:
a) hybridizing a first sequencing primer to a polynucleotide immobilized to a solid support, wherein said polynucleotide comprises a primer binding sequence and a template nucleic acid sequence comprising a plurality of cleavable sites;
b) contacting said first sequencing primer hybridized to said polynucleotide with a first detectable nucleotide analogue and a polymerase to form a first polymerase complex, detecting the first polymerase complex, and identifying said first detectable nucleotide analogue;
c) incorporating one or more nucleotides into said first sequencing primer hybridized to said polynucleotide with a polymerase to create an extension strand hybridized to said polynucleotide;
d) cleaving the at least one cleavable site, thereby generating a polynucleotide fragment immobilized to the solid support and at least one non-immobilized polynucleotide fragment, wherein said polynucleotide fragment immobilized to the solid support is hybridized to said extension strand;
e) removing the one or more non-immobilized polynucleotide fragments; and
f) hybridizing a second sequencing primer to said extension strand and contacting said second sequencing primer hybridized to said extension strand with a second detectable nucleotide analogue and a polymerase to form a second polymerase complex, detecting the second polymerase complex, and identifying said second detectable nucleotide analogue.