US 12,286,453 B2
Methods of sequencing using nucleotides with 3′ acetal blocking group
Antoine Francais, Cambridge (GB); Elena Cressina, Cambridge (GB); Angelica Mariani, Cambridge (GB); Adam Culley, Cambridge (GB); Anno Koetje, Cambridge (GB); and Xiaohai Liu, Cambridge (GB)
Assigned to Illumina Cambridge Limited, Cambridge (GB)
Filed by ILLUMINA CAMBRIDGE LIMITED, Cambridge (GB)
Filed on Sep. 28, 2023, as Appl. No. 18/477,379.
Application 18/477,379 is a continuation of application No. 17/353,512, filed on Jun. 21, 2021, granted, now 11,787,831.
Claims priority of provisional application 63/042,240, filed on Jun. 22, 2020.
Prior Publication US 2024/0132532 A1, Apr. 25, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C07H 19/073 (2006.01); C07H 19/173 (2006.01); C12Q 1/6869 (2018.01)
CPC C07H 19/073 (2013.01) [C07H 19/173 (2013.01); C12Q 1/6869 (2013.01)] 22 Claims
 
1. A method of determining the sequence of a plurality of different target single-stranded polynucleotides, comprising:
(a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;
(b) contacting the solid support with an aqueous incorporation solution comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein the nucleotides each comprises a 3′ blocking group, and at least one type of incorporated nucleotides each comprises a detectable label attached via a cleavable linker,
(c) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides;
(d) removing the 3′ blocking group from the nucleotides incorporated into the extended copy polynucleotides;
(e) washing the extended copy polynucleotides with a post cleavage wash solution after the removal of the 3′ blocking group from the incorporated nucleotides; and
(f) repeating steps (b) to (e) until the sequences of at least a portion of the target polynucleotides are determined;
wherein the one or more of four different types of nucleotides in the aqueous incorporation solution has a structure selected from Formula (Ia), (Ia′), (Ib), (Ib′), (Ib″), (Ic), (Ic′) or (Id):

OG Complex Work Unit Chemistry
wherein R4 is H, and —OR6 is a triphosphate.