Polynucleotide sequencing

Patent metadata

Field	Value
Publication number	US-12480157-B2
Application number	US-202217952414-A
Country	US
Kind code	B2
Filing date	Sep 26, 2022
Priority date	Sep 30, 2021
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

A polynucleotide sequencing method includes a wash step that employs a composition including a polymerase. The composition may also include a plurality of nucleotides. The composition may be configured to prevent the polymerase from incorporating one of the plurality of nucleotides into a copy polynucleotide strand. The composition may be substantially free of Mg 2+ .

First claim

What is claimed is:

1 . A polynucleotide sequencing method comprising:
(a) incubating an incorporation composition comprising a first polymerase and a plurality of blocked, labeled nucleotides with a template polynucleotide strand such that the first polymerase incorporates one of the plurality of blocked, labeled nucleotides into a copy polynucleotide strand complementary to at least a portion of the template polynucleotide strand; (b) identifying the blocked, labeled nucleotide incorporated into the copy polynucleotide strand; (c) removing a label and a blocking moiety from the blocked, labeled nucleotide incorporated into the copy polynucleotide strand; (d) washing the removed label and the blocking moiety away from the copy polynucleotide strand with a first wash composition comprising a second polymerase wherein the first wash composition is substantially free of Mg.sup.2+ to inhibit the second polymerase from incorporating a nucleotide into the copy polynucleotide strand; and (e) repeating steps (a) to (d) until at least a partial sequence of the template polynucleotide strand is determined.

2 . The method of claim 1 , wherein the first wash composition comprises a plurality of nucleotides.

3 . The method of claim 2 , wherein the plurality of nucleotides comprises blocked nucleotides.

4 . The method of claim 2 , wherein the plurality of nucleotides comprises nucleotides labeled with a detectable moiety.

5 . The method of claim 1 , wherein the template polynucleotide strand is immobilized on a solid support.

6 . The method of claim 1 , wherein the template polynucleotide is immobilized on a surface of a flow cell.

7 . A polynucleotide sequencing method comprising:
(a) incubating an incorporation composition comprising a first polymerase and a plurality of blocked, labeled nucleotides with a template polynucleotide strand such that the first polymerase incorporates one of the plurality of blocked, labeled nucleotides into a copy polynucleotide strand complementary to at least a portion of the template polynucleotide strand; (b) identifying the blocked, labeled nucleotide incorporated into the copy polynucleotide strand; (c) contacting the copy polynucleotide strand into which the blocked, labeled nucleotide is incorporated with a first wash composition comprising a second polymerase wherein the first wash composition is substantially free of Mg.sup.2+ to inhibit the second polymerase from incorporating a nucleotide into the copy polynucleotide strand; (d) removing a label and a blocking moiety from the blocked, labeled nucleotide incorporated into the copy polynucleotide strand; (e) washing the removed label and the blocking moiety away from the copy polynucleotide strand with a second wash composition; and (f) repeating steps (a) to (e) until at least a partial sequence of the template polynucleotide strand is determined.

8 . The method of claim 7 , wherein the first wash composition comprises a plurality of nucleotides.

9 . The method of claim 8 , wherein the plurality of nucleotides comprises blocked nucleotides.

10 . The method of claim 8 , wherein the plurality of nucleotides comprises nucleotides labeled with a detectable moiety.

11 . The method of claim 7 , wherein the template polynucleotide strand is immobilized on a solid support.

12 . The method of claim 7 , wherein the template polynucleotide is immobilized on a surface of a flow cell.

Assignees

• Illumina Inc

Inventors

• Wu Xiaolin
• Mccauley Patrick

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01L2200/16

  Reagents, handling or storing thereof · CPC title

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title

• B01L2200/04

  Exchange or ejection of cartridges, containers or reservoirs · CPC title

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title