Incorporation and imaging mixes

Patent metadata

Field	Value
Publication number	US-2022154273-A1
Application number	US-202117525530-A
Country	US
Kind code	A1
Filing date	Nov 12, 2021
Priority date	Nov 16, 2020
Publication date	May 19, 2022
Grant date	—

Abstract

An example of an incorporation mix includes a liquid carrier, a complex, and a labeled nucleotide. The complex includes a polymerase and a plasmonic nanostructure linked to the polymerase. The labeled nucleotide includes a nucleotide, a 3′ OH blocking group attached to a sugar of the nucleotide, and a dye label attached to a base of the nucleotide.

First claim

What is claimed is:

1 . An incorporation mix, comprising:
a liquid carrier; a complex, including:
a polymerase; and
a plasmonic nanostructure linked to the polymerase; and
a labeled nucleotide, including:
a nucleotide;
a 3′ OH blocking group attached to a sugar of the nucleotide; and
a dye label attached to a base of the nucleotide.

2 . The incorporation mix as defined in claim 1 , wherein the plasmonic nanostructure is selected from the group consisting of a gold nanostructure, a silver nanostructure, a tin nanostructure, a rhodium nanostructure, a ruthenium nanostructure, a palladium nanostructure, an osmium nanostructure, an iridium nanostructure, a platinum nanostructure, a chromium nanostructure, a copper nanostructure, a gallium arsenide nanostructure, a doped silicon nanostructure, an aluminum nanostructure, a magnesium nanostructure, a silver and gold composite nanostructure, and combinations thereof.

3 . The incorporation mix as defined in claim 1 , wherein the plasmonic nanostructure is chemically conjugated to an amine or a cysteine of the polymerase.

4 . The incorporation mix as defined in claim 1 , wherein:
an oligonucleotide is attached to the polymerase; and the oligonucleotide is hybridized to a complementary oligonucleotide tether that is attached to the plasmonic nanostructure.

5 . The incorporation mix as defined in claim 1 , wherein:
an oligonucleotide is attached to the polymerase; and the oligonucleotide is hybridized to a complementary portion of an oligonucleotide tether that also includes an additional portion that is wrapped around the plasmonic nanostructure.

6 . The incorporation mix as defined in claim 1 , wherein:
the plasmonic nanostructure is functionalized with a first member of a binding pair; and the polymerase includes or is functionalized with a second member of the binding pair.

7 . The incorporation mix as defined in claim 6 , wherein the first member and the second member include a NiNTA ligand and a histidine tag, or streptavidin and biotin, or a spytag and a spycatcher, or maleimide and cysteine, or azide and dibenzocyclooctyne.

8 . A method, comprising:
introducing an incorporation mix to a flow cell including clusters of template strands, the incorporation mix including:
a liquid carrier;
a plurality of complexes, each complex including:
a polymerase; and
a plasmonic nanostructure linked to the polymerase; and

a plurality of labeled nucleotides, each labeled nucleotide including:
a nucleotide;
a 3′ OH blocking group attached to a sugar of the nucleotide; and
a dye label attached to a base of the nucleotide;

whereby at least one the polymerases i) incorporates an individual one of the labeled nucleotides into a nascent strand along one the template strands, and ii) maintains its linked plasmonic nanostructure within proximity of the individual one of the labeled nucleotides; and
 optically imaging the incorporation while the plasmonic nanostructure is maintained.

9 . A kit, comprising:
an incorporation mix, including:
a liquid carrier;
a polymerase; and
a labeled nucleotide including:
a nucleotide;
a 3′ OH blocking group attached to a sugar of the nucleotide; and
a dye label attached to a base of the nucleotide; and

an imaging mix, including:
a second liquid carrier; and
a plasmonic nanostructure functionalized to associate itself within proximity of the labeled nucleotide after an incorporation event involving the labeled nucleotide.

10 . The kit as defined in claim 9 , wherein the plasmonic nanostructure is selected from the group consisting of a gold nanostructure, a silver nanostructure, a tin nanostructure, a rhodium nanostructure, a ruthenium nanostructure, a palladium nanostructure, an osmium nanostructure, an iridium nanostructure, a platinum nanostructure, a chromium nanostructure, a copper nanostructure, a gallium arsenide nanostructure, a doped silicon nanostructure, an aluminum nanostructure, a magnesium nanostructure, a silver and gold composite nanostructure, and combinations thereof.

11 . The kit as defined in claim 9 , wherein the plasmonic nanostructure is functionalized with a second polymerase.

12 . The kit as defined in claim 11 , wherein the plasmonic nanostructure is chemically conjugated to an amine or a cysteine of the second polymerase.

13 . The kit as defined in claim 11 , wherein:
an oligonucleotide is attached to the second polymerase; and the oligonucleotide is hybridized to a complementary oligonucleotide tether that is attached to the plasmonic nanostructure.

14 . The kit as defined in claim 11 , wherein:
an oligonucleotide is attached to the second polymerase; and the oligonucleotide is hybridized to a complementary oligonucleotide tether that includes a portion that is wrapped around the plasmonic nanostructure.

15 . The kit as defined in claim 9 , wherein:
the plasmonic nanostructure is functionalized with a first member of a binding pair; and the polymerase includes or is functionalized with a second member of the binding pair.

16 . The kit as defined in claim 15 , wherein the first member and the second member include a NiNTA ligand and a histidine tag, or streptavidin and biotin, or a spytag and a spycatcher, or maleimide and cysteine, or azide and dibenzocyclooctyne.

17 . The kit as defined in claim 15 , wherein the polymerase further comprises a DNA binding domain attached to a surface thereof.

18 . The kit as defined in claim 15 , wherein the polymerase further comprises:
a surface tether attached to a surface thereof; and a flow cell surface binding agent attached to the surface tether.

19 . The kit as defined in claim 9 , wherein:
the plasmonic nanostructure is functionalized with streptavidin; and the labeled nucleotide is biotinylated.

20 . A method, comprising:
introducing an incorporation mix to a flow cell including clusters of template strands, the incorporation mix including:
a liquid carrier;
a plurality of polymerases; and
a plurality of labeled nucleotides, each labeled nucleotide including:
a nucleotide;
a 3′ OH blocking group attached to a sugar of the nucleotide; and
a dye label attached to a base of the nucleotide;

whereby at least one the polymerases incorporates an individual one of the labeled nucleotides into a nascent strand along one the template strands;
introducing an imaging mix into the flow cell, the imaging mix including:
a second liquid carrier; and
a plurality of functionalized plasmonic nanostructures;

whereby at least one of the functionalized plasmonic nanostructures associates itself within proximity of the individual one of the labeled nucleotides; and
optically imaging the incorporation while the at least one of the functionalized plasmonic nanostructures is associated with the individual one of the labeled nucleotides.

21 . The method as defined in claim 20 , wherein:
each of the functionalized plasmonic nanostructures is functionalized with a second polymerase; and the method further comprises removing the incorporation mix prior to introducing the imaging mix.

22 . The method as defined in claim 20 , wherein:
each of the functionalized plasmonic nanostructures is functionalized with a first member of a binding pair; each of the polymerases includes a second member of the binding pair; and the method further comprises removing the incorporation mix prior to introducing the ima

Assignees

• Illumina Inc
• Illumina Cambridge Ltd

Inventors

• Artioli Gianluca Andrea
• Lessard-Viger Mathieu
• Mather Brian D
• Mcdonald Seth M
• Pugliese Kaitlin M
• Von Hatten Xavier

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• B01L3/502738

  characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746) · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• B01L2200/0621

  Control of the sequence of chambers filled or emptied · CPC title

• B01L2200/026

  Fluid interfacing between devices or objects, e.g. connectors, inlet details · CPC title