Nucleic acid sequencing using affinity reagents

Patent metadata

Field	Value
Publication number	US-10851410-B2
Application number	US-201815862566-A
Country	US
Kind code	B2
Filing date	Jan 4, 2018
Priority date	Jan 4, 2017
Publication date	Dec 1, 2020
Grant date	Dec 1, 2020

Abstract

The invention provides compositions and methods for sequencing nucleic acids and other applications. In sequencing by synthesis, unlabeled reversible terminators are incorporated by a polymerase in each cycle, then labeled after incorporation by binding to the reversible terminator a directly or indirectly labeled antibody or other affinity reagent.

First claim

We claim:

1. A method for detecting of a 3′-O-reversible terminator deoxyribonucleotide incorporated at the 3′ end of a primer extension product, wherein the reversible terminator deoxyribonucleotide comprises a nucleobase, a sugar moiety, and a cleavable blocking group, said method comprising
(a) providing the primer extension product comprising the incorporated reversible terminator deoxyribonucleotide;
(b) combining the primer extension product from (a) with a first affinity reagent that binds to the incorporated reversible terminator deoxyribonucleotide, wherein the first affinity reagent binds to the nucleobase, the cleavable blocking group, or both, or to the nucleobase and 3′-OH moiety of the 3′-OH deoxyribonucleotide extension product, and
(c) detecting binding of the first affinity reagent to the incorporated reversible terminator deoxyribonucleotide.

2. The method of claim 1 where the first affinity reagent binds to an epitope comprising the sugar moiety.

3. The method of claim 1 wherein the nucleobase is a natural nucleobase selected from adenine (A), guanine (G), thymine (T) cytosine (C), and uracil (U).

4. The method of claim 3 wherein the first affinity reagent discriminates
a) an incorporated reversible terminator comprising adenine (A) from an incorporated reversible terminator comprising deoxyribonucleotide guanine (G), thymine (T), or cytosine (C);
b) an incorporated reversible terminator comprising G from an incorporated reversible terminator comprising T, C, or A;
c) an incorporated reversible terminator comprising T from an incorporated reversible terminator comprising G, C, or A; or
d) an incorporated reversible terminator comprising C from an incorporated reversible terminator comprising guanine G, T, or A.

5. The method of claim 1 wherein the nucleobase is an adenine analog (A′), a guanine analog (G′), a thymine analog (T′) or a cytosine analog (C′) and the first affinity reagent preferentially binds an incorporated nucleotide comprising the analog compared to binding an incorporated nucleotide comprising the corresponding natural nucleobase.

6. The method of claim 5 wherein the first affinity reagent binds to the cleavable blocking group.

7. The method of claim 1 wherein in step (c) the primer extension product is combined with 2, 3 or 4 affinity reagents with different binding specificities and one affinity reagent binds to the incorporated reversible terminator deoxyribonucleotide.

8. The method of claim 7 wherein the binding by said one affinity reagent identifies the nucleobase.

9. The method of claim 1 further comprising (d) removing the cleavable blocking group after step (c) to produce a 3′-OH deoxyribonucleotide.

10. The method of claim 1 wherein the first affinity reagent is an antibody or an aptamer.

11. The method of claim 1 wherein the first affinity reagent is conjugated to or bound to a detectable label, and detecting binding of the affinity reagent to the incorporated reversible terminator deoxyribonucleotide in step (c) comprises detecting a signal from the detectable label.

12. The method of claim 1 wherein the first affinity reagent is not labeled and the step of detecting binding of the first affinity reagent to the incorporated reversible terminator deoxyribonucleotide comprises (i) binding one or more secondary affinity reagents to the first affinity reagent, wherein the secondary affinity reagents comprise a detectable label, and (ii) detecting binding of the first affinity reagent to the incorporated reversible terminator deoxyribonucleotide comprises detecting a signal from the detectable label.

13. The method of claim 12 where the first affinity reagent and the secondary affinity reagent are combined prior to step (b).

14. The method of claim 1 wherein binding is detected in step (d) by detecting a fluorescence or chemiluminescence signal.

15. A method for performing a sequencing-by-synthesis reaction, said method comprising the steps of:
(a) providing a DNA array comprising a plurality of immobilized template nucleic acids comprising a plurality of different template sequences wherein the different template sequences of the are immobilized at different positions on the array;
(b) annealing oligonucleotide primers to the template nucleic acids wherein the oligonucleotide primers hybridize to predetermined positions on the template nucleic acids;
(c) combining the template nucleic acids and primers annealed thereto with a polymerase and four different reversible terminator deoxyribonucleotides each reversible terminator deoxyribonucleotide comprising a nucleobase (N), a sugar moiety, and a cleavable blocking group,
wherein N is adenine (A) or an analog thereof (A′), guanine (G) or an analog thereof (G′), thymine (T) or an analog thereof (T′), and cytosine (C) or an analog thereof (C′),
wherein at least one of said four different reversible terminator deoxyribonucleotides is unlabeled,
under conditions in which a plurality of the oligonucleotide primers are extended by incorporation of a single reversible terminator deoxyribonucleotide each to produce a plurality of primer extension products each comprising a 3′ terminus, some of which comprise A or A′ incorporated at the 3′ terminus, some of which comprise T or T′ incorporated at the 3′ terminus, some of which comprise G or G′ incorporated at the 3′ terminus, and some of which comprise C or C′ incorporated at the 3′ terminus;
(d) contacting the plurality of primer extension products with one or more first affinity reagents under conditions wherein each of said one or more first affinity reagents binds to only one of the four different incorporated reversible terminator deoxyribonucleotides, wherein the first affinity reagents bind to the nucleobase, the sugar moiety, the cleavable blocking group or a combination thereof, of said one of four incorporated reversible terminator deoxyribonucleotides;
(e) detecting the binding of the one or more first affinity reagents, wherein the binding of a first affinity reagent to a primer extension product comprising an incorporated reversible terminator deoxyribonucleotide identifies the nucleobase of the incorporated reversible terminator deoxyribonucleotide and the nucleobase of the template nucleotide complementary to the nucleobase of the incorporated reversible terminator deoxyribonucleotide.

16. The method of claim 15 wherein all of the four different reversible terminator deoxyribonucleotides are unlabeled.

17. A method for sequencing a nucleic acid, comprising:
(a) contacting a nucleic acid template comprising the nucleic acid, a nucleic acid primer complementary to a portion of said template, a polymerase, and an unlabeled RT of Formula I:

wherein
R 1 is a 3′-O reversible cleavable blocking group;
R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof; and
R 3 consists of one or more phosphates;

under conditions suitable for extending the primer by incorporating the unlabeled RT into a sequence complementary to the nucleic acid template, thereby producing an unlabeled extension product comprising the unlabeled RT;
(b) contacting the unlabeled extension product with an affinity reagent comprising a detectable label under conditions wherein the affinity reagent binds specifically to the unlabeled RT to produce a labeled extension product comprising the RT; and
(c) identifying the unlabeled RT in the labeled extension product to ident

Assignees

• Mgi Tech Co Ltd
• Bgi Shenzhen

Inventors

• Drmanac Radoje
• Drmanac Snezana
• Li Handong
• Xu Xun
• Callow Matthew J
• Eckhardt Leon
• Yang Naibo

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C07K2317/73

  Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation · CPC title