Granted Patent B2

US 12,195,493 · App. 17/208,831 · Granted Jan 14, 2025

Inventors: Handong Li (San Jose, CA); Snezana Drmanac (Los Altos Hills, CA); Radoje Drmanac (Los Altos Hills, CA); Xun Xu (Sunnyvale, CA); Lingling Peng (San Jose, CA); Scott Gablenz (San Jose, CA)

Assignees: MGI Tech Co., Ltd.; BGI Shenzhen

C07H15/04C07H19/10C07H19/20C07H21/04C12Q1/6869

Abstract

Claims (43)

1. A method of sequencing comprising:

i) providing a reaction mixture comprising a template nucleic acid, a primer, a polymerase, and a first nucleoside analogue of Formula VI or a first nucleoside analogue of Formula VII:

wherein

R 1 is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl;

X is selected from the group consisting of O and S;

R 2 is a nucleobase;

L is a cleavable linker; and

A 1 of Formula VI comprises an affinity tag;

D of Formula VII comprises a detectable fluorescent label;

ii) extending the primer by incorporating the first nucleoside analogue of Formula VI with the polymerase;

iii) contacting the incorporated first nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with A 1 of the incorporated first nucleoside analogue, thereby specifically labeling the incorporated first nucleoside analogue; and

iv) detecting the specifically labeled incorporated first nucleoside analogue of Formula VI; or alternatively,

ii) extending the primer by incorporating the first nucleoside analogue of Formula VII with the polymerase;

iii) contacting the incorporated first nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with D of the incorporated first nucleoside analogue, thereby specifically labeling the incorporated first nucleoside analogue; and

iv) detecting the labeled incorporated first nucleoside analogue by detecting a fluorescence emission from the fluorescent label of Formula VII.

2. The method of claim 1 , wherein the detectably labeled affinity agent is fluorescently labeled, and the detection comprises detecting a fluorescence emission from the fluorescently labeled affinity agent in complex with A 1 of the incorporated first nucleoside analogue.

3. The method of claim 1 , wherein the method further comprises:

v) cleaving the reversible blocking group R 1 of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue;

vi) cleaving the linker L thereby removing the affinity tag A 1 of the incorporated first nucleoside analogue, or quenching the label of the detectably labeled affinity agent in complex with A 1 of the incorporated first nucleoside analogue;

vii) providing a second nucleoside analogue of Formula VI and a polymerase;

viii) extending the primer by incorporating the second nucleoside analogue with the polymerase;

ix) contacting the incorporated second nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with A 1 of the incorporated second nucleoside analogue, thereby specifically labeling the incorporated second nucleoside analogue; and

x) detecting the specifically labeled incorporated second nucleoside analogue.

4. The method of claim 1 , wherein after detecting the labeled incorporated first nucleoside analogue by detecting a fluorescence emission from the fluorescent label of Formula VII of step (iv), the method further comprising:

v) cleaving the reversible blocking group R 1 of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue of Formula VII;

vi) providing a second detectably labeled nucleoside analogue of Formula VII and a polymerase;

vii) extending the primer by incorporating the second nucleoside analogue of Formula VII with the polymerase; and

viii) detecting incorporation of the second nucleoside analogue of Formula VII by detecting a fluorescence emission from the labeled incorporated second nucleoside analogue.

5. A method of sequencing comprising:

i) providing a reaction mixture comprising template nucleic acid, a primer, a polymerase, and a first nucleoside analogue of Formula VII:

wherein

R 1 is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl;

X is selected from the group consisting of O and S; and

R 2 comprises a nucleobase;

L is a linker; and

D comprises a detectable fluorescent label;

ii) extending the primer by incorporating the first nucleoside analogue with the polymerase; and

iii) detecting the labeled incorporated first nucleoside analogue by detecting a fluorescence emission from the fluorescent label.

6. The method of claim 5 , wherein the method further comprises:

iv) cleaving the reversible blocking group R 1 of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue;

v) providing a second detectably labeled nucleoside analogue of Formula VII and a polymerase;

vi) extending the primer by incorporating the second nucleoside analogue with the polymerase; and

vii) detecting incorporation of the second nucleoside analogue by detecting a fluorescence emission from the labeled incorporated second nucleoside analogue.

Assignments (4)

Continuity (4)

References Cited (35)