Sequencing by synthesis using pulse read optics

• US 10,550,428 B2
• Filed: 09/16/2015
• Issued: 02/04/2020
• Est. Priority Date: 09/17/2014
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Eshoo, Mark W., Clemens, John M., Hayden, Mark A.
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/512,441
• Publication Number:
  US 20170247755A1
• Time in Patent Office: 1,602 Days
• Field of Search: None
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C40B 60/08 Integrated apparatus specially adapted for both creating and screening libraries
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Abstract

Provided herein are systems and methods for nucleic acid sequencing by synthesis in a plurality of wells using detectably labeled chain terminating nucleotides with photolabile blocking groups and pulses of photocleaving light. In certain embodiments, the systems and methods provides a plurality of deblock-scan cycles comprising an initial deblock time period followed by a scanning light period, wherein at least one of the following occurs in each deblock-scan cycle: 1) the deblock time period is shorter than the scan time period; 2) the deblock time period is only long enough to deblock the photolabile groups that are part of a primer in less than all of the plurality of wells; or 3) the deblock time period is between 25 and 150 mSec and the scan time is at least 200 mSec. Such shorter deblock time periods help prevent the addition of more than one nucleotide to the primer prior to scanning (e.g., accuracy is enhanced).

Claims

1. A system for photocleaving and scanning nucleotide analogs comprising:

a) a substrate comprising a plurality of wells which each contain a reaction mixture comprising a template nucleic acid, a polymerase, a primer hybridized to said template, and a first nucleotide analog,

wherein said primer comprises a 3′

terminal nucleotide analog with a photolabile blocking group that terminates chain extension, and

wherein said first nucleotide analog comprises;

i) a first detectable moiety, and ii) a photolabile blocking group that terminates chain extension; and

b) a light system component comprising;

i) a light source in optical communication with said plurality of wells which provides;

A) photocleaving light input that cleaves said photolabile blocking group when it is part of said primer; and

B) scanning input light that produces an optical signal from said first detectable moiety after said first nucleotide analog is added to said primer by said polymerase; and

ii) a light control component comprising a computer processor and a computer program wherein said computer program activates said light source such that a plurality of deblock-scan cycles are generated,

wherein each of said deblock-scan cycles comprise an initial deblock time period wherein at least a part of said photocleaving light input is passed into said plurality of wells, and a following scan time period wherein at least part of said scanning light input is passed into said plurality of wells, and

wherein at least one of the following occurs in each of said deblock-scan cycles;

A) said deblock time period is shorter than said scan time period;

B) said deblock time period is only long enough to deblock said photolabile blocking groups that are part of said primer in less than all of said plurality of wells; and

C) said deblock time period is between 25 and 150 mSec and said scan time is at least 200 mSec.

2. The system of claim 1, further comprising c) a detector component in optical communication with said plurality of wells which detects said optical signal from said plurality of wells.

3. The system of claim 1, wherein said substrate is composed of a material selected from the group consisting of:

transparent glass, transparent plastic, silicon-titanium oxide, titanium oxide, tantalum oxide, niobium oxide, hafnium oxide, aluminum oxide, zirconium oxide, silicon nitride, aluminum nitride, titanium nitride, polycarbonate (PC), PMMA, and Su8.

4. The system of claim 1, wherein said plurality of optical sensing wells are sized to provide an internal waveguide.

5. The system of claim 4, wherein each of said wells comprises a zero-mode waveguide.

6. The system of claim 1, further comprising an external waveguide.

7. The system of claim 1, wherein said wells are optically coupled to an external waveguide.

8. The system of claim 1, wherein said template nucleic acid in each of said plurality of optical sensing wells is part of a sequencing library.

9. The system of claim 1, wherein said photolabile blocking group that is part of said primer is the same as the photolabile blocking group that is part of said first nucleotide analog.

10. The system of claim 1, wherein said photolabile blocking group that is part of said primer is different from the photolabile blocking group that is part of said first nucleotide analog.

11. The system of claim 1, wherein said photolabile blocking group is selected from the group consisting of:

an o-nitrobenzyl blocking group, nitroveratryl, 1-pyrenylmethyl, 6-nitroveratryloxycarbonyl, dimethyldimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, methyl-6-nitropiperonyloxycarbonyl, dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl, o-hydroxy-alpha-methyl cinnamoyl, and 2-oxymethylene anthriquinone.

12. The system of claim 1, wherein said light source comprises a first light generating component configured to generate said photocleaving light input, and a second light generating component configured to generate said scanning light input.

13. The system of claim 1, wherein said plurality of wells are optically coupled to an external waveguide, wherein said external waveguide is either one waveguide in optical communication with all of said plurality of wells, or wherein said external waveguide is a plurality of waveguides, one for each of said plurality of wells.

14. A method for photocleaving and detecting nucleotide analogs using a system comprising:

i) a substrate comprising a plurality of wells, wherein each well contains a reaction mixture comprising a template nucleic acid, a polymerase, a primer hybridized to said template, and a first nucleotide analog,

wherein said primer comprises a 3′

terminal nucleotide analog with a photolabile blocking group that terminates chain extension, and

wherein said first nucleotide analog comprises;

i) a first detectable moiety, and ii) a photolabile blocking group that terminates chain extension; and

ii) a light system component comprising;

A) a light source in optical communication with said plurality of wells, wherein said light source provides;

I) photocleaving light input; and

II) scanning light input; and

B) a light control component comprising a computer processor and a computer program wherein said computer program activates said light source such that a plurality of deblock-scan cycles are generated, wherein each of said deblock-scan cycles comprises an initial deblock time period wherein at least a portion of said photocleaving light input is passed into said wells, and a following scan time period wherein at least a portion of said scanning light input is passed into said wells, and

iii) a detector component in optical communication with said plurality of optical sensing wells,

wherein the method comprises;

a) activating said light control component such that said light source provides a plurality of said deblock-scan cycles which deblock said photolabile blocking groups, when said photolabile groups are part of said primer, in at least some of said wells and generates an optical signal from said detectable moiety in said plurality of wells after said first nucleotide analog is added to said primer by said polymerase, wherein at least one of the following occurs in each of said deblock-scan cycles;

A) said deblock time period is shorter than said scan time period;

B) said deblock time period is only long enough to deblock said photolabile blocking groups that are part of said primer in less than all of said plurality of wells; and

C) said deblock time period is between 25 and 150 mSec and said scan time is at least 200 mSec; and

b) detecting said optical signal from detectable moiety generated during each of said plurality of deblock-scan cycles in said plurality of wells with said detector component.

15. A method for photocleaving and detecting nucleotide analogs using a system comprising:

i) a substrate comprising a plurality of optical sensing wells, wherein each of said optical sensing wells is sized to provide an internal waveguide and/or is optically coupled to an external waveguide,

wherein each optical sensing well contains a reaction mixture comprising a template nucleic acid, a polymerase, a primer hybridized to said template, and a first nucleotide analog,

wherein said primer comprises a 3′

terminal nucleotide analog with a photolabile blocking group that terminates chain extension, and

wherein said first nucleotide analog comprises;

i) a first detectable moiety, and ii) a photolabile blocking group that terminates chain extension; and

ii) a light system component comprising;

A) a light source in optical communication with said internal and/or external waveguides, wherein said light source provides;

I) photocleaving light input that generates a first electromagnetic wave in said optical sensing wells when passed into said internal or external waveguides; and

II) scanning input light that generates a second electromagnetic wave in said optical sensing wells when passed into said internal or external waveguides; and

B) a light control component comprising a computer processor and a computer program wherein said computer program activates said light source such that a plurality of deblock-scan cycles are generated, wherein each of said deblock-scan cycles comprises an initial deblock time period wherein said photocleaving light input is passed into said internal and/or external waveguides, and a following scan time period wherein said scanning light input is passed into said internal and/or external waveguides, and

iii) a detector component in optical communication with said plurality of optical sensing wells;

wherein the method comprises;

a) activating said light control component such that said light source provides a plurality of said deblock-scan cycles which deblock said photolabile blocking groups, when they are part of said primer, in at least some of said optical sensing wells and generates an optical signal from said detectable moiety in said plurality of optical sensing wells after said first nucleotide analog is added to said primer by said polymerase,

wherein at least one of the following occurs in each of said deblock-scan cycles;

A) said deblock time period is shorter than said scan time period;

B) said deblock time period is only long enough to deblock said photolabile blocking groups that are part of said primer in less than all of said plurality of optical sensing wells; and

C) said deblock time period is between 25 and 150 mSec and said scan time is at least 200 mSec; and

b) detecting said optical signal from detectable moiety generated during each of said plurality of deblock-scan cycles in said plurality of optical sensing wells with said detector component.

16. The method of claim 15, wherein said detecting provides sequence information for at least a portion of said template nucleic acid in at least some of said plurality of optical sensing wells.