Sequencing by synthesis using pulse read optics

A system comprising a light component, wherein said light component optically interfaces with a substrate component,

• wherein said substrate component comprises a plurality of wells,
• wherein each well contains, or is configured to 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 extension, and
• wherein said first nucleotide analog comprises:
• i) a detectable moiety, and ii) a photolabile blocking group that terminates chain extension;
• and
• wherein said light component comprises:
• a) a light source in optical communication with said plurality of wells which is configured to provide:
• 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
• b) a light control component that 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 where at least part of said photocleaving light input is passed into said plurality of wells, and a following scan time period where 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:
• i) said deblock time period is shorter than said scan time period;
• ii) 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
• iii) said deblock time period is between 25 and 150 mSec and said scan time is at least 200 mSec.

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;
• B) a light control component configured to activate said light source such that a plurality of deblock-scan cycles are generated which comprise:
• I) an initial deblock time period where at least a portion of said photocleaving light input is passed into said wells, and II) a following scan time period where at least a portion of said scanning light input is passed into said wells,
• 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 component provides a plurality of said deblock-scan cycles which delock 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.

A system for photocleaving and scanning nucleotide analogs comprising:

• a) 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, or is configured to 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 internal and/or external waveguides, wherein said light source provides:
• A) photocleaving light input that, when passed into said internal or external waveguides, generates a first electromagnetic wave in said optical sensing wells that is capable of cleaving said photolabile blocking group when it is part of said primer;
• and B) scanning input light that, when passed into said internal or external waveguides, generates a second electromagnetic wave in said optical sensing wells that is capable of producing 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 that 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 where said photocleaving light input is passed into said internal and/or external waveguides, and a following scan time period where said scanning light input is passed into said internal and/or external waveguides, 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 optical sensing wells;
• and
• C) said deblock time period is between 25 and 150 mSec and said scan time is at least 200 mSec.

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;
• B) a light control component configured to activate said light source such that a plurality of deblock-scan cycles are generated which comprise:
• I) an initial deblock time period where said photocleaving light input is passed into said internal and/or external waveguides, and II) a following scan time period where said scanning light input is passed into said internal and/or external waveguides,
• 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 component provides a plurality of said deblock-scan cycles which delock 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.

A system comprising a light component, wherein said light component is configured to optically interface with a substrate component,

• wherein said substrate component comprises 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, or is configured to 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 extension, and
• wherein said first nucleotide analog comprises:
• i) a detectable moiety, and ii) a photolabile blocking group that terminates chain extension;
• and
• wherein said light component comprises:
• a) a light source in optical communication with said internal and/or external waveguides, wherein said light source provides:
• A) photocleaving light input that, when passed into said internal or external waveguides, generates a first electromagnetic wave in said optical sensing wells that cleaves said photolabile blocking group when it is part of said primer;
• and B) scanning input light that, when passed into said internal or external waveguides, generates a second electromagnetic wave in said optical sensing wells that produces an optical signal from said detectable moiety after said first nucleotide analog is added to said primer;
• and
• b) a light control component that 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 where said photocleaving light input is passed into said internal and/or external waveguides, and a following scan time period where said scanning light input is passed into said internal and/or external waveguides, 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 optical sensing wells;
• and
• C) said deblock time period is between 25 and 150 mSec and said scan time is at least 200 mSec.