Nucleic acid constructs and methods for light-triggered molecular detection - Patents.com

Abstract

The present disclosure provides a method, device, and system for simultaneous multiplex amplification reactions and real-time detection in a single reaction chamber. An embodiment of the present disclosure provides a reaction chamber comprising a NA construct comprising a photosensitive chemical moiety, the NA construct being in a first molecular state and configured to change to a second molecular state after exposure to light, at least one reagent, and at least one enzyme, the reaction chamber being configured to allow light to reach the nucleic acid construct.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 850,239, filed May 20, 2019, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Nucleic acid (NA) testing is a unique analytical technique used to detect, quantify, and identify the genetic structure of specific sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. NA testing has many applications and is widely used in both life science research and molecular diagnostics. Regardless of the application and testing location, the amount of genetic material (RNA or DNA copies) in the test sample is typically very small and cannot be detected directly, so it is very common to use physicochemical, biochemical, or enzymatic methods to enhance the target-specific signal generated to ensure a more sensitive test. Some of these methods utilize molecular amplification processes, such as polymerase chain reaction (PCR), to increase the copy number of the target NA. Such tests are classified, widely known, and are traditionally classified as nucleic acid amplification tests (NAATs). In addition, methods of amplification include, for example, strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA), and rolling circle amplification (RCA).

[0003] NAAT methods have a variety of different performance criteria, including analytical sensitivity, specificity, limit of detection (LoD), quantification range, detection dynamic range (DDR), and turnaround time (TAT). Different applications require different criteria, and there is always a compromise, depending on the method used. For example, in infectious disease applications, it is important to accurately identify the presence or absence of infectious agents in clinical specimens. Therefore, NAAT methods are needed that provide LODs for several organisms in one test, but the quantification range is less important, since the treatment of patients is less dependent on that information. On the other hand, in gene expression applications, the concentration of messenger RNA (mRNA) in clinical samples is relatively high, and the DDR becomes much more important than the LOD.

[0004] Currently, there are various NAAT methods for NA detection, which use specific enzymes, reagents, and temperature profiles to amplify and detect specific sequences. In this invention, we describe methods and molecular structures that, when included in certain NAAT methods, can improve their performance criteria. Summary of the Invention [Means for solving the problem]

[0005] The present invention describes unique nucleic acid (NA) constructs and methods that can be incorporated into molecular detection assays to improve assay detection performance, broadly defined, and reduce workflow complexity and turnaround time.

[0006] An embodiment of the present disclosure provides a reaction chamber comprising a NA construct comprising a photosensitive chemical moiety, the NA construct being in a first molecular state and configured to change to a second molecular state after exposure to light, at least one reagent, and at least one enzyme, the reaction chamber being configured to allow light to reach the nucleic acid construct.

[0007] In some embodiments of the aspects provided herein, the NA construct is an oligonucleotide primer or a probe. In some embodiments of the aspects provided herein, the at least one enzyme is a polymerase, a reverse transcriptase, a terminal transferase, an exonuclease, an endonuclease, a restriction enzyme, or a ligase. In some embodiments of the aspects provided herein, the at least one reagent comprises one or more amplification reagents. In some embodiments of the aspects provided herein, the method further comprises a target NA. In some embodiments of the aspects provided herein, the enzyme is configured to catalyze a reaction associated with the target NA, the at least one reagent, and the NA construct. In some embodiments of the aspects provided herein, the NA construct in the first molecular state is configured to be active in the reaction. In some embodiments of the aspects provided herein, the NA construct in the second molecular state is configured to be inactive in the reaction. In some embodiments of the aspects provided herein, the NA construct in the first molecular state is configured to be inactive in the reaction. In some embodiments of the aspects provided herein, the NA construct in the second molecular state is configured to be active in the reaction. In some embodiments of the aspects provided herein, the method further includes another NA construct comprising another photosensitive chemical moiety, the other NA construct being in a third molecular state, and the other NA construct being configured to change to a fourth molecular state after exposure to another light. In some embodiments of the aspects provided herein, the other light is light. In some embodiments of the aspects provided herein, the NA construct in the first molecular state is configured to be active in the reaction, and the other NA construct in the third molecular state is configured to be inactive in the reaction. In some embodiments of the aspects provided herein, the NA construct in the second molecular state is configured to be inactive in the reaction, and the other NA construct in the fourth molecular state is configured to be active in the reaction.In some embodiments of the aspects provided herein, the NA construct in the first molecular state and another NA construct in the third molecular state are configured to be active in the reaction.In some embodiments of the aspects provided herein, the NA construct in the second molecular state and another NA construct in the fourth molecular state are configured to be inactive in the reaction.In some embodiments of the aspects provided herein, the NA construct in the first molecular state and another NA construct in the third molecular state are configured to be inactive in the reaction.

[0008] In some embodiments of the aspects provided herein, the NA construct in the second molecular state and another NA construct in the fourth molecular state are configured to be active in the reaction. In some embodiments of the aspects provided herein, the enzyme is a polymerase, the reaction is a polymerase chain reaction, and the NA construct is an oligonucleotide primer. In some embodiments of the aspects provided herein, the light-sensitive chemical moiety is located at the 3' end, 5' end, or center of the NA construct. In some embodiments of the aspects provided herein, the NA construct further comprises an additional light-sensitive chemical moiety. In some embodiments of the aspects provided herein, the fifth molecular state is the first molecular state, and the sixth molecular state is the second molecular state. In some embodiments of the aspects provided herein, the reaction chamber is a closed tube reaction chamber.

[0009] Another aspect of the present disclosure provides for performing a reaction comprising activating a reaction chamber to perform the reaction, the reaction chamber containing a nucleic acid construct comprising a light-sensitive chemical moiety in a first molecular state, at least one reagent, and at least one enzyme, and activating light to reach the nucleic acid construct in the reaction chamber, thereby transforming the nucleic acid construct to a second molecular state.

[0010] In some embodiments of the aspects provided herein, the NA construct is an oligonucleotide primer or a probe. In some embodiments of the aspects provided herein, the at least one enzyme is a polymerase, a reverse transcriptase, a terminal transferase, an exonuclease, an endonuclease, a restriction enzyme, or a ligase. In some embodiments of the aspects provided herein, the at least one reagent comprises one or more amplification reagents. In some embodiments of the aspects provided herein, the reaction chamber further comprises a target nucleic acid. In some embodiments of the aspects provided herein, the enzyme catalyzes the reaction of the target nucleic acid with the at least one reagent and the nucleic acid construct. In some embodiments of the aspects provided herein, the nucleic acid construct in the first molecular state is active in the reaction. In some embodiments of the aspects provided herein, the nucleic acid construct in the first molecular state is inactive in the reaction. In some embodiments of the aspects provided herein, the nucleic acid construct in the second molecular state is active in the reaction. In some embodiments of the aspects provided herein, the reaction chamber further comprises another nucleic acid construct comprising another light-sensitive chemical moiety in a third molecular state, and the other nucleic acid construct is configured to change to a fourth molecular state after exposure to another light. In some embodiments of the aspects provided herein, the other light is light, and the step of activating the light activates the nucleic acid construct. In some embodiments of the aspects provided herein, the method further comprises a step of activating the other light to reach the other nucleic acid construct. In some embodiments of the aspects provided herein, the method further comprises a step of deactivating the nucleic acid construct in the reaction after the step of activating the light. In some embodiments of the aspects provided herein, the method further comprises a step of activating the other nucleic acid construct after the step of activating the light or after the step of activating the other light. In some embodiments of the aspects provided herein, the method further comprises a step of deactivating the other nucleic acid construct after the step of activating the light or after the step of activating the other light.In some embodiments of the aspects provided herein, the method further comprises activating the nucleic acid construct in the reaction after activating the light. In some embodiments of the aspects provided herein, the method further comprises deactivating another nucleic acid construct after activating the light or activating another light. In some embodiments of the aspects provided herein, the method further comprises activating another nucleic acid construct after activating the light or activating another light. In some embodiments of the aspects provided herein, the reaction is extension, digestion, transcription, terminal transfer, or ligation. In some embodiments of the aspects provided herein, when the reaction is performed in a reaction chamber, no external reagent is added to the reaction chamber. In some embodiments of the aspects provided herein, when the reaction is performed in a reaction chamber, none of the nucleic acid construct, the at least one reagent, or the at least one enzyme is removed from the reaction chamber, the enzyme is a polymerase, the reaction is a polymerase chain reaction, and the nucleic acid construct is an oligonucleotide primer. In some embodiments of the aspects provided herein, the light-sensitive chemical moiety is located at the 3-terminus, 5-terminus, or center of the nucleic acid construct. In some embodiments of the aspects provided herein, the nucleic acid construct further comprises an additional light-sensitive chemical moiety. In some embodiments of the aspects provided herein, the target nucleic acid comprises a major allele and a minor allele, and the reaction is a polymerase chain reaction. In some embodiments of the aspects provided herein, the nucleic acid construct comprises a sequence complementary to the major allele. In some embodiments of the aspects provided herein, the nucleic acid construct in the first molecular state is inactive in the polymerase chain reaction with respect to the generation of an amplicon of the major allele. In some embodiments of the aspects provided herein, the nucleic acid construct in the second molecular state is active in the polymerase chain reaction with respect to the generation of an amplicon of the major allele.In some embodiments of the aspects provided herein, the nucleic acid construct in the second molecular state is inactive in a polymerase chain reaction with respect to generating an amplicon of the major allele. In some embodiments of the aspects provided herein, the other nucleic acid construct is a primer for the minor allele, and the method further comprises generating an amplicon of the minor allele prior to activating the light.

[0011] An embodiment of the present disclosure provides a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photocleavable moieties, wherein each of the one or more photocleavable moieties is independently: a) located at the 3' end of the nucleic acid construct; b) located at the 5' end of the nucleic acid construct; c) located between the 3' end and the 5' end; d) located on or connected to a nucleobase; e) located on or connected to a ribose; f) located between and connected to two consecutive members of the plurality of nucleotides; or g) a combination thereof.

[0012] In some embodiments of the aspects provided herein, the nucleic acid construct is configured to be inactive in a biochemical reaction, the biochemical reaction being polymerase-catalyzed chain extension, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, or restriction digestion. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of one or more photocleavable moieties, the nucleic acid molecule being configured to be active in a biochemical reaction. In some embodiments of the aspects provided herein, the nucleic acid construct is a primer, and the biochemical reaction is polymerase-catalyzed chain extension. In some embodiments of the aspects provided herein, the one or more photocleavable moieties are located at the 3' end. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and on the selected nucleic acid base. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3'-end and the 5'-end and between two consecutive members of the plurality of nucleotides.In some embodiments of the aspects provided herein, the 3'-end is configured to be inactive in biochemical reactions.In some embodiments of the aspects provided herein, the nucleic acid construct comprises a first nucleic acid section and a second nucleic acid section that is complementary to the first nucleic acid section, and the nucleic acid construct is configured to form a hairpin structure.In some embodiments of the aspects provided herein, the first nucleic acid section and the second nucleic acid section do not comprise one or more photocleavable moieties.

[0013] An aspect of the present disclosure provides a method of performing polymerase-catalyzed chain extension using a nucleic acid construct of the present disclosure, the method comprising the steps of: a) providing a reaction mixture comprising the nucleic acid construct, at least one template nucleic acid molecule, and a polymerase, wherein the nucleic acid construct has a sequence complementary to the template nucleic acid molecule; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby performing polymerase-catalyzed chain extension.

[0014] In some embodiments of the aspects provided herein, the subjecting step in b) does not activate the performing step in c). In some embodiments of the aspects provided herein, the nucleic acid construct remains intact in the reaction mixture before the irradiating step in c). In some embodiments of the aspects provided herein, the method further comprises cleaving one or more photocleavable moieties in c). In some embodiments of the aspects provided herein, the method further comprises forming a nucleic acid molecule in c). In some embodiments of the aspects provided herein, the performing step in c) comprises using the nucleic acid molecule formed after the irradiating step in c) as a primer for polymerase-catalyzed chain extension. In some embodiments of the aspects provided herein, the reaction mixture further comprises another primer, and the other primer is active in polymerase-catalyzed chain extension. In some embodiments of the aspects provided herein, the other primer is active in polymerase-catalyzed chain extension before the irradiating step in c). In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension of b) produces an amplicon that includes another primer.In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), and the method further comprises in c) 1) carrying out polymerase-catalyzed chain extension on two or more nucleotide sequences in the presence of the nucleic acid construct of the present disclosure to produce two or more amplicons in a fluid; 2) providing an array that includes a solid surface with a plurality of nucleic acid probes at independently addressable positions, the array being configured to contact with a fluid; and 3) measuring the hybridization of the two or more amplicons to two or more of the plurality of nucleic acid probes while the fluid is contacting the array to obtain an amplicon hybridization measurement, the amplicon comprising a quencher.In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises, in c), 1) providing an array comprising a solid support having a surface and a plurality of different probes, wherein the plurality of different probes are immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences, wherein the PCR amplification is performed in a fluid, and (i) a nucleic acid construct of the present disclosure is a PCR primer for each nucleic acid sequence and comprises a quencher; and (ii) the fluid is in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize to the plurality of probes, thereby quenching a signal from the fluorescent moiety; 3) detecting a signal from the fluorescent moiety at each of the addressable locations over time; 4) using the signal detected over time to determine the amount of amplicons in the fluid; and 5) using the amount of amplicons in the fluid to determine the amount of nucleotide sequences in the sample.In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), and the method, in c), comprises the steps of: 1) providing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair, and a polymerase, wherein the primer pair has sequence complementarity with the template nucleic acid molecule, the primer pair comprising a limiting primer and an excess primer, and at least one of the limiting primer and the excess primer is a nucleic acid construct of the disclosure; 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; and 3) subjecting the reaction mixture to a polymerase. The method further includes contacting a sensor array having: (i) a substrate comprising a plurality of probes immobilized on a surface of the substrate at different individually addressable locations, the probes having sequence complementarity to the limiting primers and capable of capturing the limiting primers; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal indicating that the limiting primer is bound to an individual probe of the plurality of probes; 4) using the detector array to detect at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction; and 5) detecting the target nucleic acid molecule based on the at least one signal indicating that the limiting primer is bound to an individual probe of the plurality of probes.

[0015] An embodiment of the present disclosure is a system for assaying at least one target nucleic acid molecule using a nucleic acid construct of the present disclosure, comprising: 1) a reaction chamber including a reaction mixture including a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair having a sequence complementary to the template nucleic acid molecule, and a polymerase, wherein the primer pair includes a limiting primer and an excess primer, at least one of the limiting primer and the excess primer is the nucleic acid construct of the present disclosure, and the reaction chamber including the reaction mixture is configured to promote a nucleic acid amplification reaction on the reaction mixture to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid; and 2) (i) a polymerase comprising a polymerase and a nucleic acid sample on a surface of a substrate at different individually addressable locations. The present invention provides a system comprising: (i) a sensor array comprising a substrate comprising a plurality of immobilized probes, the probes having sequence complementarity to the limiting primers and capable of capturing the limiting primers; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal indicating that the limiting primer is bound to an individual probe of the plurality of probes; and 3) a computer processor coupled to the sensor array and programmed to: (i) subject the reaction mixture to a nucleic acid amplification reaction; and (ii) detect at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction.

[0016] An embodiment of the present disclosure provides a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photo-cleavable moieties, each of which is independently a) located between the 3' end of the nucleic acid construct and the 5' end of the nucleic acid construct, b) located on or connected to a nucleobase, c) located on or connected to a ribose, d) located between and connected to two consecutive members of the plurality of nucleotides, or e) a combination thereof.

[0017] In some embodiments of the aspects provided herein, the nucleic acid construct is configured to be active in a biochemical reaction, and the biochemical reaction is polymerase-catalyzed chain extension, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, or restriction digestion. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of one or more photocleavable moieties, and the nucleic acid molecule is inactive in a biochemical reaction. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule immediately after photocleavage of one or more photocleavable moieties, and the nucleic acid molecule is active in a biochemical reaction, and the nucleic acid molecule is located near the 3' end. In some embodiments of the aspects provided herein, the nucleic acid construct is a primer, and the biochemical reaction is polymerase-catalyzed chain extension. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3'-end and the 5'-end and on the selected nucleic acid base. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a hairpin structure in the absence of the one or more photocleavable moieties, thereby rendering it inactive as a primer in the absence of the one or more photocleavable moieties. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3'-end and the 5'-end and between two consecutive members of the plurality of nucleotides. In some embodiments of the aspects provided herein, the nucleic acid construct comprises a first sequence complementary to the template nucleic acid molecule, the first sequence being located at or near the 3'-end. In some embodiments of the aspects provided herein, the nucleic acid construct further comprises a second sequence complementary to the template nucleic acid molecule, the second sequence being located at or near the 5'-end, and at least one of the one or more photocleavable moieties being located between the first sequence and the second sequence.In some embodiments of the aspects provided herein, the one or more photocleavable moieties are separated from the first sequence and / or the second sequence by at least one nucleotide. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a hairpin loop between the first sequence and the second sequence when both the first sequence and the second sequence hybridize to the template nucleic acid molecule. In some embodiments of the aspects provided herein, the second sequence comprises a 5'-to-5' linkage to the remainder of the nucleic acid construct, and the second sequence is configured to be non-extendable in polymerase-catalyzed chain elongation.

[0018] Aspects of the present disclosure provide a method of performing polymerase-catalyzed chain extension using a nucleic acid construct of the present disclosure, the method comprising the steps of: a) providing a reaction mixture comprising a nucleic acid construct, a template nucleic acid molecule, and a polymerase, wherein the nucleic acid construct comprises at least a first sequence; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension, thereby performing polymerase-catalyzed chain extension; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby terminating the polymerase-catalyzed chain extension.

[0019] In some embodiments of the aspects provided herein, the method further comprises, in c), cleaving one or more photocleavable moieties. In some embodiments of the aspects provided herein, the method further comprises, after the irradiating step, forming a nucleic acid molecule in c), where the nucleic acid molecule dissociates from the template nucleic acid molecule. In some embodiments of the aspects provided herein, the nucleic acid molecule forms a hairpin structure, where the hairpin structure comprises at least a portion of the first sequence. In some embodiments of the aspects provided herein, the nucleic acid molecule comprises the first sequence.

[0020] An aspect of the disclosure is a method of performing a light-activated nested polymerase chain reaction (PCR), comprising the steps of: a) providing a reaction mixture comprising a first primer pair, a second primer pair, a template nucleic acid molecule comprising an internal nucleic acid sequence, and a polymerase, wherein each member of the first primer pair is independently a nucleic acid construct of the disclosure, each member of the second primer pair is independently a nucleic acid construct of the disclosure, and wherein the internal nucleic acid sequence is nested within the template nucleic acid molecule; and b) subjecting the reaction mixture to a first primer pair using the first primer pair to a nested polymerase. a) subjecting a template nucleic acid molecule to conditions for strand extension of a first primer pair to amplify the template nucleic acid molecule, thereby forming an amplicon of the template nucleic acid sequence or a complementary sequence of the template nucleic acid molecule; and c) irradiating the reaction mixture with photons of light, thereby deactivating the first primer pair, terminating the first extension, activating the second primer pair, and initiating a second strand extension using the activated second primer pair to form an amplicon of the internal nucleic acid sequence or a complementary sequence of the internal nucleic acid sequence, wherein a)-c) are performed in a closed tube format.

[0021] In some embodiments of the aspects provided herein, the light-activated PCR is quantitative polymerase chain reaction (Q-PCR), and the method further includes the steps of 1) performing light-activated PCR on two or more nucleotide sequences in the presence of a first primer pair and a second primer pair to produce two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable locations, the array being configured to contact the fluid; and 3) measuring hybridization of the two or more amplicons to two or more of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain amplicon hybridization measurements, the amplicons comprising a quencher.

[0022] In some embodiments of the aspects provided herein, the light-activated PCR is quantitative polymerase chain reaction (Q-PCR), and the method further includes the steps of: 1) providing an array comprising a solid support having a surface and a plurality of different probes, the plurality of different probes being immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences, the PCR amplification being performed in a fluid, and (i) each of a first primer pair and a second primer pair for each nucleic acid sequence comprising a quencher, and (ii) the fluid being in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize to the plurality of probes, thereby quenching a signal from the fluorescent moiety; 3) detecting a signal from the fluorescent moiety at each of the addressable locations over time; 4) determining the amount of amplicons in the fluid using the signal detected over time; and 5) determining the amount of amplicons in the sample using the amount of amplicons in the fluid.In some embodiments of the aspects provided herein, the light-activated PCR is a quantitative polymerase chain reaction (Q-PCR), and the method includes the steps of: 1) providing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair, and a polymerase, wherein the primer pair has sequence complementarity with the template nucleic acid molecule, the primer pair comprising a limiting primer and an excess primer, and at least one of the limiting primer and the excess primer being a nucleic acid construct of the disclosure; 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; and 3) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; the limiting primer has sequence complementarity with and is capable of capturing the limiting primer; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal being indicative of the limiting primer binding to an individual probe of the plurality of probes; 4) using the detector array to detect at least one signal from the one or more addressable locations at multiple time points during the nucleic acid amplification reaction; and 5) detecting the target nucleic acid molecule based on the at least one signal indicative of the limiting primer binding to an individual probe of the plurality of probes.

[0023] An embodiment of the present disclosure provides a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photo-cleavable moieties, each of which is independently a) located between the 3' end of the nucleic acid construct and the 5' end of the nucleic acid construct, b) located on or connected to a nucleobase, c) located on or connected to a ribose, d) located between and connected to two consecutive members of the plurality of nucleotides, or e) a combination thereof.

[0024] In some embodiments of the aspects provided herein, the nucleic acid construct is a probe, and the nucleic acid construct is configured to be inactive in hybridization with a target nucleic acid molecule. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of one or more photocleavable moieties, and the nucleic acid molecule is configured to be active in hybridization with a target nucleic acid molecule. In some embodiments of the aspects provided herein, the nucleic acid construct comprises one free end. In some embodiments of the aspects provided herein, the nucleic acid construct comprises a fixed end or an end that is non-extendible in polymerase-catalyzed chain elongation. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and on a selected nucleobase, and the selected nucleobase is configured to hybridize with the target nucleic acid molecule in the absence of the one or more photocleavable moieties. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and between two consecutive members of the plurality of nucleotides. In some embodiments of the aspects provided herein, the nucleic acid construct comprises a first nucleic acid section and a second nucleic acid section complementary to the first nucleic acid section, and the nucleic acid construct is configured to form a hairpin structure. In some embodiments of the aspects provided herein, the first nucleic acid section and the second nucleic acid section do not comprise one or more photocleavable moieties.

[0025] An aspect of the present disclosure provides a method of performing hybridization using a nucleic acid construct of the present disclosure, the method comprising the steps of: a) providing a reaction mixture comprising the nucleic acid construct and a target nucleic acid molecule; b) subjecting the reaction mixture to hybridization conditions; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby performing hybridization.

[0026] In some embodiments of the aspects provided herein, the providing step in b) does not activate the performing step in c). In some embodiments of the aspects provided herein, the nucleic acid construct remains intact in the reaction mixture before the irradiating step in c). In some embodiments of the aspects provided herein, the method further comprises cleaving one or more photocleavable moieties in c). In some embodiments of the aspects provided herein, the method further comprises forming a nucleic acid molecule in c). In some embodiments of the aspects provided herein, the irradiating step breaks the hairpin structure of the nucleic acid construct to form a nucleic acid molecule.

[0027] An embodiment of the present disclosure provides a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photo-cleavable moieties, each of which is independently a) located between the 3' end of the nucleic acid construct and the 5' end of the nucleic acid construct, b) located on or connected to a nucleobase, c) located on or connected to a ribose, d) located between and connected to two consecutive members of the plurality of nucleotides, or e) a combination thereof.

[0028] In some embodiments of the aspects provided herein, the nucleic acid construct is a probe, and the nucleic acid construct is configured to be active in hybridization with a target nucleic acid molecule. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of one or more photocleavable moieties, and the nucleic acid molecule is configured to be inactive in hybridization with a target nucleic acid molecule. In some embodiments of the aspects provided herein, the nucleic acid construct comprises one free end. In some embodiments of the aspects provided herein, the nucleic acid construct comprises a fixed end or an end that is non-extendible in polymerase-catalyzed chain elongation. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and between two consecutive members of the plurality of nucleotides. In some embodiments of the aspects provided herein, each of the one or more photocleavable moieties is independently located between the 3'-end and the 5'-end and on the selected nucleobase, and the selected nucleobase is configured to hybridize with another nucleobase of the nucleic acid construct in the absence of the one or more photocleavable moieties.In some embodiments of the aspects provided herein, the nucleic acid construct comprises a first nucleic acid section and a second nucleic acid section complementary to the first nucleic acid section, and the nucleic acid construct is configured to form a hairpin structure in the absence of the one or more photocleavable moieties.In some embodiments of the aspects provided herein, the first nucleic acid section or the second nucleic acid section comprises one or more photocleavable moieties.

[0029] An aspect of the present disclosure provides a method of performing hybridization using a nucleic acid construct of the present disclosure, the method comprising the steps of: a) providing a reaction mixture comprising the nucleic acid construct and a target nucleic acid molecule; b) subjecting the reaction mixture to hybridization conditions; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby terminating hybridization.

[0030] In some embodiments of the aspects provided herein, the method further comprises, in c), cleaving one or more photocleavable moieties. In some embodiments of the aspects provided herein, the method further comprises, in c), forming a nucleic acid molecule. In some embodiments of the aspects provided herein, the method further comprises, in c), forming a hairpin structure in the nucleic acid molecule. In some embodiments of the aspects provided herein, the method further comprises the step of performing polymerase-catalyzed chain extension, wherein 1) the reaction mixture further comprises a polymerase and a primer, in b), the nucleic acid construct hybridizes to the target nucleic acid molecule in b), 2) the reaction mixture is subjected to conditions for polymerase-catalyzed chain extension using the primer in b), where the polymerase-catalyzed chain extension is terminated at or near the position where the nucleic acid construct forms a duplex with the target nucleic acid molecule, and 3) after the irradiating step in c), the duplex is removed, exposing the single-stranded sequence that was previously hybridized to the nucleic acid construct, thereby allowing the polymerase-catalyzed chain extension to continue and extend through the single-stranded sequence. In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 1) performing polymerase-catalyzed chain extension on two or more nucleotide sequences comprising a target nucleic acid molecule in the presence of a nucleic acid construct of the present disclosure, thereby producing two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable locations, wherein the array is configured to contact the fluid; and 3) measuring hybridization of the two or more amplicons to two or more of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain amplicon hybridization measurements, wherein the amplicons comprise a quencher.In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises the steps of: 1) providing an array comprising a solid support having a surface and a plurality of different probes, the plurality of different probes being immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences comprising a target nucleic acid molecule, the PCR amplification being performed in a fluid comprising a nucleic acid construct of the present disclosure, (i) a PCR primer for each nucleic acid sequence comprises a quencher, (ii) the fluid is in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize to the plurality of probes, thereby quenching a signal from the fluorescent moiety, and irradiation is performed during the PCR; 3) detecting a signal from the fluorescent moiety at each of the addressable locations over time; 4) using the signals detected over time to determine the amount of amplicons in the fluid; and 5) using the amount of amplicons in the fluid to determine the amount of nucleotide sequences in the sample.In some embodiments of the aspects provided herein, the polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), and the method includes the steps of: 1) providing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule comprising a target nucleic acid molecule, a primer pair, and a polymerase, wherein the primer pair has sequence complementarity with the at least one template nucleic acid molecule, the primer pair comprising a limiting primer and an excess primer, and wherein the reaction mixture further comprises at least one of the nucleic acid constructs of the disclosure; 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain an amplification product of the template nucleic acid molecule and the limiting primer, wherein the amplicon comprises the limiting primer; and 3) subjecting the reaction mixture to (i) a quantitative polymerase chain reaction (Q-PCR) with a polymerase that is a nucleic acid sample that is a nucleic acid sample comprising at least one template nucleic acid molecule comprising a target nucleic acid molecule, the primer pair having sequence complementarity with the at least one template nucleic acid molecule, the primer pair comprising a limiting primer and an excess primer, and wherein the reaction mixture further comprises at least one of the nucleic acid constructs of the disclosure. the limiting primer has sequence complementarity with and is capable of capturing the limiting primer; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal indicating that the limiting primer is bound to an individual probe of the plurality of probes; 4) using the detector array to detect at least one signal from the one or more addressable locations at multiple time points during the nucleic acid amplification reaction; and 5) detecting the target nucleic acid molecule based on the at least one signal indicating that the limiting primer is bound to an individual probe of the plurality of probes.

[0031] An embodiment of the present disclosure provides a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photo-cleavable moieties at a 5' end of the nucleic acid construct, wherein the 5' end of the nucleic acid construct is configured to be resistant to cleavage by an exonuclease, and each of the one or more photo-cleavable moieties is independently a) located on or attached to a nucleobase, b) located on or attached to a ribose, or c) a combination thereof.

[0032] In some embodiments of the aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of one or more photocleavable moieties, and the nucleic acid molecule is not resistant to cleavage by an exonuclease. In some embodiments of the aspects provided herein, the nucleic acid construct is configured to hybridize to a target nucleic acid molecule and remain resistant to cleavage by an exonuclease.

[0033] An aspect of the present disclosure provides a method of performing polymerase-catalyzed chain extension comprising: a) providing a reaction mixture comprising a nucleic acid construct of the present disclosure, a target nucleic acid molecule, a primer, and a polymerase, wherein the target nucleic acid molecule comprises a nucleic acid sequence complementary to the nucleic acid construct; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension of the primer using the target nucleic acid molecule as a template; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby performing polymerase-catalyzed chain extension through the nucleic acid sequence.

[0034] In some embodiments of the aspects provided herein, the providing step in b) does not activate the performing step in c). In some embodiments of the aspects provided herein, the nucleic acid construct remains intact in the reaction mixture before the irradiating step in c). In some embodiments of the aspects provided herein, the method further comprises cleaving one or more photocleavable moieties in c). In some embodiments of the aspects provided herein, the method further comprises forming a nucleic acid molecule in c). In some embodiments of the aspects provided herein, the performing step in c) comprises digesting the nucleic acid molecule formed after the irradiating step in c) with an exonuclease, and the polymerase is an exonuclease. In some embodiments of the aspects provided herein, the performing step in c) comprises extending a primer through the nucleic acid sequence after the irradiating step and / or after the digesting step.

[0035] An aspect of the disclosure is a method for performing polymerase-catalyzed chain extension using a nucleic acid construct, comprising the steps of: a) providing a reaction mixture comprising a nucleic acid construct, a template nucleic acid molecule, and a polymerase, wherein the nucleic acid construct comprises at least a first sequence located at or near its 3' end and a second sequence located at or near its 5' end, the first sequence being active in polymerase-catalyzed chain extension; and b) subjecting the reaction mixture to polymerase-catalyzed chain extension conditions, thereby performing polymerase-catalyzed chain extension. and c) performing a step of cleaving the nucleic acid construct with photons of light to produce a plurality of first amplicons comprising both the first sequence and the second sequence or a sequence complementary to both the first sequence and the second sequence; and b) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby cleaving the nucleic acid construct and producing a plurality of second amplicons comprising the first sequence or a sequence complementary to the first sequence, with the proviso that each of the plurality of second amplicons does not contain the second sequence or a sequence complementary to the second sequence.

[0036] An aspect of the present disclosure provides a method of performing polymerase-catalyzed chain extension using at least one of the nucleic acid constructs of the present disclosure, comprising the steps of: a) providing a reaction mixture comprising the nucleic acid construct, a template nucleic acid molecule, and a polymerase; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby performing polymerase-catalyzed chain extension, wherein the polymerase-catalyzed chain extension is PCR, RT-PCR, QPCR, or qRT-PCR.

[0037] In some embodiments of the aspects provided herein, at least one of the nucleic acid constructs is a primer for PCR, RT-PCR, QPCR, or qRT-PCR. In some embodiments of the aspects provided herein, at least one of the nucleic acid constructs is a solution-phase probe for PCR, RT-PCR, QPCR, or qRT-PCR. In some embodiments of the aspects provided herein, at least one of the nucleic acid constructs is a fixed probe for PCR, RT-PCR, QPCR, or qRT-PCR. In some embodiments of the aspects provided herein, at least one of the nucleic acid constructs is more than two nucleic acid constructs, which are a combination of a primer for PCR, RT-PCR, QPCR, or qRT-PCR, a solution-phase probe for PCR, RT-PCR, QPCR, or qRT-PCR, and a fixed probe for PCR, RT-PCR, QPCR, or qRT-PCR, each of which is independently selected.

[0038] An aspect of the present disclosure is an automated microarray system for quantifying microarray data, comprising: a) a solid support having a surface and a plurality of different probes, the plurality of different probes being immobilized on the surface; b) a fluid volume containing an analyte, the fluid volume being in contact with the solid support, and at least one of the plurality of different probes and the analyte comprising at least one of the nucleic acid constructs of the present disclosure; c) a detector or detection assembly configured to detect signals measured at a plurality of time points from each of a plurality of spots on the solid support while the fluid volume is in contact with the solid support, the signals being optical or electrochemical signals; and d) a computer configured to convert the signals into microarray data, the system further comprising instructions configured to cause the microarray data to be processed by the computer according to a processing method, the processing method comprising: 1) determining estimates of interactions between the plurality of different probes and the analyte, including by (i) analytical representation, and (ii) calibration of the microarray using at least one standard probe on the solid support; and 2) determining hybridization, cross-hybridization, and unbound transition probabilities between states.2) generating a probability matrix that utilizes estimates in a Markov chain model including modeling the probability of a signal being generated by a detector or detector assembly; 3) obtaining affinity-based array data using a detector or detector assembly; 4) applying the affinity-based array data to the probability matrix; 5) applying an optimization algorithm selected from the group consisting of a maximum likelihood estimation algorithm, a maximum a posteriori probability criterion, a constrained least squares calculation, and any combination thereof, that utilizes and does not suppress non-specific interactions by considering them as interference rather than noise; and 6) outputting the optimized affinity-based array data to a user, wherein the optimized affinity-based array data has an improved signal-to-noise ratio compared to affinity-based array data obtained by using a detector or detector assembly.

[0039] An embodiment of the present disclosure provides an integrated biosensor array comprising, in order, a molecular recognition layer comprising at least one of the nucleic acid constructs of the present disclosure, an optical layer, and a sensor layer integrated in a sandwich configuration, where a) the molecular recognition layer comprises a plurality of different probes attached to different independently addressable locations, each of the independently addressable locations configured to receive an excitation photon flux directly from a single source located on a single side of the molecular recognition layer, the molecular recognition layer transmits light to the optical layer, one of the plurality of different probes comprises at least one of the nucleic acid constructs, b) the optical layer comprises an optical filter layer, the optical layer transmits light from the molecular recognition layer to the sensor layer, whereby the transmitted light is filtered, and the sensor layer comprises an optical sensor array that detects the filtered light transmitted through the optical layer, the sensor layer comprises sensor elements fabricated using a CMOS fabrication process, and the molecular recognition layer, the optical layer, and the sensor layer constitute an integrated structure where the molecular layer is in contact with the optical layer and the optical layer is in contact with the sensor layer.

[0040] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0041] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which sets forth illustrative embodiments, and the accompanying drawings (also referred to herein as "figure" and "FIG."), in which the principles of the invention are utilized. [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 shows examples of photocleavable groups (LG=leaving group).

[0043] [Diagram 2] FIG. 2 shows an exemplary nucleic acid molecule that contains a photocleavable bond.

[0044] [Diagram 3] FIG. 3 shows examples of reagents having a photocleavable structure.

[0045] [Figure 4] FIG. 4 shows an example of a nucleic acid construct comprising a 3′-terminal elongation inhibitor.

[0046] [Diagram 5] FIG. 5 shows examples of 3′-terminal elongation inhibitor reagents.

[0047] [Figure 6] FIG. 6 shows an example of a nucleic acid construct comprising a 5′ terminal exonuclease inhibitor.

[0048] [Figure 7] FIG. 7 shows examples of 5' terminal exonuclease inhibitors.

[0049] [Figure 8] FIG. 8 shows an example of a nucleic acid construct comprising a photocleavable base pairing inhibitor.

[0050] [Figure 9] FIG. 9 shows examples of photocleavable base pairing inhibitor reagents.

[0051] [Figure 10] FIG. 10 shows an example of a photoinitiated primer.

[0052] [Figure 11] 11A to 11D show examples of phototerminating primers.

[0053] [Figure 12] 12A-12B show examples of photoinitiated hybridization probes.

[0054] [Figure 13] FIG. 13 shows an example of a photoquenchable hybridization probe.

[0055] [Figure 14] FIG. 14 shows a schematic of an example of a 5'-terminal exonuclease protectant.

[0056] [Figure 15] FIG. 15 shows a schematic of an example of light-activated nested PCR.

[0057] [Figure 16] FIG. 16 shows a schematic diagram of another example of light-activated nested PCR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0059] The present disclosure provides chemically modified light-triggered nucleic acid (NA) constructs that have unique properties such that when the constructs are triggered in a photochemical manner by photons of light, they can transform their chemical structure, thereby changing their chemical / biochemical functions. These light-triggered property changes of the chemically modified NA constructs can be utilized in molecular detection reactions / processes.

[0060] In some embodiments, the light-triggered NA constructs can be used in NA detection assays used in life science research and molecular diagnostics.In these assays, the NA molecule is the target of the assay and / or is used as the molecular recognition element of the assay.The light-triggered NA constructs are added to the assay so that by appropriately applying photons of light to the system, the light-triggered NA constructs can improve assay detection accuracy and / or reduce the complexity of the workflow and / or reduce the required time.Other advantages are also possible.

[0061] Some exemplary detection assays are NA amplification tests (NAATs) that use a polymerase chain reaction process, NA affinity-based detection systems that utilize two-dimensional and addressable DNA microarrays, and DNA sequencing arrays that incorporate solid-phase sequencing by synthesis (SBS) methods. Light-triggered nucleic acid constructs and their use in manipulations - Patents.com

[0062] The term "light-triggered nucleic acid construct" or "NA construct" as used herein generally refers to a NA molecule that includes 1) one or more photosensitive systems or photosensitive chemical moieties that may exist in a first molecular state prior to exposure to a photon of light, and 2) one or more DNA or RNA molecules covalently or non-covalently linked to the one or more photosensitive systems or photosensitive chemical moieties. When a photon of light is applied to the one or more photosensitive systems or chemical moieties in the nucleic acid construct, the one or more photosensitive systems or photosensitive chemical moieties change from a first molecular state to a second molecular state, thereby changing the biochemical properties of the NA construct. For example, the photon of light can cause a chemical change in the NA construct by breaking or creating a chemical bond(s) of the one or more photosensitive systems or photosensitive chemical moieties.

[0063] The NA construct may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 NA molecules. The NA construct contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 NA molecules. The NA construct may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or less NA molecules.

[0064] The term "photosensitive system" or "photosensitive chemical moiety", as used herein, generally refers to a single or various chemical structures that contain a photolabile chemical bond(s). The photosensitive system or photosensitive chemical moiety can absorb wavelength-specific photons to increase the reaction rate of certain chemical reactions in which the photosensitive system or photosensitive chemical moiety may participate. Other descriptive terms such as light-sensitive, light-cleavable, light-activatable, photolabile, photoactivatable, or photocleavable may also be used interchangeably with the term photosensitive.

[0065] The term "molecular state," as used herein, generally refers to the atomic and molecular structure, and chemical, physicochemical, biochemical, electrochemical, and photochemical properties associated with one or more particular molecules, such as, for example, NA constructs.

[0066] The term "biochemical properties" as used herein generally refers to the characteristics of the NA construct in biological and chemical reactions. The biochemical properties of a nucleic acid construct may change depending on the molecular state of the NA construct. The molecular state of the NA construct may change due to the reaction of one or more photosensitive systems or photosensitive chemical moieties. In addition, the biochemical properties of the NA construct in a first molecular state may be different from those in a second molecular state.

[0067] In some embodiments, the first molecular state is an inactive molecular state of the NA construct, while the second molecular state is an active molecular state of the NA construct. In some embodiments, the first molecular state is an active molecular state of the NA construct, while the second molecular state is an inactive molecular state of the NA construct.

[0068] Each NA construct may have different biochemical properties, including different reactivity in biochemical reactions. Examples of biochemical properties may include, for example, whether the NA construct can promote, block, or participate in a particular biochemical reaction, such as a polymerase chain reaction or hybridization reaction. Different biochemical properties may be triggered by photons of light.

[0069] The biochemical properties of the NA construct may include different molecular states of the NA construct. For example, the biochemical properties of the NA construct in a first molecular state may differ from the biochemical properties of the NA construct in a second molecular state. The biochemical properties of the NA construct in the first and second molecular states may be designed such that a photon of light can initiate and / or stop a specific molecular reaction in which the NA construct may participate. Such a change in molecular state may be triggered by a photon of light. An example of a biochemical property of a primer may be an active primer or an inactive primer, etc. In some embodiments, the present disclosure describes a method and system for toggling a primer between "active" and "inactive" molecular states in an extension reaction by a photon of light. In some embodiments, the switching of the active / inactive molecular states may be actuated by cleaving a photocleavable bond in the nucleic acid construct. In this disclosure, the terms "latent", "inactivated", "inert" and "non-functional" are synonymous with the term "inactive". Similar terminology is used in describing a "probe."

[0070] The NA construct is typically present in a reaction chamber, to which photons of light can be applied by a light source system.

[0071] 1. Photosensitive systems or photosensitive chemical moieties A photosensitive system or photosensitive chemical moiety may be a single or multiple chemical structure that contains a photolabile chemical bond(s). A photosensitive system or photosensitive chemical moiety may change its structure or chemical propertied when irradiated with a photon of light. A photosensitive system or photosensitive chemical moiety may absorb wavelength-specific photons to increase the reaction rate of certain chemical reactions that the photosensitive system or photosensitive chemical moiety may facilitate or participate in. For example, these chemical reactions include: -changing the chemical structure of the photosensitive system or of the photosensitive chemical moiety; Photosensitive systems, breaking the structure of the photosensitive chemical moiety into multiple smaller structures; Adding an external chemical structure to the photosensitive system, or forming one or more intramolecular bonds within a photosensitive system or photosensitive chemical moiety; forming one or more intermolecular bonds between two or more photosensitive systems or photosensitive chemical moieties or external chemical structures (relative to the photosensitive systems and photosensitive chemical moieties); or A combination of these can be done.

[0072] In some embodiments, the photosensitive system or photosensitive chemical moiety may be incorporated into the structure of a nucleic acid molecule. For example, the photosensitive system or photosensitive chemical moiety may be The functional group(s) of the NA, e.g., located at the heteroatom of the nucleobase or at the 3′-OH of the ribose ring, as part of a linker group between two NA sequences, where in the presence of a photon of light, the linker group breaks into smaller groups, thereby separating the two previously linked NA sequences into two independent nucleic acid sequences (i.e., no longer linked); or - placed at the 5' end of the NA chain, the presence of a photosensitive system or a photosensitive chemical moiety prevents certain biochemical reactions from occurring at the 5' end of the nucleic acid chain, e.g., at the photolabile group on the 5' phosphate group of the terminal NA; located at the 3'-end of the NA chain, the presence of a photosensitive system or a photosensitive chemical moiety prevents certain biochemical reactions from taking place at the 3'-end of the NA chain, for example at the photolabile group of the 3'-OH group of the terminal NA, or ·It may be a combination of these.

[0073] Some examples of photosensitive chemical moieties can be found in Mayer, G. and Heckel, A., "Biologically active molecules with a 'light switch'," Angew. Chem., Int. Ed., 2006; 45(30), pp.4900-4921, which is incorporated herein by reference in its entirety. Some examples of photosensitive chemical moieties can include an ortho-nitrobenzyloxy linker, an ortho-nitrobenzylamino linker, an alpha-substituted ortho-nitrobenzyl linker, an ortho-nitroveratrol linker, a phenacyl linker, a para-alkoxyphenacyl linker, a benzoin linker, or a pivaloyl linker. See RJT Mikkelsen, "Photolabile Linkers for Solid-phase Synthesis," ACS Comb. Sci. 2018; 20(7):377-399; S. Peukert and B. Giese, "The Pivaloylglycol Anchor Group: A New Platform for a Photolabile Linker in Solid-Phase Synthesis," J. Org. Chem. 1998, 63(24): 9045-9051, each of which is incorporated by reference in its entirety.

[0074] For example, a nitrobenzyl-based chemical moiety can be, for example, as shown below: [ka]

[0075] Nitrobenzyl-based chemical moieties can undergo a Norrish Type II mechanism upon incident photons to yield the cleaved products shown below. [ka]

[0076] Some examples of photocleavable groups can be found in Figure 1. LG refers to the leaving group in Figure 1. Some examples include 4-methoxy-7-nitroindolinyl (MNI), I-nitrobenzyl (O-NB), 3-(4,5-dimethoxy-2-nitrophenyl) 2-butyl (DMNPB) and 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI), among others.

[0077] 2. Molecular state The term "molecular state", as used herein, generally refers to the atomic and molecular structure, as well as the chemical, physicochemical, biochemical, electrochemical, and photochemical properties associated with one or more specific molecules, such as, for example, NA constructs. For example, an NA construct may exhibit its molecular state in a given aqueous environment or under other reaction conditions of nucleic acid in the presence of other molecules. The molecular state of an NA construct may include the tendency of the NA construct to undergo certain reactions, such as, for example, ligation, coupling reaction, chain extension, chain digestion, etc.

[0078] The biochemical properties of the NA construct may include different molecular states of the NA construct. For example, the biochemical properties of the NA construct in a first molecular state may be different from the biochemical properties of the NA construct in a second molecular state. The biochemical properties of the NA construct in the first and second molecular states may be designed such that a photon of light can initiate and / or stop a specific molecular reaction in which the NA construct may participate. Such a change in molecular state may be triggered by a photon of light. For example, a photochemical reaction may change the molecular structure of a nucleic acid reagent, thereby changing the biochemical properties and reactivity of the nucleic acid reagent in a biochemical reaction.

[0079] For example, the NA construct may be an "active primer", which is a primer in the traditional PCR sense that can support nucleotide addition (i.e., extension of the growing chain) facilitated by a polymerase enzyme. In other words, an active primer may be capable of base-pairing with a complementary template sequence to form an antiparallel duplex structure under experimental conditions, and has a natural (available) 3'-hydroxyl group to which the polymerase enzyme can add another nucleotide, thus extending the primer by at least one base. An "inactive primer" may be a primer that cannot support or promote the addition of a nucleotide, either due to its inability to properly bind to the template strand (inability to base-pair) or due to the absence of an available 3'-hydroxyl group of the terminal nucleotide. For example, the polymerase reaction can be blocked by placing a photocleavable chemical moiety on the 3'-hydroxyl group of the terminal nucleotide. Upon exposure to light, the photocleavable chemical moiety on the 3'-hydroxyl group can be removed, and the resulting free 3'-hydroxyl group can be available for extension of the growing chain. A similar mechanism can be applied in ligase-catalyzed reactions in terms of blocking and deblocking ligation sites on NA. Another example of a base pairing inhibitor can be a chemical group that is placed on at least one strand of DNA (e.g., a growing strand) to prevent the DNA strand from binding to its complementary strand due to steric or other chemical reasons.

[0080] In some embodiments, the present disclosure describes methods and systems for toggling primers between "active" and "inactive" molecular states by photons of light in an extension reaction. By changing the molecular state of the primers, the present disclosure can enable novel amplification strategies, especially for "closed tube" methods (i.e., no additional reagents added after the PCR reaction has begun) and "multiplex" methods, which are highly desirable in the field of NA amplification-based diagnostics. The present disclosure describes a method to effectively change the composition (and properties, e.g., molecular state) of a primer set during an amplification reaction without adding or removing reagents or changing the reaction chamber between reactions. Thus, the "inactive" molecular state describes the status and functional state of a particular primer, but not its use. An inactive primer can become active upon exposure to light, and vice versa. The following examples show individual components for simplicity and demonstration, however, some complex multiplex assays may require up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 primers, or even more. The switching of the active / inactive molecular states can be triggered by exposure to the same light or to different lights. For example, one photocleavable chemical moiety may react with one wavelength of light, while another photocleavable chemical moiety may react with a different wavelength of light.

[0081] In some embodiments, the switching of the active / inactive molecular state can be actuated by cleaving a photocleavable bond in the NA construct, thereby cutting the original nucleic acid strand into portions. In some embodiments, the switching of the active / inactive molecular state is actuated by cleaving a photocleavable bond in the NA construct, thereby removing a blocking group from a certain nucleic acid unit of the NA construct. For example, when exposed to light, the blocking group on the base pairing inhibitor can be removed, and the NA sequence of the NA construct remains intact (i.e., the length and identity of the sequence of the NA construct remain the same before and after removal of the blocking group).

[0082] In this disclosure, the terms "latent," "inactivated," "inert," and "non-functional" are synonymous with the term "inactive." Similar terminology is used when describing "probes" that are associated with signal transduction and do not participate in polymerase-catalyzed extension, such as PCR.

[0083] 3.Biochemical properties The term "biochemical property" as used herein generally refers to the characteristics of the NA construct in biological and chemical reactions, including, for example, the tendency or ability of the NA construct to participate in a certain biological or chemical reaction. In addition, the biochemical property of the NA construct in a first molecular state may be different from that in a second molecular state. One example of such a biochemical property may be the ability of the NA construct to start or stop a molecular reaction after irradiation with a photon of light. For example, biochemical properties include: the NA construct in single-stranded form base-pairs with itself to form a hairpin structure or forms a homodimer with another copy of the NA construct or a heterodimer with another NA molecule; A DNA polymerase enzyme extends the NA construct using the template NA. An RNA polymerase enzyme extends the NA construct using the template NA. A reverse transcriptase extends the NA construct using the template NA. A terminal transferase enzyme extends the NA construct. An exonuclease enzyme digests the NA construct. - An endonuclease enzyme breaks the NA construct, A restriction enzyme breaks the NA construct at specific coordinates within its sequence, and A ligase enzyme uses the NA construct as a substrate or template These include, but are not limited to, abilities.

[0084] The biochemical properties of the nucleic acid construct may change depending on the molecular state of the NA construct. The molecular state of the NA construct may change due to the reaction of one or more photosensitive systems or photosensitive chemical moieties.

[0085] 4. Reaction Chamber The term "reaction chamber" as used herein generally refers to a physical system that contains an aqueous solution or other medium and in which the NA construct resides. The reaction chamber may allow photons of light to reach the NA construct present therein and may have temperature control to set and dynamically change the temperature within the chamber, for example, the temperature of the aqueous solution.

[0086] In some embodiments, the reaction chamber may have a volume ranging from about 0.1 nanoliters (nL) to about 10 milliliters (mL). In some cases, the reaction chamber may have a volume ranging from about 1 microliter (μL) to about 100 μL. In some embodiments, the reaction chamber is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 nL. In some embodiments, the reaction chamber is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μL. In some embodiments, the reaction chamber is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL.

[0087] The reaction chamber may have a temperature ranging from about 4° C. to about 100° C. The temperature of the reaction chamber may be controlled with an accuracy of about ±0.01° C., ±0.02° C., ±0.03° C., ±0.04° C., ±0.05° C., ±0.06° C., ±0.07° C., 0.08° C., ±0.09° C., ±0.1° C., ±0.2° C., ±0.3° C., or ±0.4° C. In some embodiments, the temperature of the reaction chamber may range from about 30° C. to about 95° C., and the accuracy of controlling the temperature may be controlled within ±0.1° C.

[0088] 5.Light The term "light", as used herein in reference to a reaction chamber, generally refers to a photon flux, limited within a particular wavelength, applied to the reaction chamber for a duration. The wavelength of the light can be from about 200 nanometers (nm) to about 2000 nm. In some embodiments, the wavelength of the light can be from about 200 nm to about 400 nm, from about 300 nm to about 500 nm, or from about 400 nm to about 600 nm. In some embodiments, the total light intensity of the light is about 0.001 mW / cm 2 ~Approx. 1,000mW / cm 2 , about 0.01mW / cm 2 ~about 100mW / cm 2 , about 0.1mW / cm 2 ~about 10mW / cm 2 , about 0.05mW / cm 2 ~about 20mW / cm 2 , or about 0.02 mW / cm 2 ~about 50mW / cm 2 The duration of exposure to light may be from about 0.1 seconds to about 10,000 seconds, from 0.25 seconds to about 5,000 seconds, from about 0.5 seconds to about 1,000 seconds, from about 0.75 seconds to about 500 seconds, or from about 1 second to about 100 seconds.

[0089] 6.Light source The term "light source system", as used herein, generally refers to a combination of devices that cooperatively generate photons of light within a predetermined wavelength and control the power applied to a nucleic acid construct. A light source system may include a photon source, which may be a light emitting diode (LED), a laser source, an incandescent lamp, or a gas discharge lamp. A light source system may include a power control device for controlling the light output power. A light source system may include a wavelength selective optical filter to ensure that its output light is within the desired wavelength. A light source system may include an optical device for concentrating and / or collimating its output photon flux.

[0090] Modification of nucleic acids to activate photosensitivity A variety of methods can be used to create NA molecules or structures with photochemical properties. For example, the method can include the use of solid support phosphoramidite chemistry. The method can include synthesizing or growing a nucleic acid sequence on a solid support to a position where modification may be desired. A special phosphoramidite can then be coupled to the growing nucleic acid molecule at the modification position. The modified nucleic acid molecule may or may not be extended after modification. Once the reaction is complete, the nucleic acid molecule can then be cleaved from the solid support. The cleaved nucleic acid molecule may or may not be subjected to additional reactions or treatments (e.g., purification, modification, etc.).

[0091] Examples of photosensitive systems or photosensitive chemical moieties can be the photocleavable group of a part of a nucleotide (either between the ribose part or the nucleic acid base part or the chemical part of a nucleic acid) or the linker part between two single-stranded nucleic acids, as described above. The linker can have two photocleavable bonds, each of which is connected to a nucleic acid segment. There can be many types of modifications of nucleic acids that can activate photosensitivity, as shown elsewhere in this disclosure. Below are some specific examples.

[0092] 1. Photocleavable structure FIG. 2 shows an exemplary NA molecule containing a photocleavable bond. In a photocleavable NA structure, two nucleic acid fragments can be linked together by a photosensitive system or a photosensitive chemical moiety, which can contain one or more photocleavable bonds. When a photocleavable NA structure molecule is exposed to light from a light source, the molecule can be cut into two or more segments due to the presence of a photocleavable bond. As a result, the original NA sequence (A) can be broken into two smaller pieces, for example, sequence (A1) and sequence (A2), as shown in FIG. 2.

[0093] In some embodiments, the photosensitive system may be designed such that after cleavage, the cleaved chemical residue remains at the 3' end of the released sequence (A1) and / or the 5' end of sequence (A2). Sequence (A) may be a single-stranded or double-stranded NA. When sequence (A) is a double-stranded NA, there may be at least one photocleavable bond in each strand. In some embodiments, the position of the photocleavable bond may be adjacent to the same paired NA, so that the cleavage may produce a blunt end in each of sequence (A1) and sequence (A2). In some embodiments, the position of the photocleavable bond may be staggered in each strand, so that after the cleavage, sequence (A1) and sequence (A2) may have a sticky end (overhang).

[0094] An exemplary compound with photocleavable bond(s) is shown in FIG. 3. As shown in FIG. 3, this compound can be used with other DMT phosphoramidite-containing monomers in the chemical nucleic acid molecule synthesis of NA constructs to insert photocleavable bond(s) into the chain of NA. In the example shown in FIG. 3, an O-nitrobenzyl photolabile blocking group can link two segments of the NA molecule. Without irradiation, the photolabile bond is intact in the NA construct. The intact NA construct can exhibit molecular state 1 of the biochemical properties of the NA construct. Then, upon exposure to a light source, the NA construct molecule can be cleaved into two separate nucleic acid fragments, resulting in two newly formed NA fragments with 3'-hydroxyl and 5'-phosphorylated ends, respectively. Due to the rupture of the photolabile bond(s), the molecular state of the original NA construct can change to a new molecular state associated with the two nucleic acid fragments. This is an example of a molecular state change triggered by light.

[0095] 2. 3'-Terminal Elongation Inhibitors In NA constructs containing 3'-end extension inhibitors, a photosensitive system or photosensitive chemical moiety can be chemically attached to the 3'-end unit of the NA sequence. Due to the presence of the 3'-end extension inhibitor, the 3' extension site is blocked from extension enzymes, including but not limited to polymerases, transcriptases, and terminal transferases, and as a result, the enzymes cannot extend the growing chain from the 3'-end terminal unit, and the extension of the growing chain by the enzymes is inhibited. However, exposure to light can remove the blockage and allow the enzymes to extend the growing chain. An example of an NA construct is shown in Figure 4, where the chemical reaction promoted by DNA polymerase is initially blocked by the presence of a photosensitive system or photosensitive chemical moiety at the 3'-end of the primer. Exposure to light can then remove the blocking group and allow the enzymes to synthesize primed DNA using the template. In this case, light can be directed at the molecule and the polymerase inhibitor can be removed from the molecule, allowing the molecule to be extended by the polymerase.

[0096] An example of a 3'-terminal terminal unit that can be inserted into a 3'-terminal polymerase extension inhibitor is shown in Figure 5. The DMT phosphoramidite monomer and this 3'-terminal terminal unit can be used in the chemical synthesis of nucleic acid molecules (oligonucleotides). When the 3'-terminal terminal unit is introduced at the 3'-terminus, in the absence of exposure to light, the O-nitrobenzyl photolabile blocking group on the 3' hydroxyl group of the ribose ring of the 3'-terminal terminal unit can prevent extension at that position by DNA polymerase. Exposure to light can remove the blocking group, revealing the bare 3' hydroxyl group on the ribose ring, restoring the extension ability of the NA construct.

[0097] 3. 5'-end exonuclease protectors In NA constructs containing 5'-terminal exonuclease protectors, a photosensitive system or photosensitive chemical moiety can be chemically attached to the terminal unit at the 5'-end of the nucleic acid sequence. Due to the presence of the 5'-terminal exonuclease protector, the 5'-end digestion of the strand by the exonuclease enzyme can be blocked, and the strand is protected from cleavage or digestion. Exposure to light can remove the blockage and allow 5' to 3' digestion of the strand. For example, such a nucleic acid construct is shown in FIG. 6, where the activity of the DNA polymerase 5'-terminal exonuclease can be initially blocked, and exposure to light and removal of the 5'-terminal blocking group can restore the activity and allow the enzyme to digest the strand as shown in FIG. 6. An example of a photosensitive system or photosensitive chemical moiety that can be used as a 5'-terminal exonuclease protector is shown in FIG. 7. The hydrophobic tail on the 5' phosphodiester of the nucleotide can block digestion of nucleic acids containing the nucleotide at the 5' terminus by exonucleases. Exposure of the nucleic acid to light can remove the blocking group on the 5'-phosphate, allowing digestion by exonucleases at the 5' terminal nucleotide.

[0098] 4. Base pairing inhibitors In NA constructs containing base pairing inhibitors, a photosensitive system or photosensitive chemical moiety can be chemically attached to one or more nucleic acid bases of the nucleotide units in the NA construct. The base pairing inhibitors can be in tandem or distributed within the nucleic acid sequence. The presence of the base pairing inhibitor can inhibit base pairing of the sequence complementary to the NA construct. Subsequent exposure to light can remove the blocking group, allowing normal base pairing to occur between the deblocked NA construct and the complementary sequence. An example of such a NA construct is shown in FIG. 8, which shows an exemplary NA molecule containing a photosensitive base pairing inhibitor. When the NA molecule is not exposed to a light source, at least a subunit of the NA molecule lacks base pairing ability due to the presence of the base pairing inhibitor. Such base pairing ability can be restored by subjecting the nucleic acid molecule to a light source for a given period of time (e.g., about 1 minute or more, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or more). Alternatively, the nucleic acid molecule can be subject to the light source until photolysis is complete.

[0099] Various compounds, such as those shown in Figure 9, can be used as photosensitive base pairing inhibitors. The reagents shown in Figure 9 and other DMT phosphoramidite monomers can be used in chemical NA molecule synthesis. When introduced, O-nitrobenzyl photolabile blocking groups can be used to prevent Watson-Crick base pairing due to steric hindrance and / or lack of hydrogen bonding. Upon exposure to a light source (e.g., UV light), the blocking groups (shown on the nucleic acid bases) can be removed to restore the base pairing ability of the nucleic acid molecule. Photolabile base pairing inhibitors comprising photocleavable chemical moieties on the nucleobases, such as the compounds shown in FIG. 9 (or other similar compounds having different nucleobases with photocleavable chemical moieties attached to heteroatoms on the nucleobases, e.g., nitrogen or oxygen), can be made according to H. Lusic, et al., "Photochemical DNA Activation," Org. Lett., 2007, 9(10): 1903-1906, U.S. Publication No. 2010 / 0099159, each of which is incorporated herein by reference in its entirety.

[0100] Using the different chemical modifications to the nucleic acids disclosed above, different types of NA constructs can be constructed for different applications, as shown below.

[0101] Types of light-triggered nucleic acid constructs and their uses Also provided herein are NA constructs with unique biochemical properties related to molecular detection that can be triggered when the NA molecule is exposed to a photon of light. These NA constructs, while used in the reaction chamber, can enhance or decrease the speed, specificity, yield, and fidelity of biochemical reactions used in common molecular detection assays. Exemplary reactions are polymerase chain reaction (PCR), polymerase-catalyzed chain extension, reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, and restriction digestion, among others. When a reaction is composed of NA components that function as targets and / or reagents and / or catalysts and / or other, the present disclosure can be used to moderate the reaction by replacing the original components with NA constructs or inserting NA constructs into the original components. Examples of nucleic acid molecules or structures with photochemical properties include, but are not limited to, primers, oligonucleotides, polynucleotides, oligonucleotide-containing molecules, nucleotides, or nucleic acid probes. Nucleic acid probes can include hybridization probes that can selectively interact with target analytes (e.g., amplicons) during or at the end of a given reaction (e.g., PCR or RT-PCR). As shown below, many different types of nucleic acid constructs can exist.

[0102] 1. Light-initiated primer Light-initiated primer is an NA sequence that cannot base-pair with complementary NA sequence template and / or cannot create the start site of nucleic acid synthesis enzyme due to the presence of photosensitive system or photosensitive chemical moiety, or the blocking group that contains or is connected to photosensitive system or photosensitive chemical moiety.When light is applied, these photo-initiated primers can remove the blocking group, and then in the presence of nucleic acid template and nucleic acid synthesis enzyme, start nucleic acid synthesis.

[0103] FIG. 10 shows examples of light-initiated primers and their applications in biochemical processes, and their corresponding exemplary sequences are listed in Table I. In one embodiment, the primers may contain an internal photocleavable bond modification in the linear NA construct (e.g., primer), where the light-initiated primer is designed with a photocleavable modification so that exposure to light can remove the blocking strand and the resulting primer can form a suitable initiation site for the polymerase to act (FIG. 10, top panel). In another embodiment, the primers may contain a polymerase blocker at the 3' end of the nucleic acid construct (e.g., primer), where the light-initiated primer is designed with a 3' end extension inhibitor modification so that application of light can remove the inhibition, thereby creating a suitable initiation site for the polymerase to act (FIG. 10, second panel from the top). In one embodiment, the primer may contain one or more base pairing inhibitors distributed within the sequence of the NA construct (e.g., primer), where the photoinitiated primer is initially designed with base pairing inhibitor modifications such that the primer containing the base pairing inhibitor cannot hybridize to the template or form a start site for the polymerase. Upon exposure to light, the inhibition can be removed and the resulting primer can create a suitable start site for the polymerase to act (Figure 10, third panel from the top). In another embodiment, the primer may contain a cleavable bond within the hairpin structure of the nucleic acid (e.g., hairpin primer), where the photoinitiated primer is designed with a photocleavable hairpin monomer structure. Initially, the 3'-end region of the primer may not be available for base pairing due to the presence of the hairpin. Upon exposure to light, the hairpin may be destroyed and the resulting primer may be available for extension (Figure 10, bottom panel). In some embodiments, the primer may not be active (i.e., in an inactive molecular state) prior to exposure to a light source due to the presence of a light-sensitive system or light-sensitive chemical moieties on the nucleic acid construct.However, when the light-initiated primer is subjected to a light source, the light from the light source may remove some or all of the inhibitors / blockers or cleave the cleavable bonds contained in the light-initiated primer, thereby restoring the ability of the primer to an active molecular state.

[0104] [Table 1]

[0105] 2. Photo-terminating primer Photo-terminated primers are NA constructs that can act as the initiation site of polymerase and promote NA synthesis in the presence of nucleic acid template. When light is applied, these photo-terminated primers can be inactive and cannot initiate further NA synthesis. Photo-terminated primers can be active before exposure to light, but can be inactive after being subjected to a light source. Figures 11A-11D show examples of photo-terminated primers and their applications, and their corresponding exemplary sequences are listed in Table II.

[0106] 11A and 11B show examples of photo-terminated cooperative primers that contain photocleavable modifications. By applying light, the NA construct can be broken, and then priming can become thermodynamically unfavorable. The primer can contain a cleavable bond between the two segments of the primer, and the two linked segments of the primer can be required to hybridize to the same template to form a stable primer-template heterodimer. The primer can be active in an enzymatic reaction before exposure to light. After exposure to light, the two segments of the cooperative primer can be separated and inactive due to the cleavage of the cleavable bond, because it can become thermodynamically unfavorable for the primer-template heterodimer of each segment to bind only one segment to the template.

[0107] Figures 11C and 11D show examples of photo-stop hybridization primers. In Figure 11C, photo-stop primers are designed with photocleavable modifications so that applying light can break the primer into separate parts, which can then reduce the strength of base pairing of the converted primer-template heterodimer, so that priming becomes thermodynamically unfavorable and the primer-template heterodimer can be broken. In Figure 11D, photo-stop primers are designed using base pairing inhibitor modifications distributed in the primer sequence. Applying light can remove inhibition, which can then create a stable hairpin structure of the converted primer. Since the converted primer forms a hairpin structure, the primer-template heterodimer can be broken because it becomes thermodynamically unfavorable for intermolecular hybridization compared to intramolecular hybridization of the hairpin structure.

[0108] [Table 2]

[0109] 3. Light-initiated hybridization probes Light-initiated hybridization probes are NA constructs that can specifically identify and base-pair with their complementary sequences only after light is applied.Before that, light-initiated hybridization probes are inactive and cannot hybridize with their complementary sequences.Examples of light-initiated hybridization probes are shown in Figures 12A and 12B, and their corresponding exemplary sequences are listed in Table III.

[0110] In Figure 12A, the photoinitiated hybridization probe is designed with base pairing inhibitor modification.Initially, the photoinitiated hybridization probe containing base pairing inhibitor cannot hybridize to its complementary sequence.By applying light, the inhibition of hybridization can be removed due to the removal of base pairing inhibitor, and base pairing can be allowed, so that the converted photoinitiated hybridization probe can hybridize to its complementary sequence.

[0111] In Figure 12B, the photoinitiated hybridization probe is designed to include a photocleavable hairpin monomer structure. Initially, it is thermodynamically unfavorable for the probe to base pair to its complementary sequence due to the presence of the hairpin monomer with intramolecular hybridization. By applying light, the hairpin is destroyed by cutting the probe into two separate parts, and the converted probe can be available for base pairing and hybridization to its complementary sequence.

[0112] [Table 3]

[0113] 4. Photo-quenchable hybridization probes Photo-terminated hybridization probes are nucleic acid constructs that can specifically identify and base pair with their complementary sequences. However, upon exposure to light, they can become inactive and no longer be able to hybridize to their complementary sequences.

[0114] Examples of photoquenched hybridization probe structures are shown in FIG. 13, and their corresponding exemplary sequences are listed in Table IV.

[0115] In the top panel of FIG. 13, the photo-stop hybridization probe is designed to include a photocleavable modification that links two segments of the photo-stop hybridization probe. Before exposure to light, both segments of the photo-stop hybridization probe are hybridized to the target NA, and therefore remain in an active molecular state. Upon exposure to light, the photo-stop hybridization probe breaks the photocleavable bond, which can result in two unlinked segments of the photo-stop nucleic acid probe, and probe-template heterodimer formation can be thermodynamically unfavorable. For example, at least one segment can be designed to have a non-complementary sequence to the target nucleic acid, which can result in a change in signal when it is not hybridized to the target nucleic acid or is separated from other segments of the photo-stop hybridization probe. At least one segment of the converted photo-stop hybridization probe can be in an inactive molecular state.

[0116] In the bottom panel of FIG. 13, the photo-stopped hybridization probe is designed to include one or more base pairing inhibitors. Before exposure to light, the presence of base pairing inhibitors can prevent the self-base pairing to form hairpin structure in the photo-stopped hybridization probe. Instead, the photo-stopped hybridization probe hybridizes to the target nucleic acid, thereby remaining in an active molecular state. Upon exposure to light, the photo-stopped hybridization probe can remove base pairing inhibition and create a stable hairpin structure of at least one segment of the converted photo-stopped hybridization probe, thereby making probe-template heterodimer formation thermodynamically unfavorable. The hairpin structure of the photo-stopped hybridization probe can be in an inactive molecular state.

[0117] [Table 4]

[0118] 5. Light-initiated 5'-end exonuclease probes A light-initiated 5'-end exonuclease probe is a NA construct that contains a 5'-end exonuclease protector modification that can be removed by light. The 5'-end exonuclease protector can be a light-sensitive system or a light-sensitive chemical moiety that is chemically attached to the terminal unit at the 5' end of the NA sequence. Due to the presence of the 5'-end exonuclease protector, the 5'-end digestion of the nucleic acid strand by the exonuclease enzyme can be blocked, and the nucleic acid strand is protected from cleavage or digestion. The light-initiated 5'-end exonuclease probe is in an inactive molecular state. Exposure to light can remove the 5'-end exonuclease protector, and 5' to 3' strand digestion can be promoted. For example, such a nucleic acid construct is shown in FIG. 14, where the DNA polymerase 5'-end exonuclease is initially blocked, and exposure to light can remove the blocking group, allowing the enzyme to digest the strand.

[0119] In general, the heteroatoms, 3'-OH, 5'-OH, and phosphate groups (either at the 3' or 5' positions) on the nucleobases can be linked to photolabile chemical moieties, such as any one of those shown in Figure 1. Photocleavable linkers can have one or more photolabile chemical moieties attached to the ends of the linker such that upon exposure to light, the one or more photolabile chemical moieties can be cleaved from the nucleic acid fragment to which they are attached. Different photocleavable chemical moieties can be used in different ways. EXAMPLES

[0120] Embodiments using light-triggered NA constructs Example 1 Photoinitiated PCR In this example, a light-initiated primer pair is used in a PCR assay, as shown in FIG. 15. As shown in FIG. 15, PCR and primer extension are initiated after light is applied, but not before light is applied. Prior to exposure to light, no polymerization or exponential amplification can occur, since the primer is inactive due to the presence of a 3'-end extension inhibitor (i.e., a polymerase inhibitor at the 3' end of the primer). The advantage of this method may be that it can reduce the presence of undesired products and / or primer dimers due to non-specific DNA amplification at room temperature (or lower temperature), for example, during introduction of the sample into the reaction or other pretreatment steps. Exposure to light can remove the 3'-end extension inhibitor, and the primer can become active in polymerase-catalyzed extension (i.e., extension, elongation of the growing strand).

[0121] This method, which may hereafter be referred to as "light-initiated PCR", may be an alternative to other PCR methods, such as hot-start PCR, which activates the amplification process by heating at high temperatures. Sharkey DJ, Scalice ER, Christy KG, Atwood SM, Daiss JL, "Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction See Bio / Technology," 1994, 12(5): 506-9; N. Paul, J. Shum, T. Le, "Hot start PCR," Methods in Molecular Biology, Humana Press, 2010, 630: 301-18. Thus, light-initiated PCR may not include reagents and molecules that act as thermolabile switches.

[0122] In some embodiments of the invention, both light-initiated and hot-start PCR methods can be used to better ensure that amplification remains inactive at low temperatures and prior to PCR.

[0123] In some embodiments of the present invention, light-initiated PCR is included in a quantitative PCR (Q-PCR) system. In some embodiments, a method using light-initiated PCR is a Q-PCR method comprising: (a) performing nucleic acid amplification on two or more nucleotide sequences in the presence of one light-initiated primer to produce two or more amplicons in a fluid; (b) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable positions, the array being configured to contact the fluid; and (c) measuring hybridization of the amplicons to the two or more nucleic acid probes while the fluid is contacting the array to obtain amplicon hybridization measurements, the amplicons comprising a quencher. In some embodiments, the amplicons are created using primers comprising light primers, the primers comprising a quencher. In some embodiments, one of the primers in a primer pair comprises a quencher. In some embodiments, both primers in a primer pair comprise a quencher. In some embodiments, the quencher is incorporated into the amplicons as they are formed. In some embodiments, the amplicon is generated using deoxynucleotide triphosphates (d-NTPs), and one or more of the d-NTPs used to generate the amplicon include a quencher. In some embodiments, the amplicon hybridization measurement is performed by measuring fluorescence from a fluorescent moiety attached to a solid surface. In some embodiments, the fluorescent moiety is covalently attached to the nucleic acid probe. In some embodiments, the fluorescent moiety is attached to a substrate and is not covalently attached to the nucleic acid probe. In some embodiments, the amplicon includes a quencher, and the step of measuring hybridization is performed by measuring a decrease in fluorescence due to hybridization of the amplicon to the nucleic acid probe.

[0124] In some embodiments, the method of using light-initiated PCR is a Q-PCR method comprising: (a) providing an array comprising a solid support having a surface and a plurality of different probes, the different probes being immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; (b) performing PCR amplification on a sample comprising a plurality of nucleotide sequences, the PCR amplification being performed in a fluid, (i) the PCR primer for each nucleic acid sequence is a light-initiated primer and comprises a quencher; (ii) the fluid is in contact with the probes, thereby allowing the amplified molecules to hybridize with the probes, thereby quenching the signal from the fluorescent moiety; (c) detecting the signal from the fluorescent moiety at the addressable locations over time; (d) using the signal detected over time to determine the amount of the amplified molecules in the fluid; and (e) using the amount of the amplified molecules in the fluid to determine the amount of the nucleotide sequences in the sample. In some embodiments, the step of determining the amount of the amplified molecules is performed during or after multiple temperature cycles of the PCR amplification. In some embodiments, more than one PCR primer for each nucleic acid sequence comprises a quencher. In some embodiments, detecting the signal from the fluorescent moiety at the addressable location over time comprises measuring the rate of hybridization of the amplified molecule with the probe. In some embodiments, the sample comprises messenger RNA or a nucleotide sequence derived from messenger RNA, and determining the amount of the nucleic acid sequence in the sample is used to determine the level of gene expression in the cell or cell population from which the sample is derived. In some embodiments, the sample comprises genomic DNA or a nucleotide sequence derived from genomic DNA, and determining the amount of the nucleic acid sequence in the sample is used to determine the genetic makeup of the cell or cell population from which the sample is derived. In some embodiments, two or more PCR primers corresponding to two or more different nucleotide sequences have different quenchers. In some embodiments, two or more different addressable locations comprise different fluorescent moieties.In some embodiments, different quenchers and / or different fluorescent moieties are used to determine cross-hybridization. In some embodiments, a diagnostic test for determining the health status of an individual, comprising performing a method on a sample from such individual, using light-initiated primers to perform a Q-PCR method.

[0125] In some embodiments, a Q-PCR method is a method for assaying at least one target nucleic acid molecule, comprising the steps of: (a) providing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair comprising said photo-initiated primer, and a polymerase, wherein the primer pair has sequence complementarity with the template nucleic acid molecule, and wherein the primer pair comprises a limiting primer and an excess primer; (b) subjecting the reaction mixture to a nucleic acid amplification reaction under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; and (c) subjecting the reaction mixture to a nucleic acid amplification reaction comprising: (i) a nucleic acid sample immobilized on a surface of a substrate at different individually addressable locations; The method includes contacting a sensor array having a substrate including a number of probes, the probes having sequence complementarity with the limiting primers and capable of capturing the limiting primers, and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal indicating that the limiting primers are bound to individual probes of the plurality of probes; (d) using the detector array to detect at least one signal from one or more addressable locations at multiple time points during a nucleic acid amplification reaction; and (e) detecting a target nucleic acid molecule based on at least one signal indicating that the limiting primers are bound to individual probes of the plurality of probes. In some embodiments, the at least one signal is generated when the probes bind to the limiting primers. In some embodiments, the reaction mixture includes multiple limiting primers having different nucleic acid sequences, and the probes specifically bind to the multiple limiting primers. In some embodiments, the reaction mixture is provided in a reaction chamber configured to hold the reaction mixture and allow the probes to bind to the limiting primers. In some embodiments, the method further comprises correlating at least one signal detected at the multiple time points with the original concentration of at least one template nucleic acid molecule by analyzing the rate at which the probe binds to the limiting primer.In some embodiments, the probes are oligonucleotides. In some embodiments, the target nucleic acid molecules form hairpin loops when hybridized to the individual probes. In some embodiments, the sensor array comprises at least about 100 integrated sensors. In some embodiments, the at least one signal is an optical signal indicative of an interaction between the energy acceptor and the energy donor. In some embodiments, the energy acceptor is linked to the excess primer and / or the limiting primer. In some embodiments, the energy acceptor is linked to the target nucleic acid molecule. In some embodiments, the energy acceptor is a quencher. In some embodiments, the energy donor is a fluorophore. In some embodiments, the at least one signal is an electrical signal indicative of an interaction between the electrode and the redox label. In some embodiments, the redox label is linked to the excess primer and / or the limiting primer. In some embodiments, the redox label is linked to the target nucleic acid molecule. In some embodiments, (d) comprises measuring an increase in at least one signal relative to background. In some embodiments, (d) comprises measuring a decrease in at least one signal relative to background. In some embodiments, the target nucleic acid molecule is detected with a sensitivity of at least about 90%. In some embodiments, at least one signal is detected while a reaction mixture containing the target nucleic acid molecule is in fluid contact with the sensor array. In some embodiments, (b) comprises generating a plurality of target nucleic acid molecules having sequence complementarity with the template nucleic acid. In some embodiments, the detector array is configured to detect a plurality of signals from the addressable location, each of the plurality of signals indicating that the limiting primer is bound to an individual probe of the plurality of probes. In some embodiments, (d) comprises detecting a plurality of signals from the addressable location at a plurality of time points using the detector array, each of the plurality of signals indicating that the limiting primer is bound to an individual probe of the plurality of probes. In some embodiments, (e) comprises identifying the limiting primer.

[0126] In some embodiments, the disclosure provides a system for assaying at least one target nucleic acid molecule, comprising: (a) a reaction chamber comprising a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair having a sequence complementary to the template nucleic acid molecule, and a polymerase, wherein the primer pair comprises a limiting primer and an excess primer, and the reaction chamber comprising the reaction mixture is configured to promote a nucleic acid amplification reaction on the reaction mixture to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid; and (b) (i) a substrate comprising a plurality of probes immobilized on a surface of the substrate at distinct individually addressable locations, wherein the probes (i) a substrate, on which the limiting primer has sequence complementarity with the limiting primer and is capable of capturing the limiting primer; and (ii) a detector array configured to detect at least one signal from the addressable locations, wherein the at least one signal indicates that the limiting primer is bound to an individual probe of the plurality of probes; and (c) a computer processor coupled to the sensor array and programmed to: (i) subject the reaction mixture to a nucleic acid amplification reaction; and (ii) detect at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction.

[0127] In some embodiments, a Q-PCR method is a method for assaying at least one template nucleic acid molecule, comprising: (a) (i) a substrate comprising a plurality of first probes immobilized to first pixels and a plurality of second probes immobilized to second pixels, where the first probes are configured to capture individual primers of a primer set and the second probes are configured to capture a control nucleic acid molecule; and (ii) a detector array configured to detect at least one first signal from the first pixels and at least one second signal from the second pixels, where a difference over time between the at least one first signal and the at least one second signal indicates that an individual primer is bound to an individual probe of the plurality of first probes. (b) subjecting a reaction mixture to a nucleic acid amplification reaction under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of a template nucleic acid molecule, the reaction mixture comprising (i) a nucleic acid sample containing or suspected to contain a template nucleic acid molecule, (ii) a primer set, (iii) a control nucleic acid molecule, and (iv) a polymerization enzyme, each primer of the primer set having sequence complementarity with the template nucleic acid molecule; (c) detecting at least one first signal and at least one second signal at multiple time points during the nucleic acid amplification reaction using a detector array; and (d) detecting the template nucleic acid molecule using the difference between the at least one first signal and the at least one second signal. In some embodiments, the at least one first signal is generated when each probe binds to each primer, and the at least one second signal is generated when an additional probe of the second probe binds to the control nucleic acid molecule. In some embodiments, the control nucleic acid molecule is not amplified in the amplification reaction. In some embodiments, the reaction mixture includes a plurality of template nucleic acid molecules, and the first probe specifically binds to a plurality of target nucleic acid molecules as amplification products of the plurality of template nucleic acid molecules.In some embodiments, the primer set comprises a plurality of individual primers having different nucleic acid sequences, and the first probe is configured to specifically bind to the plurality of individual primers. In some embodiments, the reaction mixture is provided in a reaction chamber configured to hold the reaction mixture and allow the first and second probes to bind to the individual primers and the control nucleic acid molecule. In some embodiments, the method further comprises correlating at least one first signal detected at the plurality of time points with an initial concentration of at least one template nucleic acid molecule by analyzing the rate at which the probe binds to the individual primers of the primer set. In some embodiments, the first probe or the second probe is an oligonucleotide. In some embodiments, the sensor array comprises at least about 100 integrated sensors. In some embodiments, the at least one first signal is a first optical signal indicative of a first interaction between a first energy acceptor and a first energy donor associated with the individual primers and the individual probes, and the at least one second signal is a second optical signal indicative of a second interaction between a second energy acceptor and a second energy donor associated with the control nucleic acid molecule and an additional probe of the second probe. In some embodiments, the first energy acceptor is linked to an individual primer and the second energy acceptor is linked to a control nucleic acid molecule. In some embodiments, the first energy acceptor is linked to a target nucleic acid molecule. In some embodiments, the first energy acceptor is a first quencher and the second energy acceptor is a second quencher. In some embodiments, the first energy donor is a first fluorophore and the second energy donor is a second fluorophore. In some embodiments, the first energy donor is linked to a first probe and the second energy donor is linked to a second probe. In some embodiments, the target nucleic acid molecule is detected with a sensitivity of at least about 90%. In some embodiments, the at least one first signal is detected while a reaction mixture containing the target nucleic acid molecule is in fluid contact with the sensor array.

[0128] In some embodiments, a Q-PCR system is for assaying at least one template nucleic acid molecule, comprising: (a) a reaction chamber comprising a reaction mixture, the reaction mixture comprising: (i) a nucleic acid sample containing or suspected of containing a template nucleic acid molecule; (ii) a primer set comprising individual primers; (iii) a control nucleic acid molecule; and (iv) a polymerization enzyme, wherein each primer of the primer set has sequence complementarity to the template nucleic acid molecule, the reaction chamber comprising the reaction mixture is configured to promote a nucleic acid amplification reaction with the reaction mixture under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product(s) of the template nucleic acid molecule, wherein the nucleic acid amplification reaction does not result in any amplification product of the control nucleic acid; and (b) a reaction chamber comprising: (i) a plurality of first probes immobilized on first pixels; a plurality of second probes immobilized on second pixels; The present invention comprises a sensor array including a substrate including a first probe configured to capture an individual primer of a primer set and a second probe configured to capture a control nucleic acid molecule, (ii) a detector array configured to detect at least one first signal from a first pixel and at least one second signal from a second pixel, where a difference over time between the at least one first signal and the at least one second signal indicates that an individual primer is bound to an individual probe of the plurality of first probes, and (c) a computer processor coupled to the sensor array and programmed to (i) subject the reaction mixture to a nucleic acid amplification reaction, and (ii) detect at least one first signal and at least one second signal at multiple time points during the nucleic acid amplification reaction. In some embodiments, the computer processor is programmed to detect the template nucleic acid molecule using the difference between the at least one first signal and the at least one second signal. In some embodiments, the reaction mixture includes a plurality of template nucleic acid molecules, and the first probe specifically binds to a plurality of target nucleic acid molecules as amplification products of the plurality of template nucleic acid molecules.In some embodiments, the primer set comprises a plurality of individual primers having different nucleic acid sequences, and the first probe is configured to specifically bind to the plurality of individual primers. In some embodiments, the detector array comprises an optical detector. In some embodiments, the at least one first signal is a first optical signal indicative of a first interaction between a first energy acceptor and a first energy donor associated with the individual primers and the individual probes, and the at least one second signal is a second optical signal indicative of a second interaction between a second energy acceptor and a second energy donor associated with the control nucleic acid molecule and an additional probe of the second probe. In some embodiments, the optical detector comprises a complementary metal oxide semiconductor device. In some embodiments, the detector array comprises an electrical detector. In some embodiments, the electrical detector comprises a complementary metal oxide semiconductor device. In some embodiments, the sensor array comprises at least about 100 integrated sensors.

[0129] Various techniques and technologies can be used to carry out Q-PCR using microarray or CMOS biochip.For example, some such techniques are described in U.S. Patent No. 8,048,626, U.S. Patent No. 9,499,861, and U.S. Patent No. 10,174,367, each of which is incorporated herein by reference in its entirety for all purposes.

[0130] In some embodiments of the present invention, the photoremovable block is included in a NA affinity-based detection system, such as a DNA microarray. DNA microarrays, which are essentially massively parallel affinity-based biosensors, are primarily used to measure gene expression levels, i.e., to quantify the process of transcription of DNA data into messenger RNA molecules (mRNA). The information transcribed into mRNA is further translated into proteins, the molecules that perform most of the functions in cells. Thus, by measuring gene expression levels, researchers may be able to infer important information about the functionality of a cell or an entire organism. Thus, variations from typical expression levels are often indicative of disease, and thus DNA microarray experiments may provide valuable insights into the genetic causes of disease. Indeed, one of the ultimate goals of DNA microarray technology is to enable the development of molecular diagnostics and the creation of personalized medicine.

[0131] DNA microarrays are essentially affinity-based biosensors in which binding is based on hybridization, a process in which complementary DNA strands bind specifically to each other to create structures at a lower energy state. Typically, the surface of a DNA microarray consists of an array of spots (grids), each containing a single-stranded DNA oligonucleotide capture molecule as a recognition element, whose position is fixed during the hybridization and detection process. Each single-stranded DNA capture molecule typically has a length of 25-70 bases, depending on the exact platform and application. In the DNA microarray detection process, first, the mRNA that needs to be quantified is used to generate fluorescently labeled cDNA, which is then applied to the microarray. Under appropriate experimental conditions (e.g., temperature and salt concentration), the labeled cDNA molecules that perfectly match the microarray hybridize, i.e. bind, to the complementary capture oligos. In any case, some non-specific binding is always present because cDNA may non-specifically cross-hybridize to oligonucleotides that are only partial complements (mismatches) rather than perfect matches. Furthermore, the fluorescence intensity at each spot is measured to obtain an image that correlates to the hybridization process and thus the gene expression level.

[0132] Molecular recognition assays generally involve detecting a binding event between two molecules. The strength of the binding can be referred to as "affinity". The affinity between biological molecules is influenced by non-covalent intermolecular interactions, including, for example, hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. In multiplex binding experiments, such as those contemplated herein, multiple analytes and probes are included. For example, the experiment can include testing the binding between multiple different nucleic acid molecules or different proteins. In such experiments, the analyte preferentially binds to the probe with which it has high affinity. Thus, determining that a particular probe is involved in a binding event indicates that an analyte is present in the sample that has sufficient affinity for the probe to meet the threshold level of detection of the detection system being used. Based on the specificity and strength of binding between the probe and the analyte, it may be possible to determine the identity of the binding partner.

[0133] In developing a solution from the perspective of DNA microarrays, the present invention provides (i) a process that considers cross-hybridization as interference rather than noise (similar to interference in wireless communication, cross-hybridization does in fact have signal content), (ii) a model of hybridization and cross-hybridization as a stochastic process, (iii) the use of analytical methods (e.g., melting temperature or Gibbs free energy function) to build the model and fine-tune the model using experimental data, (iv) the detection and quantification of gene expression levels as a stochastic estimation problem, and (v) the construction of optimal estimates. The present invention uses statistical signal processing techniques to optimally detect and quantitate targets in microarrays by considering and utilizing the uncertainties mentioned above.

[0134] A variety of techniques and technologies can be used to synthesize arrays of biological materials on a substrate or support.For example, some such techniques are described in U.S. Patent No. 9,223,929 and U.S. Patent No. 9,133,504, each of which is incorporated herein by reference in its entirety for all purposes.

[0135] In some embodiments of the present invention, the photoremovable block is included in the CMOS biochip system. In some embodiments, the present disclosure provides a fully integrated biosensor array that includes a molecular recognition layer, an optical layer, and a sensor layer, in sequence, including a NA construct, integrated with an additional layer, for example, another layer inserted between any of the molecular recognition layer, optical layer, and sensor layer, in a sandwich configuration or in tandem. The molecular recognition layer includes an open surface and a plurality of different probes attached to the open surface at different independently addressable locations. The molecular recognition layer can also transmit light to the optical layer. The optical layer includes an optical filter layer, which transmits light from the molecular recognition layer to the sensor layer. The transmission of light between the layers can be filtered by the optical layer. The sensor layer includes an optical sensor array that detects the filtered light transmitted through the optical layer. In addition, there can be a fluid volume that includes an analyte in fluid contact with the molecular recognition layer. The fluid volume can include the NA construct.

[0136] The integrated biosensor array of the present disclosure can measure analyte binding in real time. The integrated biosensor microarray can detect the binding kinetics of the assay and is in contact with an affinity-based assay. The biosensor array includes a molecular recognition layer that includes binding probes in optical communication with a sensor for detecting binding to the probes in real time.

[0137] An integrated fluorescence-based microarray system for real-time measurement of analyte binding to multiple probes, including a capture probe layer, a fluorescence emission filter, and an image sensor, can be constructed using standard complementary metal oxide semiconductor (CMOS) processes.

[0138] In some embodiments of the present invention, the optical sensor array of the sensor layer is part of a semiconductor-based sensor array. The semiconductor-based sensor array can be either an organic semiconductor or an inorganic semiconductor. In some embodiments, the semiconductor device is a silicon-based sensor. Examples of sensors useful in the present invention include, but are not limited to, charge-coupled devices (CCDs), CMOS devices, and digital signal processors. The semiconductor device of the sensor layer can also include an integrated in-pixel photocurrent detector. The detector can include a capacitive transimpedance amplifier (CTIA).

[0139] In another embodiment, the semiconductor device has an in-pixel analog to digital converter.In another embodiment, the optical sensor array of the sensor layer can be a photodiode array.

[0140] The sensor layer can be created using a CMOS process. The semiconductor detection platform can be an assembly of an integrated system capable of measuring the binding events of a real-time microarray (RT-μArray). In some embodiments, the integrated device system includes a transducer array placed in contact with or near the RT-μArray assay.

[0141] The semiconductor detection platform of the RT-μArray may include an independent transducer array for receiving and / or analyzing signals from the target and probe binding events of the RT-μArray platform. Multiple transducers can collectively function to measure several binding events at any individual microarray spot. For example, the individual measured signals can be summed and / or averaged by transducers dedicated to a spot.

[0142] A detection circuit connected to the optical sensor array can be embedded in the sensor layer. A signal processing circuit can also be connected to the optical sensor array and embedded in the sensor layer. In some embodiments, the transducer and / or the detection circuit and / or the analysis system are implemented using electronic components fabricated and / or embedded in a semiconductor substrate. Examples of such fabrication techniques include, but are not limited to, silicon fabrication processes, microelectromechanical surface micromachining, CMOS fabrication processes, CCD fabrication processes, silicon-based bipolar fabrication processes, and gallium arsenide fabrication processes.

[0143] The transducer array can be an image sensor array.Examples of such image array include but are not limited to CMOS image sensor array, CMOS linear optical sensor, CCD image sensor, and CCD linear optical sensor.Image sensor can be used to detect the activity of probe / analyte interaction in the integrated biosensor array platform.

[0144] A variety of techniques and technologies can be used to make and / or use a CMOS biochip system. For example, some such techniques are described in U.S. Patent No. 8,637,436 and U.S. Patent No. 8,969,781.

[0145] Example 2 Light-activated nested PCR In this example, two primer pairs are used, as shown in Figure 16. One pair is light-initiated and the other is light-terminated. At a specific point in the PCR cycle, light is applied to inactivate the light-terminated primer pair and activate the light-initiated pair.

[0146] In some embodiments, the photo-terminated primer pair is adjacent to the photo-initiated primer pair such that the amplicon generated by the active form of the photo-terminated primer pair is used as a template for the active form of the photo-initiated primer pair (see FIG. 16). The advantage of this system is that it can increase the specificity and sensitivity of the amplification by reducing non-specific amplicons and products that may be produced due to the amplification of unexpected primer binding sites in the template.

[0147] This method, which may hereafter be referred to as "light-activated nested PCR", may be an alternative to the traditional nested PCR method, in which two PCR amplifications are carried out in tandem in two different reaction chambers. See G. Bein, R. Glaeser, & H. Kirchner, "Rapid HLA-DRB1 genotyping by nested PCR amplification. Tissue antigens," 1992, 39(2): 68-73; M. Pfeffer, B. Linssen, MD Parker, and RM Kinney, "Specific detection of Chikungunya virus using a RT-PCR / nested PCR combination," Journal of Veterinary Medicine, Series B, 2002, 49(1): 49-54. The advantage of light-activated nested PCR, however, is that both amplifications can occur in the same reaction and in a closed tube format.

[0148] In some embodiments of the present invention, light-activated nested PCR is included in Q-PCR system.The device, system and method disclosed in Example 1 can be modified and applied herein by using suitable NA construct as light-initiated primer pair and / or light-stopped primer pair in light-activated nested PCR, and irradiating reaction mixture to start or stop specific PCR process in the process of carrying out light-activated nested PCR.

[0149] In some embodiments of the invention, the photoremovable block is included in a NA affinity-based detection system, for example a DNA microarray.

[0150] In some embodiments of the present invention, the photoremovable block is included in a CMOS biochip system.

[0151] Example 3 Light-removable blocking In this embodiment, photo-terminated hybridization probes are used as sequence-selective blocking agents in polymerase chain reaction or other primer-initiated molecular amplification reactions. See PL Dominguez, and MS Kolodney, "Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens," Oncogene, 2005, 24(45):.6830-6834. JF Huang, et al., "Single-tubed wild-type blocking quantitative PCR detection assay for the sensitive detection of codon 12 and 13 KRAS mutations," PloS one, 2015, 10(12).

[0152] In some embodiments, photo-stop hybridization probe inhibits PCR amplification of wild-type sequence and allows synthesis of mutant sequence at the same time.By doing this, the ratio of wild-type amplicon to mutant amplicon decreases as amplification proceeds.This facilitates better detection of mutant at the end of PCR.The existence of photo-stop construct type further allows removal of blocking agent by light, and produces clean PCR product without interference of hybridization probe.

[0153] In some embodiments of the present invention, the photoremovable block is included in the Q-PCR system.The device, system and method disclosed in Example 1 can be modified and applied herein by using a suitable NA construct as a photoremovable block probe in tandem with the light-initiated PCR process, and irradiating the reaction mixture in the process of carrying out the light-initiated PCR to start or stop a specific PCR process.

[0154] In some embodiments of the present invention, the photoremovable blocking is included in a NA affinity-based detection system, such as a DNA microarray.The device, system, and method disclosed in Example 1 can be modified and applied herein by using a suitable NA construct as a photoremovable blocking probe in a NA affinity-based detection system, such as a DNA microarray.When using a NA affinity-based detection system to detect, for example, a target nucleic acid, the photoremovable blocking probe can interact with the target nucleic acid, a fixed probe, or a solution-based probe, or a combination thereof.In the process of carrying out the NA affinity-based detection system, by irradiating the reaction mixture, different amplicons can be produced and / or different hybridization events can be detected by the NA affinity-based detection system.

[0155] In some embodiments of the present invention, the photoremovable blocking is included in a CMOS biochip system. The device, system and method disclosed in Example 1 can be modified and applied herein by using a suitable NA construct as a photoremovable blocking probe in a CMOS biochip system. When using a CMOS biochip system to detect, for example, a target nucleic acid, the photoremovable blocking probe can interact with the target nucleic acid, the fixed probe, or the solution-based probe, or a combination thereof. By irradiating the reaction mixture in the process of implementing the CMOS biochip system, and by irradiating the reaction mixture in the process of implementing the NA affinity-based detection system, different amplicons can be produced and / or different hybridization events can be detected by the CMOS biochip system.

[0156] Example 4 Light anchoring primer In this example, phototerminateable primers are used to alter the effective length of the primers during PCR.

[0157] In some embodiments, the photo-terminated primer is cleaved after a certain number of PCR cycles into two parts: an inactive part derived from the original 5' end of the primer, and an active (extendable) part derived from the original 3' end that allows PCR to continue after photocleavage. This results in a high melting temperature (T M By exposing the primer to light, the length of the primer increases by 100%. M The application of this method involves the use of high T to accommodate mismatches within the template in early PCR cycles and / or to overcome secondary structures in either the RNA or DNA templates. M Primer design is included.

[0158] In some embodiments of the present invention, the light anchored primer is included in the Q-PCR system. The device, system and method disclosed in Example 1 can be modified and applied herein by using a suitable NA construct as the light anchored primer in the light anchored PCR process. Before exposure to light, the generated amplicon can include a full-length light anchored primer. By irradiating the reaction mixture, new primer pairs can be produced. Each new primer is shorter in length than the corresponding full-length light anchored primer. Thus, the amplicon produced using the new primer pair can be shorter in length than before exposure to light. Two sets of amplicons with different lengths can be produced using the same template nucleic acid molecule.

[0159] In some embodiments of the invention, the photoanchored primers are included in a NA affinity-based detection system, for example a DNA microarray.

[0160] In some embodiments of the present invention, the photo-anchored primers are included in a CMOS biochip system.

[0161] Other terms used in this disclosure The term "quantitative PCR" or "Q-PCR" as used herein generally refers to a polymerase chain reaction (PCR) process that can be used for qualitative and quantitative determination of nucleic acid sequences. In some cases, Q-PCR is synonymous with real-time PCR. Q-PCR involves measuring the amount of amplification product (or amplicon) as a function of amplification cycles, and such information can be used to determine the amount of nucleic acid sequence corresponding to the amplicon that was present in the original sample.

[0162] The term "reverse transcription polymerase chain reaction" or "RT-PCR" as used herein generally refers to a variant of polymerase chain reaction (PCR) in which a ribonucleic acid (RNA) strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using an enzyme called reverse transcriptase. The resulting cDNA is then amplified using conventional PCR. RT-PCR utilizes a pair of primers that are complementary to a predetermined sequence in each of the two strands of the cDNA. These primers are then extended by DNA polymerase, and copies of the strands are made after each PCR cycle, resulting in exponential amplification. The term "quantitative reverse transcription polymerase chain reaction" or "qRT-PCR" as used herein refers to real-time detection of an RT-PCR reaction, as is done in a Q-PCR reaction.

[0163] In this disclosure, all methods or systems disclosed for QPCR can be applied to qRT-PCR after making corresponding modifications known to those skilled in the art.

[0164] The term "probe", as used herein, generally refers to a molecular species or other marker that can bind to a specific target nucleic acid sequence. A probe can be any type of molecule or particle. A probe can include a molecule and can be attached to a substrate or other solid surface directly or via a linker molecule.

[0165] The term "detector," as used herein, generally refers to a device, generally containing optical and / or electronic components, that is capable of detecting a signal.

[0166] The term "mutation," as used herein, generally refers to a genetic mutation or sequence variation, such as a point mutation, single nucleotide polymorphism (SNP), insertion, deletion, substitution, transition, translocation, copy number variation, or another genetic mutation, alteration, or sequence variation.

[0167] The terms "about" or "approximately" as used herein generally refer to within ±15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a specified amount.

[0168] The term "label," as used herein, refers to a specific molecular structure that can be attached to a target molecule to provide a unique characteristic not inherent in the target molecule, thereby making the target molecule distinguishable and trackable.

[0169] The term "limited," as used herein in the context of a chemical or biological reaction, generally refers to a species that is in a limited (e.g., stoichiometrically limited) amount in a given reaction volume, such that upon completion of the chemical or biological reaction (e.g., PCR), the species cannot be present in the reaction volume.

[0170] The term "excess," as used herein in the context of a chemical or biological reaction, generally refers to a species that is in excess (e.g., stoichiometrically limited) amount in a given reaction volume such that the species may be present in the reaction volume upon completion of the chemical or biological reaction (e.g., PCR).

[0171] The term "nucleotide" as used herein refers to a molecule that can function as a monomer or subunit of a nucleic acid, e.g., deoxyribonucleic acid (DNA) or ribonucleic acid RNA). A nucleotide can be a deoxynucleotide triphosphate (dNTP) or an analog thereof, e.g., a molecule having multiple phosphates in the phosphate chain, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphates. Nucleotides can generally include adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide can include any subunit that can be incorporated into a growing chain of nucleic acid. Such subunits can be A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T, or U, or is complementary to a purine (i.e., A or G, or variants thereof) or pyrimidine (i.e., C, T, or U, or variants thereof). The subunits can allow individual nucleobases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or their uracil counterparts) to be degraded. Nucleotides can be labeled or unlabeled. Labeled nucleotides can produce a detectable signal, e.g., an optical, electrostatic, or electrochemical signal.

[0172] The Q-PCR process can be described in the following non-limiting example: A PCR reaction is carried out using a primer pair designed to amplify a given nucleic acid sequence in a sample. Appropriate enzymes and nucleotides, such as deoxynucleotide triphosphates (dNTPs), are added to the reaction, and the reaction is subjected to several amplification cycles. The amount of amplicon generated from each cycle is detected, but in early cycles, the amount of amplicon may be below the detection threshold. Amplification can occur in two phases: an exponential phase, followed by a non-exponential plateau phase. During the exponential phase, the amount of PCR product approximately doubles with each cycle. As the reaction progresses, however, reaction components are consumed, and eventually one or more of the components become limiting. At this point, the reaction slows down and enters a plateau phase. Initially, the amount of amplicon remains at or below background levels, and no increase is detectable even as amplicon products accumulate exponentially. Eventually, enough amplification product accumulates to produce a detectable signal. The number of cycles at which this occurs is called the threshold cycle or C t It is called. C t Because the C value is measured during the exponential phase when reagent is not limiting, Q-PCR can be used to reliably and accurately calculate the initial amount of template present in the reaction. t may be determined primarily by the amount of nucleic acid sequence corresponding to the amplicon present at the beginning of the amplification reaction. If a large number of templates are present at the beginning of the reaction, relatively few amplification cycles may be required for sufficient product to accumulate to obtain a signal above background. Thus, the reaction may exhibit a low or early C t In contrast, if low amounts of template are present at the beginning of the reaction, more amplification cycles may be required for the fluorescent signal to rise above background. Thus, the reaction may have a high or late C tThe methods and systems provided herein allow for the measurement of the accumulation of multiple amplicons in a single fluid in a single amplification reaction, and thus the determination of the amount of multiple nucleic acid sequences in the same sample by the above-mentioned Q-PCR technique.

[0173] As used herein, the term "real-time" generally refers to measuring the status of a reaction while it is occurring, either in a transition phase or biological equilibrium. A real-time measurement is performed simultaneously to monitor, measure, or observe an ongoing event, as opposed to a measurement made after the reaction has finished. Thus, a "real-time" assay or measurement generally contains not only the result that is measured and quantified, such as fluorescence, but also expressing this at various time points, i.e., nanoseconds, microseconds, milliseconds, seconds, minutes, hours, etc. "Real-time" may include detection of the kinetic product of a signal, including taking multiple readings to characterize the signal over a period of time. For example, a real-time measurement may include determining the rate of increase or decrease in the amount of analyte. Measuring a signal in real time may be useful to determine the rate by measuring the change in the signal, although in some cases, measuring the absence of change in the signal may also be useful. For example, the lack of change in the signal over time may indicate that the reaction (e.g., binding, hybridization) has reached a steady state.

[0174] As used herein, the terms "polynucleotide," "oligonucleotide," "nucleotide," "nucleic acid," and "nucleic acid molecule" generally refer to polymeric forms of nucleotides (polynucleotides) of various lengths (e.g., 20 bases to 5000 kilobases), either ribonucleotides (RNA) or deoxyribonucleotides (DNA). The term may refer only to the primary structure of the molecule. Thus, the term may include triple-, double-, and single-stranded DNA, as well as triple-, double-, and single-stranded RNA. It may also include modifications, such as by methylation and / or capping, as well as unmodified forms of polynucleotides.

[0175] Nucleic acids may include phosphodiester bonds (i.e., natural nucleic acids). Nucleic acids may include nucleic acid analogs that may have alternative backbones, including, for example, phosphoramides (see, for example, Beaucage et al., Tetrahedron 49(10):1925 (1993) and U.S. Pat. No. 5,644,048), phosphorodithioates (see, for example, Briu et al., J. Am. Chem. Soc. 11 1:2321 (1989), O-methyl phosphoramidite linkages (see, for example, Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid (PNA) backbones and linkages (see, for example, Carlsson et al., Nature 380:207 (1996)).Nucleic acids may be prepared using a cationic backbone (see, e.g., Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)), a nonionic backbone (see, e.g., U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863, Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988), Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994), Chapters 2 and 3, ASC Symposium Series 580, Other analog nucleic acids may be included, including those with non-ribose backbones (see, e.g., U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghui and P. Dan Cook, Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)). Nucleic acids may contain one or more carbocyclic sugars (see, e.g., U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghui and P. Dan Cook). These modifications of the ribose-phosphate backbone can facilitate the addition of labels or increase the stability and half-life of such molecules in physiological environments.

[0176] As used herein, the term "amplicon" generally refers to a molecular species generated by amplification of a nucleotide sequence, such as by PCR. An amplicon may be a polynucleotide, such as RNA or DNA, or a mixture thereof, and the sequence of nucleotides in an amplicon may correlate with the sequence of the nucleotide sequence from which it was generated (i.e., either corresponding to or complementary to the sequence). An amplicon may be either single-stranded or double-stranded. In some cases, an amplicon may be generated by using one or more primers incorporated into the amplicon. In some cases, an amplicon may be generated in a polymerase chain reaction or PCR amplification, where two primers may be used to produce a pair of complementary single-stranded amplicons or a double-stranded amplicon.

[0177] As used herein, the term "probe" generally refers to a molecular species or marker that can bind to a nucleic acid sequence. A probe can be any type of molecule or particle. A probe can include a molecule and can be attached to a substrate or surface directly or via a linker molecule. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0178] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. The present invention has been described with reference to the foregoing specification, but the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific expressions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It is understood that various alternatives to the embodiments of the present invention described herein can be used in the practice of the present invention. It is therefore contemplated that the present invention should cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, are covered thereby. In certain embodiments, for example, the following items are provided: (Item 1) a) a nucleic acid construct comprising a photosensitive chemical moiety, the nucleic acid construct being in a first molecular state and configured to change to a second molecular state after exposure to light; b) at least one reagent; c) at least one enzyme; A reaction chamber comprising: A reaction chamber configured to allow the light to reach the nucleic acid construct. (Item 2) 2. The reaction chamber according to item 1, wherein the nucleic acid construct is an oligonucleotide primer or probe. (Item 3) 3. The reaction chamber according to any one of items 1 to 2, wherein the at least one enzyme is a polymerase, a reverse transcriptase, a terminal transferase, an exonuclease, an endonuclease, a restriction enzyme, or a ligase. (Item 4) 4. The reaction chamber according to any one of items 1 to 3, wherein the at least one reagent comprises one or more amplification reagents. (Item 5) 5. The reaction chamber according to any one of items 1 to 4, further comprising a target nucleic acid. (Item 6) 6. The reaction chamber of claim 5, wherein the enzyme is configured to catalyze a reaction involving the target nucleic acid, the at least one reagent, and the nucleic acid construct. (Item 7) 7. The reaction chamber of item 6, wherein the nucleic acid construct in the first molecular state is configured to be active in the reaction. (Item 8) 8. The reaction chamber of item 7, wherein the nucleic acid construct in the second molecular state is configured to be inactive in the reaction. (Item 9) 7. The reaction chamber of item 6, wherein the nucleic acid construct in the first molecular state is configured to be inactive in the reaction. (Item 10) 10. The reaction chamber of item 9, wherein the nucleic acid construct in the second molecular state is configured to be active in the reaction. (Item 11) 11. The reaction chamber of any one of items 1 to 10, further comprising another nucleic acid construct comprising another light-sensitive chemical moiety, wherein the another nucleic acid construct is in a third molecular state, and the another nucleic acid construct is configured to change to a fourth molecular state after exposure to a different light. (Item 12) 12. The reaction chamber of claim 11, wherein the other light is the light. (Item 13) 13. The reaction chamber of claim 11 or 12, wherein the nucleic acid construct in the first molecular state is configured to be active in the reaction and the other nucleic acid construct in the third molecular state is configured to be inactive in the reaction. (Item 14) 14. The reaction chamber according to any one of items 11 to 13, wherein the nucleic acid construct in the second molecular state is configured to be inactive in the reaction and the other nucleic acid construct in the fourth molecular state is configured to be active in the reaction. (Item 15) 13. The reaction chamber of claim 11 or 12, wherein the nucleic acid construct in the first molecular state and the other nucleic acid construct in the third molecular state are configured to be active in the reaction. (Item 16) 16. The reaction chamber of any one of items 11, 12, and 15, wherein the nucleic acid construct in the second molecular state and the other nucleic acid construct in the fourth molecular state are configured to be inactive in the reaction. (Item 17) 13. The reaction chamber of claim 11 or 12, wherein the nucleic acid construct in the first molecular state and the other nucleic acid construct in the third molecular state are configured to be inactive in the reaction. (Item 18) 18. The reaction chamber of any one of items 11, 12, and 17, wherein the nucleic acid construct in the second molecular state and the another nucleic acid construct in the fourth molecular state are configured to be active in the reaction. (Item 19) 19. The reaction chamber according to any one of items 6 to 18, wherein the enzyme is the polymerase, the reaction is a polymerase chain reaction, and the nucleic acid construct is the oligonucleotide primer. (Item 20) 20. The reaction chamber according to any one of items 1 to 19, wherein the photosensitive chemical moiety is located at the 3-terminus, 5-terminus, or center of the nucleic acid construct. (Item 21) 21. The reaction chamber according to any one of items 1 to 20, wherein the nucleic acid construct further comprises an additional photosensitive chemical moiety. (Item 22) 22. The reaction chamber according to any one of items 1 to 21, wherein the fifth molecular state is the first molecular state and the sixth molecular state is the second molecular state. (Item 23) 23. The reaction chamber according to any one of items 1 to 22, which is a closed tube reaction chamber. (Item 24) A method of carrying out a reaction, comprising the steps of: Activating a reaction chamber to carry out a reaction, the reaction chamber comprising: a) a nucleic acid construct comprising a photosensitive chemical moiety in a first molecular state; b) at least one reagent; c) at least one enzyme; and activating light to reach the nucleic acid construct in the reaction chamber, thereby transforming the nucleic acid construct into a second molecular state; A method comprising: (Item 25) 25. The method of claim 24, wherein the nucleic acid construct is an oligonucleotide primer or probe. (Item 26) 26. The method of claim 24 or 25, wherein the at least one enzyme is a polymerase, reverse transcriptase, terminal transferase, exonuclease, endonuclease, restriction enzyme, or ligase. (Item 27) 27. The method of any one of items 24 to 26, wherein the at least one reagent comprises one or more amplification reagents. (Item 28) 28. The method according to any one of items 24 to 27, wherein the reaction chamber further comprises a target nucleic acid. (Item 29) 30. The method of claim 28, wherein the enzyme catalyzes the reaction of the target nucleic acid with the at least one reagent and the nucleic acid construct. (Item 30) 30. The method according to any one of items 24 to 29, wherein the nucleic acid construct in the first molecular state is active in the reaction. (Item 31) 31. The method according to any one of items 24 to 30, wherein the nucleic acid construct in the second molecular state is inactive in the reaction. (Item 32) 30. The method according to any one of items 24 to 29, wherein the nucleic acid construct in the first molecular state is inactive in the reaction. (Item 33) 33. The method of any one of items 24 to 29 and 32, wherein the nucleic acid construct in the second molecular state is active in the reaction. (Item 34) 25. The method of claim 24, wherein the reaction chamber further comprises another nucleic acid construct comprising another light-sensitive chemical moiety in a third molecular state, the other nucleic acid construct being configured to transform to a fourth molecular state after exposure to another light. (Item 35) 35. The method of claim 34, wherein the other light is the light, and the activating step activates the nucleic acid construct. (Item 36) 36. The method of claim 34 or 35, further comprising activating the other light to reach the other nucleic acid construct. (Item 37) 37. The method according to any one of items 34 to 36, further comprising the step of inactivating the nucleic acid construct in the reaction after the step of activating the light. (Item 38) 35. The method of claim 34, further comprising the step of activating the other nucleic acid construct after the step of activating the light or after the step of activating the other light. (Item 39) 35. The method of claim 34, further comprising the step of deactivating the other nucleic acid construct after the step of activating the light or the step of activating the other light. (Item 40) 37. The method according to any one of claims 34 to 36, further comprising the step of activating the nucleic acid construct in the reaction after the step of activating the light. (Item 41) 31. The method of claim 30, further comprising the step of deactivating the other nucleic acid construct after the step of activating the light or the step of activating the other light. (Item 42) 42. The method of claim 41, further comprising the step of activating the other nucleic acid construct after the step of activating the light or the step of activating the other light. (Item 43) 43. The method according to any one of items 34 to 42, wherein the reaction is extension, digestion, transcription, terminal transfer, or ligation. (Item 44) 44. The method according to any one of items 34 to 43, wherein when the reaction is carried out in the reaction chamber, no external reagents are added to the reaction chamber. (Item 45) 45. The method according to any one of items 34 to 44, wherein when the reaction is carried out in the reaction chamber, none of the nucleic acid construct, the at least one reagent, or the at least one enzyme is removed from the reaction chamber. (Item 46) 46. ​​The method according to any one of items 24 to 45, wherein the enzyme is the polymerase, the reaction is a polymerase chain reaction, and the nucleic acid construct is the oligonucleotide primer. (Item 47) 47. The method according to any one of items 24 to 46, wherein the photosensitive chemical moiety is located at the 3-terminus, 5-terminus, or center of the nucleic acid construct. (Item 48) 48. The method of any one of items 24 to 47, wherein the nucleic acid construct further comprises an additional photosensitive chemical moiety. (Item 49) 49. The method of any one of items 28 to 48, wherein the target nucleic acid comprises a major allele and a minor allele, and the reaction is a polymerase chain reaction. (Item 50) 50. The method of claim 49, wherein the nucleic acid construct comprises a sequence complementary to the major allele. (Item 51) 51. The method of claim 50, wherein the nucleic acid construct in the first molecular state is inactive in the polymerase chain reaction with respect to generating an amplicon of the major allele. (Item 52) 52. The method of claim 51, wherein the nucleic acid construct in the second molecular state is active in the polymerase chain reaction for producing the amplicon of the major allele. (Item 53) 52. The method of claim 51, wherein the nucleic acid construct in the second molecular state is inactive in the polymerase chain reaction with respect to producing the amplicon of the major allele. (Item 54) 54. The method of any one of items 49 to 53, wherein the other nucleic acid construct is a primer for the minor allele, and the method further comprises the step of producing an amplicon for the minor allele prior to the step of activating the light. (Item 55) a) a plurality of nucleotides; b) one or more photocleavable moieties; A nucleic acid construct comprising: Each of the one or more photocleavable moieties independently comprises: a) located at the 3' end of the nucleic acid construct; b) located at the 5' end of the nucleic acid construct; c) located between the 3' end and the 5' end; d) located on or connected to a nucleobase; e) located on or attached to ribose; f) located between and adjacent to two consecutive members of said plurality of nucleotides; or g) Nucleic acid constructs which are combinations thereof. (Item 56) 56. The nucleic acid construct of claim 55, wherein the nucleic acid construct is configured to be inert in a biochemical reaction, the biochemical reaction being polymerase-catalyzed chain extension, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, or restriction digestion. (Item 57) 57. The nucleic acid construct of claim 55 or 56, wherein the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of the one or more photocleavable moieties, and the nucleic acid molecule is configured to be active in the biochemical reaction. (Item 58) 58. The nucleic acid construct according to any one of items 55 to 57, wherein the nucleic acid construct is a primer and the biochemical reaction is polymerase-catalyzed chain extension. (Item 59) 59. The nucleic acid construct of item 58, wherein the one or more photocleavable moieties are located at the 3' end. (Item 60) 59. The nucleic acid construct of item 58, wherein each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and on a selected nucleobase. (Item 61) 59. The nucleic acid construct of item 58, wherein each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and between the two consecutive members of the plurality of nucleotides. (Item 62) 62. The nucleic acid construct of item 61, wherein the 3' end is configured to be inert in the biochemical reaction. (Item 63) 62. The nucleic acid construct of claim 61, comprising a first nucleic acid section and a second nucleic acid section complementary to the first nucleic acid section and configured to form a hairpin structure. (Item 64) 64. The nucleic acid construct of claim 63, wherein the first nucleic acid section and the second nucleic acid section do not comprise the one or more photocleavable moieties. (Item 65) 65. A method for carrying out polymerase-catalyzed chain extension using the nucleic acid construct according to any one of Items 55 to 64, comprising: a) providing a reaction mixture comprising the nucleic acid construct, at least one template nucleic acid molecule, and a polymerase, wherein the nucleic acid construct has a sequence complementary to the template nucleic acid molecule; b) subjecting the reaction mixture to conditions for chain extension catalyzed by the polymerase; c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby effecting chain extension catalyzed by the polymerase; A method comprising: (Item 66) Item 66. The method according to Item 65, wherein the providing step of item b) does not activate the performing step of item c). (Item 67) 67. The method of claim 65 or 66, wherein the nucleic acid construct remains intact in the reaction mixture prior to the irradiating step of c). (Item 68) 68. The method according to any one of items 65 to 67, further comprising a step of cleaving the one or more photocleavable moieties in c). (Item 69) 69. The method according to any one of items 65 to 68, further comprising in c) forming the nucleic acid molecule. (Item 70) 70. The method according to any one of items 65 to 69, wherein the carrying out step of c) comprises using the nucleic acid molecule formed after the irradiating step in c) as a primer for chain extension catalyzed by the polymerase. (Item 71) 71. The method of any one of items 65 to 70, wherein the reaction mixture further comprises another primer, the another primer being active in chain extension catalyzed by the polymerase. (Item 72) 72. The method of claim 71, wherein the other primer is active in the polymerase-catalyzed chain extension prior to the irradiating step of c). (Item 73) 73. The method of claim 71 or 72, wherein the polymerase-catalyzed chain extension of b) produces an amplicon comprising the additional primer. (Item 74) The polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), and the method further comprises in step c) 1) performing polymerase-catalyzed chain extension of two or more nucleotide sequences in the presence of the nucleic acid construct according to any one of items 55 to 64 to produce two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable locations, said array configured to be contacted with said fluid; 3) measuring hybridization of the two or more amplicons to two or more nucleic acid probes of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain amplicon hybridization measurements, wherein the amplicons include a quencher; 74. The method according to any one of items 65 to 73, further comprising: (Item 75) The polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), and the method further comprises in step c) 1) providing an array comprising a solid support having a surface and a plurality of different probes immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences, the PCR amplification being performed in a fluid, (i) the nucleic acid construct according to any one of items 55 to 64 being a PCR primer for each nucleic acid sequence and comprising a quencher, and (ii) the fluid being in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize to the plurality of probes, thereby quenching the signal from the fluorescent moiety; 3) detecting the signal from the fluorescent moiety at each of the addressable locations over time; and 4) determining the amount of said amplicon in said fluid using said signal detected over time; 5) using the amount of the amplicon in the fluid to determine the amount of the nucleotide sequence in the sample; 74. The method according to any one of items 65 to 73, further comprising: (Item 76) The polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), and the method further comprises in step c) 1) preparing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair, and a polymerase, wherein the primer pair has sequence complementarity with the template nucleic acid molecule, the primer pair comprises a limiting primer and an excess primer, and at least one of the limiting primer and the excess primer is the nucleic acid construct according to any one of Items 55 to 64; 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; 3) contacting the reaction mixture with a sensor array having: (i) a substrate comprising a plurality of probes immobilized on a surface of the substrate at distinct, individually addressable locations, the probes having sequence complementarity with the limiting primers and capable of capturing the limiting primers; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal being indicative of binding of the limiting primer to an individual probe of the plurality of probes; 4) detecting the at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction using the detector array; 5) detecting the target nucleic acid molecule based on the at least one signal indicative of binding of the limiting primer to the individual probe of the plurality of probes; 74. The method according to any one of items 65 to 73, further comprising: (Item 77) A system for assaying at least one target nucleic acid molecule using the nucleic acid construct according to any one of items 55 to 64, comprising: 1) A reaction chamber containing a reaction mixture including a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair having a sequence complementary to the template nucleic acid molecule, and a polymerase, wherein the primer pair includes a limiting primer and an excess primer, and at least one of the limiting primer and the excess primer is the nucleic acid construct according to any one of items 55 to 64, and the reaction chamber containing the reaction mixture is configured to promote a nucleic acid amplification reaction for the reaction mixture to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid; 2) a sensor array comprising: (i) a substrate comprising a plurality of probes immobilized on a surface of the substrate at distinct individually addressable locations, the probes having sequence complementarity with the limiting primers and capable of capturing the limiting primers; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal being indicative of binding of the limiting primer to an individual probe of the plurality of probes; 3) a computer processor coupled to the sensor array and programmed to: (i) subject the reaction mixture to the nucleic acid amplification reaction; and (ii) detect the at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction. A system comprising: (Item 78) a) a plurality of nucleotides; b) one or more photocleavable moieties; A nucleic acid construct comprising: Each of the one or more photocleavable moieties independently comprises: a) located between the 3' end of the nucleic acid construct and the 5' end of the nucleic acid construct; b) located on or connected to a nucleobase; c) located on or attached to ribose; d) located between and connected to two consecutive members of said plurality of nucleotides; or e) Nucleic acid constructs which are combinations thereof. (Item 79) 79. The nucleic acid construct of claim 78, wherein the nucleic acid construct is configured to be active in a biochemical reaction, the biochemical reaction being polymerase-catalyzed chain extension, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, or restriction digestion. (Item 80) 80. The nucleic acid construct of claim 78, wherein the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of the one or more photocleavable moieties, and the nucleic acid molecule is inactive in the biochemical reaction. (Item 81) 80. The nucleic acid construct of claim 78, wherein the nucleic acid construct is configured to form a nucleic acid molecule upon photocleavage of the one or more photocleavable moieties, the nucleic acid molecule being active in the biochemical reaction, and the nucleic acid molecule being located near the 3' end. (Item 82) 82. The nucleic acid construct according to any one of items 78 to 81, wherein the nucleic acid construct is a primer and the biochemical reaction is polymerase-catalyzed chain extension. (Item 83) 83. The nucleic acid construct of claim 82, wherein each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and on a selected nucleobase. (Item 84) 84. The nucleic acid construct of item 83, wherein in the absence of the one or more photo-cleavable moieties, the nucleic acid construct is configured to form a hairpin structure, thereby rendering the nucleic acid construct inactive as a primer in the absence of the one or more photo-cleavable moieties. (Item 85) 83. The nucleic acid construct of item 82, wherein each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and between the two consecutive members of the plurality of nucleotides. (Item 86) 86. The nucleic acid construct according to any one of items 82 to 85, wherein the nucleic acid construct comprises a first sequence complementary to a template nucleic acid molecule, the first sequence being located at or near the 3' end. (Item 87) 87. The nucleic acid construct of claim 86, further comprising a second sequence complementary to the template nucleic acid molecule, wherein the second sequence is located at or near the 5' end, and wherein at least one of the one or more photocleavable moieties is located between the first and second sequences. (Item 88) 88. The nucleic acid construct of any one of items 87, wherein the one or more photocleavable moieties are separated from the first sequence and / or the second sequence by at least one nucleotide. (Item 89) 89. The nucleic acid construct of claim 87 or 88, wherein the first sequence and the second sequence are configured to form a hairpin loop between the first sequence and the second sequence when both sequences hybridize to the template nucleic acid molecule. (Item 90) 90. The nucleic acid construct of any one of items 87 to 89, wherein the second sequence comprises a 5' to 5' linkage connecting to the remainder of the nucleic acid construct, wherein the second sequence is configured to be non-extendable in the polymerase-catalyzed chain extension. (Item 91) A method for carrying out polymerase-catalyzed chain extension using the nucleic acid construct according to any one of items 78 to 90, comprising: a) providing a reaction mixture comprising the nucleic acid construct, the template nucleic acid molecule, and a polymerase, wherein the nucleic acid construct comprises at least the first sequence; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension, thereby effecting polymerase-catalyzed chain extension; c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby terminating the polymerase-catalyzed chain extension. A method comprising: (Item 92) 92. The method of claim 91, further comprising the step of cleaving the one or more photocleavable moieties in c). (Item 93) 93. The method of claim 91 or 92, further comprising, in c), after the irradiating step, forming the nucleic acid molecule, wherein the nucleic acid molecule dissociates from the template nucleic acid molecule. (Item 94) 94. The method of claim 93, wherein the nucleic acid molecule forms a hairpin structure, and the hairpin structure comprises at least a portion of the first sequence. (Item 95) 94. The method of claim 93, wherein the nucleic acid molecule comprises the first sequence. (Item 96) 1. A method for performing a light-activated nested polymerase chain reaction (PCR), comprising: a) providing a reaction mixture comprising a first primer pair, a second primer pair, a template nucleic acid molecule comprising an internal nucleic acid sequence, and a polymerase, wherein each member of the first primer pair is independently the nucleic acid construct according to any one of items 78 to 90, each member of the second primer pair is independently the nucleic acid construct according to any one of items 55 to 64, and the internal nucleic acid sequence is nested within the template nucleic acid molecule; b) subjecting the reaction mixture to first strand extension conditions using the first primer pair to amplify the template nucleic acid molecule, thereby forming an amplicon of the template nucleic acid or a complementary sequence of the template nucleic acid molecule; c) irradiating the reaction mixture with photons of light, thereby deactivating the first primer pair, terminating the first extension, activating the second primer pair, and initiating a second strand extension using the activated second primer pair to form an amplicon of the internal nucleic acid sequence or a complementary sequence of the internal nucleic acid sequence; Including, a) to c) are carried out in a closed tube format; method. (Item 97) The light-activated PCR is a quantitative polymerase chain reaction (Q-PCR), and the method comprises: 1) performing the light-activated PCR on two or more nucleotide sequences in the presence of the first primer pair and a second primer pair to produce two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable locations, said array configured to be contacted with said fluid; 3) measuring hybridization of the two or more amplicons to two or more nucleic acid probes of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain amplicon hybridization measurements, wherein the amplicons include a quencher; Item 97. The method of item 96, further comprising: (Item 98) The light-activated PCR is a quantitative polymerase chain reaction (Q-PCR), and the method comprises: 1) providing an array comprising a solid support having a surface and a plurality of different probes immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences, the PCR amplification being performed in a fluid, (i) each of the first primer pair and the second primer pair for each nucleic acid sequence comprises a quencher, and (ii) the fluid is in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize to the plurality of probes, thereby quenching signals from the fluorescent moiety; 3) detecting the signal from the fluorescent moiety at each of the addressable locations over time; and 4) determining the amount of said amplicon in said fluid using said signal detected over time; 5) using the amount of the amplicon in the fluid to determine the amount of the nucleotide sequence in the sample; Item 97. The method of item 96, further comprising: (Item 99) The light-activated PCR is a quantitative polymerase chain reaction (Q-PCR), and the method comprises: 1) preparing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair, and a polymerase, wherein the primer pair has sequence complementarity with the template nucleic acid molecule, the primer pair comprises a limiting primer and an excess primer, and at least one of the limiting primer and the excess primer is the nucleic acid construct according to any one of Items 55 to 64; 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; 3) contacting the reaction mixture with a sensor array having: (i) a substrate comprising a plurality of probes immobilized on a surface of the substrate at distinct, individually addressable locations, the probes having sequence complementarity with the limiting primers and capable of capturing the limiting primers; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal being indicative of binding of the limiting primer to an individual probe of the plurality of probes; 4) detecting the at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction using the detector array; 5) detecting the target nucleic acid molecule based on the at least one signal indicative of binding of the limiting primer to the individual probe of the plurality of probes; Item 97. The method of item 96, further comprising: (Item 100) a) a plurality of nucleotides; b) one or more photocleavable moieties; A nucleic acid construct comprising: Each of the one or more photocleavable moieties independently comprises: a) located between the 3' end of the nucleic acid construct and the 5' end of the nucleic acid construct; b) located on or connected to a nucleobase; c) located on or attached to ribose; d) located between and connected to two consecutive members of said plurality of nucleotides; or e) Nucleic acid constructs which are combinations thereof. (Item 101) 101. The nucleic acid construct of item 100, which is a probe and is configured to be inactive upon hybridization with a target nucleic acid molecule. (Item 102) 102. The nucleic acid construct of claim 100 or 101, wherein the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of the one or more photocleavable moieties, and the nucleic acid molecule is configured to be active in the hybridization with the target nucleic acid molecule. (Item 103) 103. The nucleic acid construct according to any one of items 100 to 102, comprising one free end. (Item 104) 104. The nucleic acid construct according to any one of items 100 to 103, comprising a fixed end or an end that is non-extendible in polymerase-catalyzed chain elongation. (Item 105) 105. The nucleic acid construct according to any one of items 102 to 104, wherein each of the one or more photo-cleavable moieties is independently located between the 3' end and the 5' end and on a selected nucleobase, and the selected nucleobase is configured to hybridize with the target nucleic acid molecule in the absence of the one or more photo-cleavable moieties. (Item 106) 105. The nucleic acid construct of any one of items 102 to 104, wherein each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and between the two consecutive members of the plurality of nucleotides. (Item 107) 107. The nucleic acid construct of Item 106, comprising a first nucleic acid section and a second nucleic acid section complementary to the first nucleic acid section, and configured to form a hairpin structure. (Item 108) 108. The nucleic acid construct of claim 107, wherein the first nucleic acid section and the second nucleic acid section do not comprise the one or more photocleavable moieties. (Item 109) A method for performing the hybridization using the nucleic acid construct according to any one of Items 100 to 108, comprising: a) providing a reaction mixture comprising the nucleic acid construct and the target nucleic acid molecule; b) subjecting said reaction mixture to said hybridization conditions; c) irradiating said reaction mixture or said nucleic acid construct with photons of light, thereby effecting said hybridization; A method comprising: (Item 110) Item 109. The method of claim 109, wherein the providing step of item b) does not activate the performing step of item c). (Item 111) 111. The method of claim 109 or 110, wherein the nucleic acid construct remains intact in the reaction mixture prior to the irradiating step of c). (Item 112) 112. The method according to any one of items 109 to 111, further comprising a step of cleaving said one or more photocleavable moieties in c). (Item 113) 113. The method according to any one of items 109 to 112, further comprising in c) forming the nucleic acid molecule. (Item 114) Item 114. The method of item 113, wherein the irradiating step breaks the hairpin structure of the nucleic acid construct and forms the nucleic acid molecule. (Item 115) a) a plurality of nucleotides; b) one or more photocleavable moieties; A nucleic acid construct comprising: Each of the one or more photocleavable moieties independently comprises: a) located between the 3' end of the nucleic acid construct and the 5' end of the nucleic acid construct; b) located on or connected to a nucleobase; c) located on or attached to ribose; d) located between and connected to two consecutive members of said plurality of nucleotides; or e) Nucleic acid constructs which are combinations thereof. (Item 116) 116. The nucleic acid construct of item 115, which is a probe and is configured to be active in hybridization with a target nucleic acid molecule. (Item 117) Item 117. The nucleic acid construct of item 115 or item 116, wherein the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of the one or more photocleavable moieties, and the nucleic acid molecule is configured to be inactive upon hybridization with the target nucleic acid molecule. (Item 118) 118. The nucleic acid construct according to any one of items 115 to 117, comprising one free end. (Item 119) 119. The nucleic acid construct of any one of items 115 to 118, comprising a fixed end or an end that is non-extendible in polymerase-catalyzed chain extension. (Item 120) 120. The nucleic acid construct of any one of items 115 to 119, wherein each of the one or more photocleavable moieties is independently located between the 3' end and the 5' end and between the two consecutive members of the plurality of nucleotides. (Item 121) 120. The nucleic acid construct according to any one of items 115 to 119, wherein each of the one or more photo-cleavable moieties is independently located between the 3' end and the 5' end and on a selected nucleobase, and the selected nucleobase is configured to hybridize with another nucleobase of the nucleic acid construct in the absence of the one or more photo-cleavable moieties. (Item 122) 117. The nucleic acid construct of any one of Items 115-119 and 112, comprising a first nucleic acid section and a second nucleic acid section complementary to the first nucleic acid section, and configured to form a hairpin structure in the absence of the one or more photocleavable moieties. (Item 123) 123. The nucleic acid construct of item 122, wherein the first nucleic acid section or the second nucleic acid section comprises the one or more photocleavable moieties. (Item 124) A method for performing the hybridization using the nucleic acid construct according to any one of Items 115 to 123, comprising: a) providing a reaction mixture comprising the nucleic acid construct and the target nucleic acid molecule; b) subjecting said reaction mixture to said hybridization conditions; c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby terminating the hybridization. A method comprising: (Item 125) 125. The method of claim 124, further comprising the step of cleaving the one or more photocleavable moieties in c). (Item 126) 126. The method of claim 124 or 125, further comprising in c) forming the nucleic acid molecule. (Item 127) 127. The method of claim 126, further comprising the step of forming the hairpin structure in the nucleic acid molecule in c). (Item 128) further comprising the step of performing polymerase-catalyzed chain extension; 1) the reaction mixture further comprises a polymerase and a primer, and in b) the nucleic acid construct hybridizes to the target nucleic acid molecule in b); 2) in b), subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension using the primer, wherein the polymerase-catalyzed chain extension terminates at or near the position where the nucleic acid construct forms a duplex with the target nucleic acid molecule; 3) After the irradiating step of c), the double strands are removed to expose single stranded sequences previously hybridized to the nucleic acid construct, thereby allowing polymerase-catalyzed strand extension to continue and extend through the single stranded sequences. (Item 129) The polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), the method comprising: 1) performing polymerase-catalyzed chain extension of two or more nucleotide sequences comprising the target nucleic acid molecule according to item 128 in the presence of the nucleic acid construct according to any one of items 115 to 123, thereby producing two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable locations, said array configured to be contacted with said fluid; 3) measuring hybridization of the two or more amplicons to two or more nucleic acid probes of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain amplicon hybridization measurements, wherein the amplicons include a quencher; Item 129. The method of item 128, further comprising: (Item 130) The polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), the method comprising: 1) providing an array comprising a solid support having a surface and a plurality of different probes immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences comprising the target nucleic acid molecule, the PCR amplification being carried out in a fluid comprising the nucleic acid construct according to any one of items 115 to 123, (i) a PCR primer for each nucleic acid sequence comprises a quencher, (ii) the fluid is in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize with the plurality of probes, thereby quenching the signal from the fluorescent moiety, and the irradiating step is carried out during the PCR; 3) detecting the signal from the fluorescent moiety at each of the addressable locations over time; and 4) determining the amount of said amplicon in said fluid using said signal detected over time; 5) using the amount of the amplicon in the fluid to determine the amount of the nucleotide sequence in the sample; Item 129. The method of item 128, further comprising: (Item 131) The polymerase-catalyzed chain extension is a quantitative polymerase chain reaction (Q-PCR), the method comprising: 6) preparing the reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule comprising the target nucleic acid molecule, a primer pair, and a polymerase, wherein the primer pair has sequence complementarity with the at least one template nucleic acid molecule, the primer pair comprises a limiting primer and an excess primer, and the reaction mixture further comprises at least one of the nucleic acid constructs according to any one of items 115 to 123; 7) subjecting the reaction mixture to Q-PCR under conditions sufficient to obtain an amplification product of the template nucleic acid molecule and the limiting primer, wherein the amplicon comprises the limiting primer; and 8) contacting the reaction mixture with a sensor array comprising: (i) the substrate comprising a plurality of probes immobilized on a surface of the substrate at distinct, individually addressable locations, the probes having sequence complementarity with the limiting primers and capable of capturing the limiting primers; and (ii) a detector array configured to detect at least one signal from the addressable locations, the at least one signal being indicative of binding of the limiting primer to an individual probe of the plurality of probes; 9) detecting the at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction using the detector array; 10) detecting the target nucleic acid molecule based on the at least one signal indicative of binding of the limiting primer to the individual probe of the plurality of probes; Item 129. The method of item 128, further comprising: (Item 132) a) a plurality of nucleotides; b) one or more photo-cleavable moieties at the 5' end of the nucleic acid construct, wherein the 5' end of the nucleic acid construct is configured to be resistant to cleavage by an exonuclease; A nucleic acid construct comprising: Each of the one or more photocleavable moieties independently comprises: a) located on or connected to a nucleobase; b) located on or connected to ribose, or c) A nucleic acid construct which is a combination thereof. (Item 133) 133. The nucleic acid construct of claim 132, wherein the nucleic acid construct is configured to form a nucleic acid molecule after photocleavage of the one or more photocleavable moieties, and the nucleic acid molecule is not resistant to the cleavage by the exonuclease. (Item 134) 134. The nucleic acid construct of claim 132 or 133, configured to hybridize to a target nucleic acid molecule and remain resistant to said cleavage by said exonuclease. (Item 135) 1. A method for performing polymerase-catalyzed chain extension, comprising: a) preparing a reaction mixture comprising the nucleic acid construct according to any one of items 132 to 134, the target nucleic acid molecule, a primer, and a polymerase, wherein the target nucleic acid molecule comprises a nucleic acid sequence complementary to the nucleic acid construct; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension of the primer using the target nucleic acid molecule as a template; c) irradiating said reaction mixture or said nucleic acid construct with photons of light, thereby effecting said polymerase-catalyzed chain extension through said nucleic acid sequence; A method comprising: (Item 136) Item 136. The method of claim 135, wherein the providing step of item b) does not activate the performing step of item c). (Item 137) 137. The method of claim 135 or 136, wherein the nucleic acid construct remains intact in the reaction mixture prior to the irradiating step of c). (Item 138) 138. The method according to any one of items 135 to 137, further comprising a step of cleaving said one or more photocleavable moieties in c). (Item 139) 139. The method according to any one of items 135 to 138, further comprising in c) forming the nucleic acid molecule. (Item 140) 140. The method according to any one of items 135 to 139, wherein the carrying out step of c) comprises digesting the nucleic acid molecule formed after the irradiating step in c) with the exonuclease, and the polymerase is the exonuclease. (Item 141) 141. The method according to any one of items 135 to 140, wherein the step of carrying out c) comprises extending the primer through the nucleic acid sequence after the irradiating step and / or after the digesting step. (Item 142) A method for carrying out polymerase-catalyzed chain extension using the nucleic acid construct according to any one of items 78, 79, 81, 82, and 85 to 88, comprising: a) providing a reaction mixture comprising the nucleic acid construct, the template nucleic acid molecule, and a polymerase, wherein the nucleic acid construct comprises at least the first sequence located at or near the 3' end and the second sequence located at or near the 5' end, and the first sequence is active in chain extension catalyzed by the polymerase; b) subjecting the reaction mixture to conditions for polymerase-catalyzed chain extension, thereby effecting the polymerase-catalyzed chain extension and producing a plurality of first amplicons comprising sequences complementary to both the first sequence and the second sequence, or to both the first sequence and the second sequence; c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby cleaving the nucleic acid construct and producing a plurality of second amplicons comprising the first sequence or a sequence complementary to the first sequence, with the proviso that each of the plurality of second amplicons does not contain the second sequence or a sequence complementary to the second sequence; A method comprising: (Item 143) A method for polymerase-catalyzed chain extension using at least one of the nucleic acid constructs according to any one of items 55 to 64, 78 to 90, 100 to 108, and 115 to 123, comprising: a) providing a reaction mixture comprising the nucleic acid construct, the template nucleic acid molecule, and a polymerase; b) subjecting the reaction mixture to conditions for chain extension catalyzed by the polymerase; c) irradiating the reaction mixture or the nucleic acid construct with photons of light; thereby performing chain extension catalyzed by said polymerase. Including, The method, wherein said polymerase-catalyzed chain extension is PCR, RT-PCR, QPCR, or qRT-PCR. (Item 144) the at least one of the nucleic acid constructs independently comprises: a) primers for the PCR, RT-PCR, QPCR, or qRT-PCR; b) a solution phase probe for said PCR, RT-PCR, QPCR, or qRT-PCR; or c) The method according to claim 143, wherein the PCR, RT-PCR, QPCR, or qRT-PCR is a fixed probe. (Item 145) 1. An automated microarray system for quantifying microarray data, comprising: a) a solid support having a surface and a plurality of different probes, said plurality of different probes being immobilized on said surface; b) a fluid volume containing a sample, the fluid volume being in contact with the solid support, and the sample containing at least one of the plurality of different probes and at least one of the nucleic acid constructs according to any one of items 55 to 64, 78 to 90, 100 to 108, and 115 to 123; c) a detector or detection assembly configured to detect a signal measured at multiple time points from each of multiple spots on the solid support while the fluid volume is in contact with the solid support, the signal being an optical signal or an electrochemical signal; and d) a computer configured to convert the signals into microarray data, the computer further comprising instructions configured to cause the microarray data to be processed by the computer according to a processing method, the processing method comprising: 1) determining an estimate of an interaction between the plurality of different probes and the analyte, including by (i) an analytical representation, and (ii) a calibration of the microarray using at least one standard probe on the solid support; 2) generating a probability matrix utilizing estimates in a Markov chain model that includes modeling hybridization, cross-hybridization, and unbound transition probabilities between states; 3) obtaining affinity-based array data using said detector or said detector assembly; and 4) applying the affinity-based array data to the probability matrix; 5) applying an optimization algorithm selected from the group consisting of a maximum likelihood estimation algorithm, a maximum a posteriori probability criterion, a constrained least squares computation, and any combination thereof, that exploits and does not suppress non-specific interactions by considering them as interference rather than noise; 6) outputting optimized affinity-based array data to a user, said optimized affinity-based array data having an improved signal-to-noise ratio compared to said affinity-based array data obtained by using said detector or said detector assembly; Including computers and A system comprising: (Item 146) In order, A molecular recognition layer comprising at least one of the nucleic acid constructs according to any one of items 55 to 64, 78 to 90, 100 to 108, and 115 to 123; An optical layer; The sensor layer is assembled in a sandwich configuration. 1. An integrated biosensor array comprising: a) the molecular recognition layer comprises a plurality of different probes attached to different independently addressable locations, each of the independently addressable locations configured to receive an excitation photon flux directly from a single source located on a single side of the molecular recognition layer, the molecular recognition layer transmits light to the optical layer, and one of the plurality of different probes comprises said at least one of the nucleic acid constructs; b) the optical layer includes an optical filter layer, the optical layer transmitting light from the molecular recognition layer to the sensor layer, thereby filtering the transmitted light; c) an integrated biosensor array, wherein the sensor layer comprises an optical sensor array that detects the filtered light transmitted from the optical layer, the sensor layer comprising sensor elements fabricated using a CMOS fabrication process, and wherein the molecular recognition layer, the optical layer and the sensor layer form an integrated structure in which the molecular layer is in contact with the optical layer and the optical layer is in contact with the sensor layer.

Claims

1. A composition comprising a phototerminated primer pair and a photoinitiated primer pair for nested polymerase chain reaction (PCR), each member of the phototerminated primer pair and the photoinitinated primer pair independently a) a plurality of nucleotides; b) one or more photocleavable moieties; A nucleic acid construct comprising: the nucleic acid construct of the photo-terminating primer pair is configured such that it is active in the nested PCR and the nucleic acid construct of the photo-terminating primer pair is inactive in the nested PCR in the absence of the one or more photo-cleavable moieties on the photo-terminating primer pair; the nucleic acid construct of the light-initiated primer pair is configured to be inactive in the nested PCR, and the nucleic acid construct of the light-initiated primer pair is configured to be active in the nested PCR in the absence of the one or more photo-cleavable moieties on the light-initiated primer pair; each of the one or more photocleavable moieties independently: a) located at the 3' end of the nucleic acid construct; b) located at the 5' end of the nucleic acid construct; c) located between the 3' end and the 5' end; d) located on or connected to a nucleobase of a nucleotide of said plurality of nucleotides; e) located on or connected to the ribose of said nucleotide; f) located between and connected to said nucleotide and another nucleotide of said plurality of nucleotides; or g) combinations thereof; The composition, wherein the light-terminated primer pair is adjacent to the light-initiated primer pair such that an amplicon produced by the active form of the light-terminated primer pair is used as a template for the active form of the light-initiated primer pair.

2. The composition described in claim 1, wherein the nucleic acid construct of the phototerminating primer pair is configured to form a hairpin structure in the absence of the one or more photocleavable moieties on the phototerminating primer pair.

3. The composition described in claim 1 or 2, wherein the one or more photocleavable portions of the phototerminating primer pair are located at the 5' end of the phototerminating primer.

4. The composition of claim 1, wherein the nucleic acid construct of the photo-terminated primer pair is configured to form a nucleic acid molecule following photo-cleavage of the one or more photo-cleavable moieties on the photo-terminated primer pair, and the nucleic acid molecule is inactive in the nested PCR.

5. The composition of claim 1, wherein the nucleic acid construct of the light-initiated primer pair is configured to form a nucleic acid molecule following photocleavage of the one or more photocleavable moieties on the light-initiated primer pair, and the nucleic acid molecule is configured to be active in the nested PCR.

6. The composition described in claim 5, wherein the one or more photocleavable portions of the photoinitiated primer pair are located at the 3' end.

7. The composition of claim 5, wherein each of the photocleavable portions of the photoinitiated primer pair is located between the 3' end and the 5' end and on a nucleic acid base.

8. The composition described in claim 5, wherein the photo-cleavable portions of the one or more photo-cleavable portions of the photo-initiated primer pair are independently located between the 3' end and the 5' end of the nucleic acid construct of the photo-initiated primer and between the two consecutive members of the plurality of nucleotides.

9. The composition described in claim 7, wherein each of the nucleic acid constructs of the photoinitiated primer pair comprises a first nucleic acid section and a second nucleic acid section complementary to the first nucleic acid section and forms another hairpin structure.

10. The composition described in claim 9, wherein the first nucleic acid section and the second nucleic acid section of the photoinitiated primer pair do not contain the one or more photocleavable moieties.

11. The composition of any one of claims 1 to 10, wherein each member of the photo-terminating primer pair has a first sequence complementary to a template nucleic acid molecule, and the first sequence complementary to the template nucleic acid molecule is located at the 3' end of each member of the photo-terminating primer pair.

12. The composition of claim 11, wherein each member of the photo-initiated primer pair has a second sequence complementary to the template nucleic acid molecule, and the first sequence is adjacent to the second sequence with respect to the template nucleic acid molecule.

13. A method for performing the nested PCR using the composition according to any one of claims 1 to 12, comprising: a) providing a reaction mixture comprising the composition comprising the photo-terminated primer pair and the photo-initiated primer pair, at least one template nucleic acid molecule comprising an internal nucleic acid sequence, and a polymerase, wherein the internal nucleic acid sequence is nested within the template nucleic acid molecule; b) subjecting the reaction mixture to first strand extension conditions using the phototerminating primer pair to amplify the template nucleic acid molecule, thereby forming an amplicon of the template nucleic acid molecule or a complementary sequence of the template nucleic acid molecule; c) irradiating the reaction mixture with photons of light, thereby inactivating the photo-terminated primer pair to terminate the first strand extension and activating the photo-initiated primer pair to initiate a second strand extension using the activated photo-initiated primer pair to form an amplicon of the internal nucleic acid sequence or a complementary sequence of the internal nucleic acid sequence; wherein a)-c) are carried out in a closed tube format. 1) performing the nested PCR on two or more nucleotide sequences in the presence of the light-terminated primer pair and the light-initiated primer pair to produce two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently addressable locations, said array configured to be contacted with said fluid; 3) measuring hybridization of the two or more amplicons to two or more nucleic acid probes of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain amplicon hybridization measurements, the amplicons comprising a quencher; The method of claim 13 further comprising:

15. The method of claim 1, wherein the nested PCR is a quantitative polymerase chain reaction (Q-PCR), 1) providing an array comprising a solid support having a surface and a plurality of different probes immobilized on the surface at different addressable locations, each addressable location comprising a fluorescent moiety; 2) performing a PCR amplification on a sample comprising a plurality of nucleotide sequences; the PCR amplification is performed in a fluid, (i) each of the light-terminated primer pair and the light-initiated primer pair for each nucleic acid sequence comprises a quencher; and (ii) the fluid is in contact with the plurality of different probes, and amplicons produced in the PCR amplification hybridize to the plurality of probes, thereby quenching signals from the fluorescent moiety; 3) detecting the signal from the fluorescent moiety at each of the addressable locations over time; and 4) determining the amount of the amplicon in the fluid using the signal detected over time; and 5) using the amount of the amplicon in the fluid to determine the amount of the nucleotide sequence in the sample. The method of claim 13 further comprising:

16. The method of claim 1, wherein the nested PCR is a quantitative polymerase chain reaction (Q-PCR), 1) the light-initiated primer pair comprises a limiting primer and an excess primer; 2) subjecting the reaction mixture to the Q-PCR under conditions sufficient to obtain at least one target nucleic acid molecule as an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule comprises the limiting primer; and 3) contacting the reaction mixture with a sensor array having: (i) a substrate comprising a plurality of probes immobilized on a surface of the substrate at different individually addressable locations, the probes having sequences complementary to the limiting primers and capable of capturing the limiting primers; and (ii) an array of detectors configured to detect at least one signal from the addressable locations, the at least one signal being indicative of binding of the limiting primer to an individual probe of the plurality of probes; 4) detecting the at least one signal from one or more of the addressable locations at multiple time points during the nucleic acid amplification reaction using the array of detectors; 5) detecting the target nucleic acid molecule based on the at least one signal indicative of binding of the limiting primer to the individual probes of the plurality of probes; The method of claim 13 further comprising:

Images