Photo-triggered nucleic acid constructs and methods for molecular detection

Abstract

The present disclosure provides methods, apparatuses, and systems for implementing simultaneous multiplexed amplification reactions and real-time detection in a single reaction chamber.

Description

[0001] Cross-reference

[0002] This application claims the priority of U.S. Provisional Patent Application No. 62 / 850,239, filed on May 20, 2019, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] Nucleic acid (NA) detection is a unique analytical technique for detecting, quantifying, and identifying the genetic structure of specific sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. NA detection has many applications and is widely used in life science research and molecular diagnostics. Irrespective of the application and testing venue, the amount of genetic material (RNA or DNA copies) in the test sample is usually very small and cannot be directly detected; thus, it is very common to use physical-chemical, biochemical, or enzymatic methods to enhance the generated target-specific signals to ensure more sensitive detection. Some of these methods utilize molecular amplification processes such as polymerase chain reaction (PCR) to increase the copy number of the target NA. These detections are classified and well-known and are generally categorized as nucleic acid amplification tests (NAATs). In addition, amplification methods include, for example: strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and rolling circle amplification (RCA).

[0004] NAAT methods have a variety of different performance criteria, including analytical sensitivity, specificity, limit of detection (LoD), quantification range, dynamic detection range (DDR), and turnaround time (TAT). Different applications require different criteria, and there are always trade-offs depending on the method used. For example, in infectious disease applications, accurately identifying the presence of infectious pathogens in clinical specimens is crucial. Thus, one needs NAAT methods that provide an LoD for some organisms per test, while the quantification range is less important because patient treatment is less dependent on this information. On the other hand, in gene expression applications, the concentration of messenger RNA (mRNA) in clinical samples is relatively large, and the DDR is much more important than the LoD.

[0005] Today, there are a variety of NAAT methods for NA detection that use specific enzymes, reagents, and temperature curves to amplify and detect specific sequences. In the present invention, we describe methods and molecular structures that, once included in a specific NAAT method, can improve its performance criteria. Summary of the Invention

[0006] In the present invention, unique nucleic acid (NA) constructs and methods are described that, by incorporating them into molecular detection assays, can improve the analytical detection performance defined in a broad sense and reduce the complexity of the workflow and its turnaround time.

[0007] Aspects of the present disclosure provide a reaction chamber comprising: an NA construct comprising a photosensitive chemical moiety, wherein the NA construct is in a first molecular state and is configured to change to a second molecular state upon exposure to light; at least one reagent; and at least one enzyme; wherein the reaction chamber is configured to allow light to reach the nucleic acid construct.

[0008] In some embodiments of the aspects provided herein, the NA construct is an oligonucleotide primer or probe. In some embodiments of the aspects provided herein, the at least one enzyme is a polymerase, reverse transcriptase, terminal transferase, exonuclease, endonuclease, restriction enzyme, or 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 related to 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 comprises another NA construct comprising another photosensitive chemical moiety, wherein the other NA construct is in a third molecular state and is configured to change to a fourth molecular state upon exposure to another light. In some embodiments of the aspects provided herein, the other light is the 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 the other 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 the other NA construct in the fourth molecular state are configured to be inactive. In some embodiments of the aspects provided herein, the NA construct in the first molecular state and the other NA construct in the third molecular state are configured to be inactive in the reaction.

[0009] In some embodiments of aspects provided herein, a NA construct in a second molecular state and another NA construct in a fourth molecular state are configured to be active in a reaction. In some embodiments of 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 aspects provided herein, the photosensitive chemical moiety is located at the 3'-end, 5'-end, or middle of the NA construct. In some embodiments of aspects provided herein, the NA construct further comprises an additional photosensitive chemical moiety. In some embodiments of 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 aspects provided herein, the reaction chamber is a closed-tube reaction chamber

[0010] Another aspect of the present disclosure provides a method of performing a reaction, comprising: activating a reaction chamber to perform a reaction, the reaction chamber comprising: a nucleic acid construct comprising a photosensitive 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 changing the nucleic acid construct to a second molecular state.

[0011] In some embodiments of the aspects provided herein, the NA construct is an oligonucleotide primer or probe. In some embodiments of the aspects provided herein, the at least one enzyme is a polymerase, reverse transcriptase, terminal transferase, exonuclease, endonuclease, restriction enzyme, or ligase. In some embodiments of the aspects provided herein, wherein the at least one reagent includes one or more amplification reagents. In some embodiments of the aspects provided herein, the reaction chamber further includes a target NA. In some embodiments of the aspects provided herein, the enzyme catalyzes the reaction of the nuclear 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 includes another nucleic acid construct containing another photosensitive chemical moiety in the third molecular state, wherein the another nucleic acid construct is configured to change to the fourth molecular state after exposure to another light. In some embodiments of the aspects provided herein, the another light is the light, and wherein the activation light activates the nucleic acid construct. In some embodiments of the aspects provided herein, the method further includes: activating another light to reach another nucleic acid construct. In some embodiments of the aspects provided herein, the method further includes: inactivating the nucleic acid construct in the reaction after the activation light. In some embodiments of the aspects provided herein, the method further includes: activating another nucleic acid construct after the activation light or after activating another light. In some embodiments of the aspects provided herein, the method further includes: inactivating another nucleic acid construct after the activation light or after activating another light. In some embodiments of the aspects provided herein, the method further includes: inactivating the nucleic acid construct in the reaction after the activation light. In some embodiments of the aspects provided herein, the method further includes: inactivating another nucleic acid construct after the activation light or after activating another light. In some embodiments of the aspects provided herein, the method further includes: activating another nucleic acid construct after the activation light or after 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 carried out in the reaction chamber, no external reagents are added to the reaction chamber. In some embodiments of the aspects provided herein, when the reaction is carried out in the reaction chamber, there is no nucleic acid construct, the enzyme is a polymerase, the reaction is a polymerase chain reaction, and the nucleic acid construct is an oligonucleotide primer. At least one reagent or at least one enzyme is removed from the reaction chamber. In some embodiments of the aspects provided herein, the photosensitive chemical moiety is located at the 3-terminus, 5-terminus, or middle of the nucleic acid construct.In some embodiments of aspects provided herein, the nucleic acid construct further comprises additional photosensitive chemical moieties. In some embodiments of aspects provided herein, the target nucleic acid comprises a major allele and a minor allele, and wherein the reaction is a polymerase chain reaction. In some embodiments of aspects provided herein, the nucleic acid construct comprises a sequence complementary to the major allele. In some embodiments of aspects provided herein, the nucleic acid construct in a first molecular state is inactive in a polymerase chain reaction for generating an amplicon of the major allele. In some embodiments of aspects provided herein, the nucleic acid construct in a second molecular state is active in a polymerase chain reaction for generating an amplicon of the major allele. In some embodiments of aspects provided herein, the nucleic acid construct in a second molecular state is inactive in a polymerase chain reaction for generating an amplicon of the major allele. In some embodiments of aspects provided herein, another nucleic acid construct is a primer for the minor allele, and the method further comprises generating an amplicon of the minor allele before activating the light.

[0012] Aspects of the present disclosure provide 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 located at: a) the 3'-end of the nucleic acid construct; b) the 5'-end of the nucleic acid construct; c) between the 3'-end and the 5'-end; d) on or linked to a nucleobase; e) on or linked to a ribose; f) between two consecutive members of the plurality of nucleotides and linked to the two consecutive members of the plurality of nucleotides; or g) a combination thereof.

[0013] In some embodiments of aspects provided herein, the nucleic acid construct is configured to be inactive in a biochemical reaction, where the biochemical reaction is polymerase-catalyzed strand extension, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, or restriction enzyme digestion. In some embodiments of aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photolysis of one or more photocleavable moieties, and wherein the nucleic acid molecule is configured to be active in a biochemical reaction. In some embodiments of aspects provided herein, the nucleic acid construct is a primer, and wherein the biochemical reaction is polymerase-catalyzed strand extension. In some embodiments of aspects provided herein, one or more photocleavable moieties are located at the 3'-end. In some embodiments of 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. In some embodiments of 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 a plurality of nucleotides. In some embodiments of aspects provided herein, the 3'-end is configured to be inactive in a biochemical reaction. In some embodiments of 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, wherein the nucleic acid construct is configured to form a hairpin structure. In some embodiments of aspects provided herein, the first nucleic acid section and the second nucleic acid section do not comprise one or more photocleavable moieties.

[0014] Aspects of the present disclosure provide methods of performing polymerase-catalyzed strand extension using the nucleic acid constructs of the present disclosure, comprising: a) providing a reaction mixture comprising a 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 strand extension; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby performing polymerase-catalyzed strand extension.

[0015] In some embodiments of the aspects provided herein, the performance in b) cannot achieve the performance in c). In some embodiments of the aspects provided herein, the nucleic acid construct remains intact in the reaction mixture prior to the irradiation in c). 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 performance in c) comprises using the nucleic acid molecule formed in c) after irradiation as a primer for polymerase-catalyzed strand extension. In some embodiments of the aspects provided herein, the reaction mixture further comprises another primer, wherein the another primer is active in polymerase-catalyzed strand extension. In some embodiments of the aspects provided herein, prior to the irradiation in c), the another primer is active in polymerase-catalyzed strand extension. In some embodiments of the aspects provided herein, the polymerase-catalyzed strand extension in b) produces an amplicon comprising the another primer. In some embodiments of the aspects provided herein, the polymerase-catalyzed strand extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: in c), 1) performing polymerase-catalyzed strand 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 comprising a solid surface having a plurality of nucleic acid probes at independently locatable positions, the array being configured to contact the fluid; and 3) measuring the hybridization of two or more amplicons with two or more of the plurality of nucleic acid probes when the fluid contacts the array to obtain an amplicon hybridization measurement, wherein the amplicon comprises a quencher. In some embodiments of the aspects provided herein, the polymerase-catalyzed strand 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, the plurality of different probes being immobilized on the surface at different addressable positions, each addressable position comprising a fluorescent moiety; 2) performing PCR amplification on a sample comprising a plurality of nucleotide sequences; the PCR amplification is performed in a fluid, wherein: (i) the nucleic acid construct of the present disclosure is a PCR primer for each nucleic acid sequence and comprises a quencher; and (ii) the fluid contacts the plurality of different probes, wherein the amplicons generated in the PCR amplification hybridize with the plurality of probes, thereby quenching the signal from the fluorescent moiety; 3) detecting the signal from the fluorescent moiety at each addressable position over time; 4) using the signal detected over time and determining 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, polymerase-catalyzed chain extension is quantitative polymerase chain reaction (Q-PCR), and the method further includes, in c): 1) providing a reaction mixture comprising a nucleic acid sample, a primer pair, and a polymerase, the nucleic acid sample comprising at least one template nucleic acid molecule, 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, wherein at least one of the limiting primer and the excess primer is a nucleic acid construct of the present disclosure; 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid molecule and the limiting primer, wherein 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 at different individually locatable positions on the surface of the substrate, wherein the probes have sequence complementarity with the limiting primer and are capable of capturing the limiting primer, and (ii) a detector array configured to detect at least one signal from the locatable positions, wherein the at least one signal indicates binding of the limiting primer to a single probe among the plurality of probes; 4) detecting at least one signal from one or more locatable positions using the detector array 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 binding of the limiting primer to a single probe among the plurality of probes.

[0016] Aspects of the present disclosure provide a system for analyzing at least one target nucleic acid molecule using a nucleic acid construct of the present disclosure, comprising: 1) a reaction chamber containing 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, wherein at least one of the limiting primer and the excess primer is a nucleic acid construct of the present disclosure, and wherein the reaction chamber containing the reaction mixture is configured to facilitate a nucleic acid amplification reaction of the reaction mixture to produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid; 2) a sensor array comprising (i) a substrate comprising a plurality of probes immobilized at different individually locatable positions on the surface of the substrate, wherein the probes have sequence complementarity with the limiting primer and are capable of capturing the limiting primer, and (ii) a detector array configured to detect at least one signal from the locatable positions, wherein the at least one signal indicates binding of the limiting primer to a single probe among 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 locatable positions at multiple time points during the nucleic acid amplification reaction.

[0017] Aspects of the present disclosure provide 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 located at: a) between the 3'-end and the 5'-end of the nucleic acid construct; b) on or attached to a nucleobase; c) on or attached to a ribose; d) between two consecutive members of the plurality of nucleotides and attached to the two consecutive members of the plurality of nucleotides; or e) a combination thereof.

[0018] In some embodiments of aspects provided herein, the nucleic acid construct is configured to be active in a biochemical reaction, where the biochemical reaction is polymerase-catalyzed strand extension, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligation, terminal transferase extension, hybridization, exonuclease digestion, endonuclease digestion, or restriction enzyme digestion. In some embodiments of aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photolysis of one or more photocleavable moieties, and where the nucleic acid molecule is inactive in a biochemical reaction. In some embodiments of aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule shortly after photolysis of one or more photocleavable moieties, and where the nucleic acid molecule is active in a biochemical reaction, where the nucleic acid molecule is located near the 3'-end. In some embodiments of aspects provided herein, the nucleic acid construct is a primer, and where the biochemical reaction is polymerase-catalyzed strand extension. In some embodiments of 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. In some embodiments of aspects provided herein, the nucleic acid construct is configured to form a hairpin structure in the absence of one or more photocleavable moieties, thereby losing activity as a primer in the absence of one or more photocleavable moieties. In some embodiments of 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 a plurality of nucleotides. In some embodiments of aspects provided herein, the nucleic acid construct comprises a first sequence complementary to a template nucleic acid molecule, and where the first sequence is located at or near the 3'-end. In some embodiments of aspects provided herein, the nucleic acid construct further comprises a second sequence complementary to the template nucleic acid molecule, where the second sequence is located at or near the 5'-end, and where at least one of the one or more photocleavable moieties is located between the first sequence and the second sequence. In some embodiments of 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 aspects provided herein, the nucleic acid construct is configured to form a hairpin loop between the first sequence and the second sequence when the first sequence and the second sequence hybridize to the template nucleic acid molecule. In some embodiments of aspects provided herein, the second sequence comprises a 5' to 5' linkage to the remainder of the nucleic acid construct, and where the second sequence is configured to be non-extendable in polymerase-catalyzed strand extension.

[0019] Aspects of the present disclosure provide methods for polymerase-catalyzed strand extension using the nucleic acid constructs of the present disclosure, including: 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 strand extension to effect polymerase-catalyzed strand extension; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light to terminate polymerase-catalyzed strand extension.

[0020] 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 after irradiation, wherein 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, and wherein 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.

[0021] Aspects of the present disclosure provide a method for performing light-enabled nested polymerase chain reaction (PCR), including: 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 present disclosure, wherein each member of the second primer pair is independently a nucleic acid construct of the present disclosure, and wherein the internal nucleic acid sequence is nested within the template nucleic acid molecule; b) using the first primer, subjecting the reaction mixture to conditions for first strand extension to amplify the template nucleic acid molecule to form an amplicon of the template nucleic acid molecule or a complementary sequence of the template nucleic acid molecule; and c) irradiating the reaction mixture with photons of light to inactivate the first pair of primers and terminate the first extension, activate the second pair of primers and initiate a second strand extension using the activated second pair of primers, and 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 manner.

[0022] In some embodiments of the aspects provided herein, the light-enabled PCR is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 1) performing light-enabled PCR on two or more nucleotide sequences in the presence of the first primer pair and the second primer pair to generate two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently locatable positions, the array being configured to contact the fluid; and 3) measuring the hybridization of two or more amplicons with 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 quencher.

[0023] In some embodiments of the aspects provided herein, opto-PCR is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 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 locatable positions, each locatable position comprising a fluorescent moiety; 2) performing PCR amplification on a sample comprising a plurality of nucleotide sequences; the PCR amplification is carried out in a fluid, wherein: (i) each of the first pair of primers and the second pair of primers for each nucleic acid sequence comprises a quencher; and (ii) the fluid is contacted with the plurality of different probes, wherein the amplicons generated in the PCR amplification hybridize with the plurality of probes, thereby quenching the signal from the fluorescent moiety; 3) detecting over time the signal from the fluorescent moiety at each locatable position; 4) using the signal detected over time and determining the amount of amplicon in the fluid; and 5) using the amount of amplicon in the fluid to determine the amount of nucleotide sequence in the sample. In some embodiments of the aspects provided herein, opto-PCR is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 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, and wherein the primer pair comprises a limiting primer and an excess primer, wherein at least one of the limiting primer and the excess primer is a nucleic acid construct of the present disclosure;

[0024] 2) subjecting the reaction mixture to Q-PCR under conditions sufficient to produce at least one target nucleic acid molecule that is 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 at different individually locatable positions on the surface of the substrate, wherein the probes have sequence complementarity with the limiting primer and are capable of capturing the limiting primer, and (ii) a detector array configured to detect at least one signal from the locatable position, wherein the at least one signal indicates binding of the limiting primer to a single probe among the plurality of probes; 4) detecting at multiple time points during the nucleic acid amplification reaction using the detector array at least one signal from one or more locatable positions; and 5) detecting the target nucleic acid molecule based on the at least one signal indicating binding of the limiting primer to a single probe among the plurality of probes.

[0025] Aspects of the present disclosure provide 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 located at: a) between the 3'-end and the 5'-end of the nucleic acid construct; b) on or attached to a nucleobase; c) on or attached to a ribose; d) between two consecutive members of the plurality of nucleotides and attached to the two consecutive members of the plurality of nucleotides; or e) a combination thereof.

[0026] In some embodiments of aspects provided herein, the nucleic acid construct is a probe, and wherein the nucleic acid construct is configured to be inactive in hybridization with a target nucleic acid molecule. In some embodiments of aspects provided herein, the nucleic acid construct is configured to form a nucleic acid molecule after photolysis of one or more photocleavable moieties, and wherein the nucleic acid molecule is configured to be active in hybridization with a target nucleic acid molecule. In some embodiments of aspects provided herein, the nucleic acid construct comprises a free end. In some embodiments of aspects provided herein, the nucleic acid construct includes a fixed end or an end that is non-extendable in polymerase-catalyzed strand extension. In some embodiments of 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, wherein the selected nucleobase is configured to hybridize with a target nucleic acid molecule in the absence of the one or more photocleavable moieties. In some embodiments of 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 a plurality of nucleotides. In some embodiments of aspects provided herein, the nucleic acid construct comprises a first nucleic acid portion and a second nucleic acid portion complementary to the first nucleic acid portion, wherein the nucleic acid construct is configured to form a hairpin structure. In some embodiments of aspects provided herein, the first nucleic acid portion and the second nucleic acid portion do not comprise one or more photocleavable moieties.

[0027] Aspects of the present disclosure provide methods of hybridization using the nucleic acid constructs of the present disclosure, comprising: a) providing a reaction mixture comprising a nucleic acid construct and a target nucleic acid molecule; b) subjecting the reaction mixture to conditions for hybridization; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light, thereby effecting hybridization.

[0028] In some embodiments of aspects provided herein, the subjecting in b) does not enable the effecting in c). In some embodiments of aspects provided herein, the nucleic acid construct remains intact in the reaction mixture prior to the irradiation in c). In some embodiments of aspects provided herein, the method further comprises: in c), cleaving one or more photocleavable moieties. In some embodiments of aspects provided herein, the method further comprises: in c), forming a nucleic acid molecule. In some embodiments of aspects provided herein, the irradiation disrupts the hairpin structure of the nucleic acid construct and forms a nucleic acid molecule.

[0029] Aspects of the present disclosure provide 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 located at: a) between the 3'-end and the 5'-end of the nucleic acid construct; b) on or attached to a nucleobase; c) on or attached to a ribose; d) between two consecutive members of the plurality of nucleotides and attached to the two consecutive members of the plurality of nucleotides; or e) a combination thereof.

[0030] In some embodiments of the aspects provided herein, the nucleic acid construct is a probe, and wherein 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 upon photocleavage of one or more photocleavable moieties, and wherein 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 a free end. In some embodiments of the aspects provided herein, the nucleic acid construct includes a fixed end or an end that is non-extendable in polymerase-catalyzed strand 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 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 a selected nucleobase, wherein the selected nucleobase is configured to hybridize to 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 portion and a second nucleic acid portion complementary to the first nucleic acid portion, wherein 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 portion or the second nucleic acid portion does not comprise the one or more photocleavable moieties.

[0031] Aspects of the present disclosure provide a method of hybridization using the nucleic acid construct of the present disclosure, comprising: a) providing a reaction mixture comprising the nucleic acid construct and a target nucleic acid molecule; b) subjecting the reaction mixture to conditions for hybridization; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light to terminate hybridization.

[0032] In some embodiments of aspects provided herein, the method further comprises: in c), cleaving one or more photocleavable moieties. In some embodiments of aspects provided herein, the method further comprises: in c), forming a nucleic acid molecule. In some embodiments of aspects provided herein, the method further comprises: in c), forming a hairpin structure in the nucleic acid molecule. In some embodiments of aspects provided herein, the method further comprises performing polymerase-catalyzed strand extension, wherein: 1) the reaction mixture further comprises a polymerase and a primer, wherein in b) the nucleic acid construct hybridizes to the target nucleic acid molecule in b); 2) the reaction mixture in b) is subjected to conditions for polymerase-catalyzed strand extension using the primer, wherein the polymerase-catalyzed strand extension stalls at or near the position where the nucleic acid construct hybridizes to the target nucleic acid molecule to form a duplex; and 3) after the radiation in c), the duplex is removed and the single-stranded sequence that was previously hybridized to the nucleic acid construct is exposed, thereby allowing the polymerase-catalyzed strand extension to continue and extend through the single-stranded sequence. In some embodiments of aspects provided herein, the polymerase-catalyzed strand extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 1) performing polymerase-catalyzed strand extension on two or more nucleotide sequences comprising the target nucleic acid molecule in the presence of the nucleic acid construct of the present disclosure, thereby generating two or more amplicons in a fluid;

[0033] 2) Providing an array comprising a solid surface having a plurality of nucleic acid probes at independently locatable positions, the array being configured to contact a fluid; and 3) Measuring the hybridization of two or more amplicons with two or more of the plurality of nucleic acid probes while the fluid is in contact with the array to obtain an amplicon hybridization measurement, wherein the amplicon comprises a quencher. In some embodiments of the aspects provided herein, the polymerase-catalyzed strand extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 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 locatable positions, each locatable position comprising a fluorescent moiety; 2) Performing PCR amplification on a sample comprising a plurality of nucleotide sequences containing a target nucleic acid molecule; the PCR amplification is performed in a fluid comprising the nucleic acid construct of the present disclosure, wherein: (i) Each PCR primer of each nucleic acid sequence comprises a quencher; and (ii) The fluid is in contact with the plurality of different probes, wherein the amplicons generated during the PCR amplification hybridize with the plurality of probes, thereby quenching the signal from the fluorescent moiety; wherein radiation occurs during the PCR; 3) Detecting over time the signal from the fluorescent moiety at each locatable position; 4) Using the signal detected over time and determining the amount of amplicon in the fluid; and 5) Using the amount of amplicon in the fluid to determine the amount of nucleotide sequence in the sample. In some embodiments of the aspects provided herein, the polymerase-catalyzed strand extension is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 1) Providing a reaction mixture comprising a nucleic acid sample, a primer pair, and a polymerase, the nucleic acid sample containing at least one template nucleic acid molecule containing a target nucleic acid molecule, wherein the primer pair has sequence complementarity with the at least one template nucleic acid molecule, wherein the primer pair comprises a limiting primer and an excess primer, and wherein the reaction mixture further comprises at least one of the nucleic acid constructs of the present disclosure; 2) Subjecting the reaction mixture to Q-PCR under conditions sufficient to produce an amplification product of the template nucleic acid molecule and the limiting primer, the amplicon comprising the limiting primer; 3) Contacting the reaction mixture with a sensor array having (i) a substrate comprising a plurality of probes immobilized on the surface of the substrate at different individually locatable positions, wherein the probes have sequence complementarity with the limiting primer and are capable of capturing the limiting primer, and (ii) a detector array configured to detect at least one signal from the locatable positions, wherein the at least one signal indicates the binding of the limiting primer to a single probe among the plurality of probes; 4) Detecting at multiple time points during the nucleic acid amplification reaction at least one signal from one or more locatable positions using the detector array; and 5) Detecting the target nucleic acid molecule based on the at least one signal indicating the binding of the limiting primer to a single probe among the plurality of probes.

[0034] Aspects of the present disclosure provide a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photocleavable 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; wherein each of the one or more photocleavable moieties is independently located at: a) on or linked to a nucleobase; b) on or linked to a ribose; or c) a combination thereof.

[0035] 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 wherein 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.

[0036] Aspects of the present disclosure provide a method for performing polymerase-catalyzed strand 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 primer strand extension 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 strand extension through the nucleic acid sequence.

[0037] In some embodiments of the aspects provided herein, the subjecting in b) does not enable the performing in c). In some embodiments of the aspects provided herein, the nucleic acid construct remains intact in the reaction mixture prior to the irradiation in c). 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 performing in c) comprises digesting the nucleic acid molecule formed in c) by an exonuclease after the irradiation, wherein the polymerase is the exonuclease. In some embodiments of the aspects provided herein, the performing in c) comprises extending the primer through the nucleic acid sequence after the irradiation and / or after the digestion.

[0038] Aspects of the present disclosure provide methods for polymerase-catalyzed strand extension using nucleic acid constructs, including: 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 the 3'-end and a second sequence located at or near the 5'-end, and wherein the first sequence is active in polymerase-catalyzed strand extension; b) subjecting the reaction mixture to conditions for polymerase-catalyzed strand extension to effect polymerase-catalyzed strand extension and generate a plurality of first amplicons, the first amplicons comprising the sequences of the first and second sequences or the complementary sequences of the first and second sequences; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light to cleave the nucleic acid construct and generate a plurality of second amplicons, the second amplicons comprising the first sequence or the complementary sequence of the first sequence, provided that each of the plurality of second amplicons does not comprise the second sequence or the complementary sequence of the second sequence.

[0039] Aspects of the present disclosure provide methods for polymerase-catalyzed strand extension using at least one of the nucleic acid constructs of the present disclosure, including: a) providing a reaction mixture comprising a nucleic acid construct, a template nucleic acid molecule, and a polymerase, b) subjecting the reaction mixture to conditions for polymerase-catalyzed strand extension; and c) irradiating the reaction mixture or the nucleic acid construct with photons of light to effect polymerase-catalyzed strand extension, wherein the polymerase-catalyzed strand extension is PCR, RT-PCR, QPCR, or qRT-PCR.

[0040] 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 liquid-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 solid-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 more than two nucleic acid constructs and is a combination of a primer for PCR, RT-PCR, QPCR, or qRT-PCR, a liquid-phase probe for PCR, RT-PCR, QPCR, or qRT-PCR, and a solid-phase probe for PCR, RT-PCR, QPCR, or qRT-PCR, wherein each is independently selected.

[0041] Aspects of the present disclosure provide an automated microarray system for quantifying microarray data, comprising: a) a solid support having a surface and a plurality of different probes, wherein the plurality of different probes are immobilized on the surface; b) a fluid volume containing an analyte, wherein the fluid volume is in contact with the solid support, wherein at least one of the plurality of different probes and the analyte comprises at least one of the nucleic acid constructs of the present disclosure; c) a detector or detection assembly configured to detect signals measured at multiple time points from each of a plurality of spots on the solid support when the fluid solvent is in contact with the solid support, wherein the signal is an optical signal or an electrochemical signal; d) a computer configured to convert the signals into microarray data, wherein the computer further comprises instructions configured to cause the computer to process the microarray data according to a processing method, the processing method comprising: 1) determining an estimate of the interaction between the plurality of different probes and the analyte, including (i) analyzing an expression and (ii) calibrating the microarray by using at least one standard probe on the solid support; 2) generating a stochastic matrix that utilizes the estimate in a Markov chain model, the Markov chain model including modeling hybridization, cross-hybridization, and transition probabilities between states; 3) using the detector or detector assembly to obtain affinity-based array data; 4) using the affinity-based array data for the stochastic matrix; 5) applying an optimization algorithm selected from maximum likelihood estimation algorithms, maximum a posteriori probability criteria, constrained least squares calculations, and any combination thereof, the optimization algorithm exploiting and not suppressing non-specific interactions by treating non-specific interactions 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 the affinity-based array data obtained by using the detector or detector assembly.

[0042] Aspects of the present disclosure provide an integrated biosensor array sequentially comprising 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, wherein: a) the molecular recognition layer comprises a plurality of different probes attached to different independently positionable locations, each independently positionable location being configured to receive an excitation photon flux directly from a single source located on one side of the molecular recognition layer, wherein the molecular recognition layer transmits light to the optical layer, wherein at least one of the plurality of different probes comprises at least one of the nucleic acid constructs; b) the optical layer comprises a filter layer, wherein the optical layer transmits light from the molecular recognition layer to the sensor layer, thereby filtering the transmitted light; and the sensor layer comprises an optical sensor array that detects the filtered light transmitted through the optical layer, the sensor layer comprising sensor elements fabricated using a CMOS manufacturing process; wherein the molecular recognition layer, the optical layer, and the sensor layer comprise an integrated structure, wherein the molecular layer is in contact with the optical layer and the optical layer is in contact with the sensor layer.

[0043] Incorporation by reference

[0044] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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 invention will be obtained by reference to the following detailed description of illustrative embodiments that make use of the principles of the invention, and to the accompanying drawings (also referred to herein as "FIGURES"), wherein:

[0046] Figure 1 Examples of photocleavable groups are shown (LG = leaving group);

[0047] Figure 2 Examples of nucleic acid molecules containing photocleavable bonds are shown;

[0048] Figure 3 Examples of reagents having a photocleavable structure are shown;

[0049] Figure 4 Examples of nucleic acid constructs containing 3'-end extension inhibitors are shown;

[0050] Figure 5 Examples of reagents for 3'-end extension inhibitors are shown;

[0051] Figure 6 Examples of nucleic acid constructs containing 5'-end extension inhibitors are shown;

[0052] Figure 7 Examples of 5'-end extension inhibitors are shown;

[0053] Figure 8 Examples of nucleic acid constructs containing photocleavable base pairing inhibitors are shown;

[0054] Figure 9 Examples of reagents for photocleavable base pairing inhibitors are shown;

[0055] Figure 10 Examples of photoinitiated primers are shown;

[0056] Figures 11A - 11D Examples of photoresistant primers are shown;

[0057] Figures 12A - 12B Examples of photoinitiated hybridization probes are shown;

[0058] Figure 13 Examples of photoresistant hybridization probes are shown;

[0059] Figure 14 Shows examples of 5'-terminal exonuclease protectants;

[0060] Figure 15 Schematically shows an example of photo-induced nested PCR; and

[0061] Figure 16 Schematically shows another example of photo-induced nested PCR. Detailed Description

[0062] Although various embodiments of the present invention have been shown and described herein, it will be readily apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, various changes, alterations, and substitutions may be contemplated by those skilled in the art. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0063] The present disclosure provides chemically modified and photo-triggered nucleic acid (NA) constructs with unique properties such that the constructs can have their chemical structure transformed when triggered photochemically by photons of light, thereby altering their chemical / biochemical functions. These photo-triggered change properties of the chemically modified NA constructs can be used in molecular detection reactions / processes.

[0064] In some embodiments, the photo-triggered NA constructs can be used in NA detection assays used in life science research and molecular diagnostics. In these assays, the NA molecules are the targets of the assay and / or are used as molecular recognition elements of the assay. The photo-triggered NA constructs are added to the assay such that by appropriately applying photons of light to the system, the photo-triggered NA constructs can improve assay detection accuracy and / or reduce workflow complexity and / or shorten turnaround time. Other advantages are possible.

[0065] Some example detection assays are NA amplification testing (NAAT) using the polymerase chain reaction process; NA affinity-based detection systems utilizing two-dimensional locatable DNA microarrays; and DNA sequencing arrays that incorporate the solid-phase synthesis sequencing (SBS) method.

[0066] Light-triggered nucleic acid constructs and their use in surgery

[0067] As used herein, the term "light-triggered nucleic acid construct" or "NA construct" generally refers to an NA molecule that includes 1) one or more photosensitive systems or photosensitive chemical moieties that can be in a first molecular state prior to exposure to photons 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 photons of light are 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 the first molecular state to a second molecular state, which in turn alters the biochemical properties of the NA construct. For example, photons of light can cause a chemical change in the NA construct by breaking or forming one or more chemical bonds in the one or more photosensitive systems or photosensitive chemical moieties.

[0068] The NA construct can comprise 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 comprises 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 can comprise no more than 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.

[0069] As used herein, the term "photosensitive system" or "photosensitive chemical moiety" generally refers to a single or multiple chemical structures that contain a photo-labile chemical bond. The photosensitive system or photosensitive chemical moiety can absorb photons of a specific wavelength to increase the reaction rate of certain chemical reactions in which the photosensitive system or photosensitive chemical moiety can participate. Other descriptive terms, such as photosensitive, photocleavable, photoactivatable, photo-labile, photoactivatable or photocleavable, can be used interchangeably with the term photosensitive.

[0070] As used herein, the term "molecular state" generally refers to the atomic and molecular structures and chemical, physico-chemical, biochemical, electro-chemical and photochemical properties associated with one or more specific molecules (such as an NA construct).

[0071] As used herein, the term "biochemical property" generally refers to the characteristics of a NA construct in biological and chemical reactions. The biochemical properties of a nucleic acid construct can change depending on the molecular state of the NA construct. The molecular state of a NA construct can be changed by the reaction of one or more photosensitive systems or photosensitive chemical moieties. Additionally, the biochemical properties of a NA construct in a first molecular state can be different from those in a second molecular state.

[0072] 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.

[0073] Each NA construct can have different biochemical properties, including different reactivities in biochemical reactions. Examples of biochemical properties can include, for example, whether the NA construct can promote, block, or participate in a specific biochemical reaction, such as a polymerase chain reaction or a hybridization reaction. Different biochemical properties can be triggered by photons of light.

[0074] The biochemical properties of a NA construct can include different molecular states of the NA construct. For example, the biochemical properties of a NA construct in a first molecular state can be different from those in a second molecular state. The biochemical properties of the NA construct in the first and second molecular states can be designed such that photons of light can initiate and / or terminate specific molecular reactions that the NA construct can participate in. This change in molecular state can be triggered by photons of light. Examples of biochemical properties of a primer can be an active primer and an inactive primer, etc. In some embodiments, the present disclosure describes methods and systems for using photons of light to switch a primer between "active" and "inactive" molecular states in an extension reaction. In some embodiments, the active / inactive molecular state conversion can be achieved by cleaving a photocleavable bond within the nucleic acid construct. In the present disclosure, the terms "latent", "inactivated", "inert", and "non-functional" are synonymous with the term "inactive". Similar terms are used when describing a "probe".

[0075] A NA construct is typically maintained in a reaction chamber, and photons of light can be applied to the reaction chamber by a light source system.

[0076] 1. Photosensitive systems or photosensitive chemical moieties

[0077] A photosensitive system or a photosensitive chemical moiety can be one or more chemical structures that contain photosensitive chemical bonds. When irradiated with photons of light, the photosensitive system or the photosensitive chemical moiety can change its structure or chemical properties. The photosensitive system or the photosensitive chemical moiety can absorb photons of a specific wavelength to increase the reaction rate of certain chemical reactions that the photosensitive system or the photosensitive chemical moiety can participate in. For example, these chemical reactions can:

[0078] · change the chemical structure of the photosensitive system or the photosensitive chemical moiety;

[0079] · break the structure of the photosensitive system or the photosensitive chemical moiety into multiple smaller structures;

[0080] · add an external structure to the photosensitive system; or

[0081] · form one or more intramolecular bonds within the photosensitive system or the photosensitive chemical moiety;

[0082] · form one or more intermolecular bonds between two or more photosensitive systems or photosensitive chemical moieties or external chemical structures (relative to the photosensitive system and the photosensitive chemical moiety); or

[0083] · a combination thereof.

[0084] In some embodiments, the photosensitive system or the photosensitive chemical moiety can be incorporated into the structure of a nucleic acid molecule. For example, the photosensitive system or the photosensitive chemical moiety can be:

[0085] · placed on one or more functional groups of the NA, for example, on the heteroatom of a nucleobase or on the 3'-OH of a ribose ring;

[0086] · used as part of a linker group between two NA sequences, where, in the presence of photons of light, the linker group can break into smaller groups, thereby separating two previously linked NA sequences into two independent nucleic acid sequences (i.e., they are no longer linked);

[0087] · placed at the 5'-end of the NA strand, where the presence of the photosensitive system or the photosensitive chemical moiety prevents certain biochemical reactions from occurring at the 5'-end of the nucleic acid strand (e.g., a photosensitive group on the 5'-phosphate group of the terminal NA);

[0088] · placed at the 3'-end of the NA strand, where the presence of the photosensitive system or the photosensitive chemical moiety prevents certain biochemical reactions from occurring at the 3'-end of the NA strand (e.g., a photosensitive group on the 3'-OH group of the terminal NA); or

[0089] · a combination thereof.

[0090] Examples of some 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. Examples of some photosensitive chemical moieties can include o-nitrobenzyloxy linkers, o-nitrobenzylamino linkers, α-substituted o-nitrobenzyl linkers, o-nitroveratryl linkers, benzoyl linkers, p-alkoxybenzoyl linkers, benzoin linkers or pivaloyl linkers. R.J.T. 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 herein by reference in its entirety.

[0091] For example, nitrobenzyl-based chemical moieties can be, for example, those shown below:

[0092] R is not H

[0093]

[0094] NA is a nucleic acid, nucleotide, nucleobase, 5’ phosphate, or part of a linker

[0095] Nitrobenzyl-based chemical moieties can undergo Norrish type II mechanism by incident photons to provide cleavage products as shown below:

[0096]

[0097] Some examples of photocleavable groups can be found in Figure 1 . Figure 1 where LG refers to the leaving group. Some examples among them are 4-methoxy-7-nitroindolinyl (MNI), I-nitrobenzyl (O-NB), 3-(4,5-dimethoxy-2-nitrophenyl) 2-butyl (DMNPB) 4-carboxymethoxy-5,7-dinitroindolyl (CDNI).

[0098] 2. Molecular states

[0099] As used herein, the term "molecular state" generally refers to the atomic and molecular structures and chemical, physicochemical, biochemical, electrochemical, and photochemical properties associated with one or more specific molecules (e.g., NA constructs). For example, an NA construct may display its molecular state in a defined aqueous environment or under other reaction conditions of nucleic acids 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 (e.g., ligation, coupling reactions, chain extension, chain digestion, etc.).

[0100] The biochemical properties of an NA construct may include different molecular states of the NA construct. For example, the biochemical properties of an NA construct in a first molecular state may be different from those in a second molecular state. The biochemical properties of the NA constructs in the first and second molecular states can be designed such that photons of light can initiate and / or terminate specific molecular reactions in which the NA construct can participate. Such changes in molecular state can be triggered by photons of light. For example, a photochemical reaction can alter the molecular structure of a nucleic acid reagent, thereby changing the biochemical properties and reactivity of the nucleic acid reagent in a biochemical reaction.

[0101] For example, an NA construct can be an "active primer" which is a primer in the traditional PCR sense and can support polymerase-facilitated nucleotide addition (i.e., extension of the growing chain). In other words, an active primer is capable of base pairing with a complementary template sequence under experimental conditions to form an antiparallel duplex structure and may have a native (usable) 3'-hydroxyl group to which a polymerase can add another nucleotide, thereby extending the primer by at least one base. An "inactive primer" can be a primer that cannot support or facilitate nucleotide addition because it cannot bind the template strand sufficiently (cannot base pair) or the terminal nucleotide lacks a usable 3'-hydroxyl group. For example, placing a photocleavable chemical moiety on the 3'-hydroxyl group of the terminal nucleotide can block the polymerase reaction. 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 used to extend the growing chain. Similar mechanisms can be applied to ligase-catalyzed reactions to block and unblock ligation sites on the NA. Other examples of base pairing inhibitors can be chemical groups placed on at least one strand of the DNA (e.g., the growing strand) such that they prevent the DNA strand from binding to its complementary strand due to steric or other chemical reasons.

[0102] In some embodiments, the present disclosure describes methods and systems that utilize photons of light to switch primers between "active" and "inactive" molecular states during an extension reaction. By changing the molecular state of the primers, the present disclosure enables new amplification strategies, particularly with respect to "closed-tube" methods (i.e., no additional reagents are added after the PCR reaction has started) and "multiplexing" methods, which are highly desirable in the field of nucleic acid (NA)-based amplification diagnostics. The present disclosure describes methods for effectively changing the composition (and properties, such as molecular state) of a primer set during an amplification reaction without adding or removing reagents between reactions or changing the reaction chamber. Thus, the "inactive" molecular state describes the state and functional state of a particular primer, rather than its use. An inactive primer can become active when exposed to light, and vice versa. Although the following examples show individual components for simplicity and illustration, some complex multiplex assays may require up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 primers, or even more. The active / inactive molecular state transitions can be triggered by the same light exposure or different light exposures. For example, one photocleavable chemical moiety can react at one wavelength of light, while another photocleavable chemical moiety can react at another wavelength of light.

[0103] In some embodiments, the active / inactive molecular state transition can be achieved by cleaving a photocleavable bond in an NA construct, thereby cleaving the original nucleic acid strand into parts. In some embodiments, the active / inactive molecular state transition can be achieved by cleaving a photocleavable bond in an NA construct, thereby removing a blocking group from certain nucleic acid units of the NA construct. For example, upon exposure to light, a blocking group on a 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 are the same before and after the removal of the blocking group).

[0104] In the present disclosure, the terms "latent," "inactivated," "inert," and "non-functional" are synonymous with the term "inactive." Similar terms are used when describing "probes" that are related to signal transduction and do not participate in polymerase-catalyzed extension (e.g., PCR).

[0105] 3. Biochemical properties

[0106] As used herein, the term "biochemical property" generally refers to the characteristics of an NA construct in biological and chemical reactions, including, for example, the tendency or ability of the NA construct to participate in certain biochemical or chemical reactions. In addition, the biochemical properties of an NA construct in a first molecular state can be different from those in a second molecular state. An example of such a biochemical property can be the ability of an NA construct to initiate or terminate a molecular reaction upon irradiation with photons of light. For example, biochemical properties can include, but are not limited to, the ability to:

[0107] · The single-stranded form of the NA construct base pairs with itself and forms a hairpin structure, or forms a homodimer with another replicant of the NA construct and a heterodimer with another NA molecule;

[0108] · DNA polymerase extends the NA construct using a template NA;

[0109] · RNA polymerase extends the NA construct using a template NA;

[0110] · Reverse transcriptase extends the NA construct using a template NA;

[0111] · Terminal transferase extends the NA construct;

[0112] · Exonuclease digests the NA construct;

[0113] · Endonuclease cleaves the NA construct;

[0114] · Restriction enzyme cleaves the NA construct at specific coordinates of its sequence; and

[0115] · Ligase uses the NA construct as a substrate or template.

[0116] The biochemical properties of the nucleic acid construct can be altered according to the molecular state of the NA construct. The molecular state of the NA construct can be altered by the reaction of one or more photosensitive systems or photosensitive chemical moieties.

[0117] 4. Reaction chambers

[0118] As used herein, the term "reaction chamber" generally refers to a physical system that confines an aqueous solution or other medium and in which the NA construct is present. The reaction chamber may allow photons of light to reach the NA construct located inside and may have a temperature controller to set and dynamically change the temperature inside the chamber, such as the temperature of the aqueous solution.

[0119] In some embodiments, the reaction chamber may have a volume of from about 0.1 nanoliter (nL) to about 10 milliliters (mL). In some cases, the reaction chamber may have a volume of 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.

[0120] The reaction chamber may have a temperature in the range of from about 4 °C to about 100 °C. The temperature of the reaction chamber can 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 range of the reaction chamber can be from about 30 °C to about 95 °C, and the accuracy of controlling the temperature can be controlled within ±0.1 °C.

[0121] 5. Light

[0122] As used herein, the term "light" with respect to the reaction chamber generally refers to a photon flux that is restricted within a specific wavelength range and applied to the reaction chamber for a period of time. 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 optical power of the light can be from about 0.001 mW / cm 2 to about 1,000 mW / cm 2 、from about 0.01 mW / cm 2 to about 100 mW / cm 2 、from about 0.1 mW / cm 2 to about 10 mW / cm 2 、from about 0.05 mW / cm 2 to about 20 mW / cm2 、 or about 0.02 mW / cm 2 to about 50 mW / cm 2 。 The duration of exposure to light can be from about 0.1 second (sec) to about 10,000 sec, from about 0.25 sec to about 5,000 sec, from about 0.5 sec to about 1,000 sec, from about 0.75 sec to about 500 sec, from about 1 sec to about 100 sec.

[0123] 6. Light sources

[0124] As used herein, the term "light source system" generally refers to a combination of devices that together generate photons of light within a defined wavelength and control the power applied to the nucleic acid construct. The light source system can include a photon source (which can be a light-emitting diode (LED)), a laser source, an incandescent lamp, or a gas discharge lamp. The light source system can include a power control device for controlling the light output power. The light source system can include a wavelength selection filter to ensure that its output light is within the desired wavelength. The light source system can include optical devices for focusing and / or collimating its output photon flux.

[0125] Modification of nucleic acids to achieve photosensitivity

[0126] Various methods can be used to prepare NA molecules or structures with photochemical properties. For example, the method can include using 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. Next, a special phosphoramidite can 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 be cleaved from the solid support. The cleaved nucleic acid molecule may or may not be subjected to additional reactions or treatments (such as purification, modification, etc.).

[0127] Examples of the photosensitive system or photosensitive chemical moiety as described above can be a photocleavable group on a partial nucleotide (between the ribose moiety or the nucleobase moiety or any chemical moiety of the nucleic acid), or as part of a linker between two single-stranded nucleic acids. The linker can have two photocleavable bonds, each of which binds to a nucleic acid segment. There can be various types of nucleic acid modifications that can achieve photosensitivity as shown elsewhere in this disclosure. Some specific examples are given below.

[0128] 1. Photocleavable structures

[0129] Figure 2Shows an example 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 may include one or more photocleavable bonds. When the photocleavable NA structure molecule is exposed to light from a light source, due to the presence of the photocleavable bond, the molecule may be cleaved into two or more segments. As a result, the original NA sequence (A) can be broken into, for example, two smaller sequences (A1) and sequence (A2), as Figure 2 shown.

[0130] In some embodiments, the photosensitive system can be designed such that after cleavage, the cleaved chemical residues remain at the 3'-end of sequence (A1) and / or the 5'-end of sequence (A2). Sequence (A) can be single-stranded or double-stranded NA. When sequence (A) is double-stranded NA, there may be at least one photocleavable bond on each strand. In some embodiments, the position of the photocleavable bond can be adjacent to the same paired NA, such that cleavage can generate blunt ends in sequences (A1) and (A2) respectively. In some embodiments, the position of the photocleavable bond can be staggered on each strand, such that after cleavage, sequences (A1) and (A2) can have sticky ends (overhangs).

[0131] Figure 3 An example compound having a photocleavable bond is shown in Figure 3 As shown, in the chemical nucleic acid molecule synthesis of an NA construct, this compound can be used together with other DMT phosphoramidite-containing monomers to insert a photocleavable bond into the NA chain. In the example shown in Figure 3 an o-nitrobenzyl photo-labile blocking group can link two segments of an NA molecule. In the absence of radiation, the photo-labile bond in the NA construct is intact. The intact NA construct can exhibit the 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, and 3'-hydroxyl and 5'-phosphorylated ends can be generated in the two newly formed NA fragments respectively. Due to the cleavage of the photo-labile bond, the molecular state of the original NA construct can be changed to a new molecular state associated with the two nucleic acid fragments. This is an example of a light-triggered change in molecular state.

[0132] 2. 3'-end extension inhibitors

[0133] In an NA construct containing a 3'-end extension inhibitor, a photosensitive system or a photosensitive chemical moiety can be chemically attached to the 3'-terminal terminal unit of the NA sequence. Due to the presence of the 3'-end extension inhibitor, the 3'-extension site is blocked by extension enzymes (including but not limited to polymerases, transcriptases, terminal transferases, etc.), such that the enzyme cannot extend the growing chain from the 3'-terminal terminal unit, and the extension of the growing chain by the enzyme is inhibited. However, exposure to light can remove the block and allow the enzyme to extend the growing chain. Figure 4 An example of an NA construct is shown, where a chemical reaction promoted by DNA polymerase is initially blocked by a photosensitive system or a photosensitive chemical moiety at the 3'-end of the primer. Then, exposure to light can remove the blocking group, and the enzyme can synthesize primer DNA using the template. In this case, light is directed to the molecule, and the polymerase inhibitor can be removed from the molecule, such that the molecule can be extended by the polymerase.

[0134] Figure 5 Examples of 3'-terminal terminal units into which a 3'-end polymerase extension inhibitor can be inserted are shown. The DMT phosphoramidite monomer and this 3'-terminal terminal unit can be used for the chemical synthesis of nucleic acid molecules (oligonucleotides). Once the 3'-terminal terminal unit is installed at the 3'-end and in the absence of light exposure, the o-nitrobenzyl photo-labile blocking group on the 3'-hydroxy group of the ribose ring of the 3'-terminal terminal unit can prevent DNA polymerase from extending at this position. Upon light exposure, the blocking group can be removed to expose the naked 3'-hydroxy group on the ribose ring and restore the extension ability of the NA construct.

[0135] 3. 5’-end exonuclease protectants

[0136] In an NA construct containing a 5'-end exonuclease protector, a photosensitive system or a photosensitive chemical moiety can be chemically attached to the 5'-terminal terminal unit of the nucleic acid sequence. Due to the presence of the 5'-end exonuclease protector, the digestion of the 5'-end of the chain by the exonuclease can be blocked, and the chain can be protected from cleavage or digestion. Exposure to light can remove the block and allow 5'-to-3' chain digestion. For example, Figure 6 An example of such a nucleic acid structure is shown, where the activity of the DNA polymerase 5'-end exonuclease can be initially blocked, and after removing the 5'-end blocking group upon exposure to light, the activity can be restored and the enzyme can be allowed to digest the chain, as Figure 6 shown. Examples of photosensitive systems or photosensitive chemical moieties that can be used as 5'-end exonuclease protectors are shown as Figure 7 shown. The hydrophobic tail on the 5'-phosphodiester of the nucleotide can block the digestion of the nucleic acid containing the 5'-terminal nucleotide by the exonuclease. Exposure of the nucleic acid to light can remove the blocking group on the 5'-phosphate and allow the exonuclease to digest the 5'-terminal nucleotide.

[0137] 4. Base pairing inhibitors

[0138] In an NA construct comprising a base pairing inhibitor, a photosensitive system or a photosensitive chemical moiety can be chemically attached to one or more nucleobases of nucleotide units within the NA construct. The base pairing inhibitor can be in series within the nucleic acid sequence or can be distributed within the nucleic acid sequence. The presence of the base pairing inhibitor can inhibit base pairing between the complementary sequence and the NA construct. Subsequent exposure to light can remove the blocking group and allow normal base pairing to occur between the de-blocked NA construct and the complementary sequence. Examples of such NA constructs are shown in Figure 8 which shows exemplary NA molecules comprising a photosensitive base pairing inhibitor. When the NA molecule is not exposed to a light source, at least the subunits of the NA molecule lack base pairing ability due to the presence of the base pair inhibitor. This base pairing ability can be restored by subjecting the nucleic acid molecule to a light source for a given period of time (e.g., greater than or equal to about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min or more). Optionally, the nucleic acid molecule can be subjected to a light source until photolysis is complete.

[0139] A variety of compounds can be used as photosensitive base pairing inhibitors, such as Figure 9 the compounds shown. Figure 9 The reagents shown and other DMT phosphoramidite monomers can be used for chemical NA molecule synthesis. After installation, the o-nitrobenzyl photolabile blocking group 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 group (shown on the nucleobase) can be removed to restore the base pairing ability of the nucleic acid molecule. Photosensitive base pairing inhibitors comprising a photocleavable chemical moiety on the nucleobase, such as Figure 9 the compounds shown in

[0140] as disclosed above, different chemical modifications on the nucleic acid can be used to construct different types of NA constructs, as shown below, for different uses.

[0141] Types of light-triggered nucleic acid constructs and their uses

[0142] The present disclosure also provides NA constructs that can be triggered when an NA molecule is exposed to photons of light and that have unique biochemical properties related to molecular detection. When used in a reaction chamber, these NA constructs can increase or decrease the rate, specificity, yield, and / or fidelity of biochemical reactions used in common molecular detection assays. Example 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. If the reaction includes NA components that act as targets and / or reagents and / or catalysts and / or otherwise, the present disclosure can be used to moderate the reaction by replacing natural components with NA constructs or inserting NA constructs into natural components. Examples of nucleic acid molecules or structures with photochemical properties can 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 a target analyte (e.g., an amplicon) during or at the end of a given reaction (e.g., PCR or RT-PCR). There can be many different types of nucleic acid constructs, as shown below.

[0143] 1. Light-initiated primers

[0144] A photoinitiated primer is an NA sequence that cannot base pair with a complementary NA sequence template and / or cannot create a nucleic acid polymerase initiation site due to the presence of a photosensitive system or a photosensitive chemical moiety, or a blocking group that contains or is linked to a photosensitive system or a photosensitive chemical moiety. When light is applied, these photoinitiated primers can remove the blocking group and can subsequently be capable of nucleic acid synthesis in the presence of a nucleic acid template and a nucleic acid polymerase.

[0145] Figure 10 Examples of photoinitiated primers and their applications in biochemical processes are shown, and the corresponding example sequences are listed in Table I. In one embodiment, the primer can include an internal photocleavable bond modification in a linear NA construct (e.g., a primer), where the photoinitiated primer is designed to have a photocleavable modification such that when exposed to light, the blocking strand can be removed, and the resulting primer can form an appropriate initiation site for polymerase action ( Figure 10 , upper figure). In another embodiment, the primer can include a polymerase blocker at the 3’ end of a nucleic acid construct (e.g., a primer), where the photoinitiated primer is designed using a 3’-end extension inhibitor modification such that when light is applied, the inhibition can be removed, thereby creating a suitable initiation site for polymerase action ( Figure 10, the second figure from the top). In one embodiment, a primer may comprise one or more base pairing inhibitors distributed within the sequence of a NA construct (e.g., a primer), wherein the design of a photoinitiated primer is modified using the base pairing inhibitor such that the primer containing the base pairing inhibitor cannot initially hybridize to a template or form a polymerase initiation site. When exposed to light, the inhibition can be removed, and the resulting primer can create a suitable initiation site for polymerase action ( Figure 10 , the third figure from the top). In another embodiment, a primer may comprise a cleavable bond within a hairpin structure of a nucleic acid (e.g., a hairpin primer), and a photoinitiated primer is designed using a photolyzable bond hairpin monomer structure. Initially, due to the presence of the hairpin, the 3'-end region of the primer may not be available for base pairing. When exposed to light, the hairpin is disrupted, and the resulting primer can be used for extension ( Figure 10 , the figure below). In some embodiments, due to the presence of a photosensitive system or a photosensitive chemical moiety on a nucleic acid construct, a primer may be inactive (i.e., in an inactive molecular state) before exposure to a light source. However, when a photoinitiated primer is subjected to a light source, the light from the light source can remove some or all of the inhibitors / blockers, or cleave the cleavable bond contained in the photoinitiated primer, thereby restoring the ability of the primer to an active molecular state.

[0146] Table I: Example Sequences of Photoinitiated Primers

[0147]

[0148]

[0149] [PC]: Photolyzable Modification

[0150] [EI]: Extension Inhibitor

[0151] [PCEI]: Photolyzable Polymerase Extension Inhibitor

[0152] N* : Nucleobase with a Photolyzable / Photoremovable Base Pairing Inhibitor

[0153] 2. Light-blocking primers

[0154] A photoreactive primer is a NA construct that can serve as a polymerase initiation site and facilitate NA synthesis in the presence of a nucleic acid template. When light is applied, these photoreactive primers can become inactive and unable to further synthesize NA. A photoreactive primer may be active before light exposure but may become inactive after being subjected to a light source. Figures 11A - 11D Examples of photoreactive primers and their applications are shown, and their corresponding example sequences are listed in Table II.

[0155] Figure 11A and 11BExamples containing photocleavable modified photoresist co - primers are shown. Applying light can break the NA construct, and subsequently render the initiation thermodynamically unfavorable. The primer may contain a cleavable bond between two segments of the primer, and it may be required that the two linked segments of the primer hybridize to the same template to form a stable primer - template heterodimer. Before light exposure, the primer may be active for enzymatic reactions. After exposure to light, the two segments of the co - primer may separate due to the cleavage of the cleavable bond and may become inactive because the binding of only one segment to the template may be thermodynamically unfavorable for the primer - template heterodimer of each segment.

[0156] Figure 11C and 11D Examples of photoresist hybridization primers are shown. In Figure 11C , the photoresist primer is designed to have a photocleavable modification such that applying light can break the primer into separate parts, and subsequently reduce the base - pairing strength of the converted primer - template heterodimer. Thus, the primer becomes thermodynamically unfavorable, and the primer - template heterodimer may break. In Figure 11D , the photoresist primer is designed using base - pairing inhibitor modifications distributed in the primer sequence. Applying light can remove the inhibition, and subsequently create a stable hairpin structure for the converted primer. Since the converted primer forms a hairpin structure, the primer - template heterodimer can be disrupted because intermolecular hybridization is thermodynamically less favorable compared to intramolecular hybridization of the hairpin structure.

[0157] Table II: Example sequences of photoresist primers

[0158]

[0159] [LK]: Non - extendable linker

[0160] [PC]: Photocleavable modification

[0161] N* : Nucleobase with photocleavable / photo - removable base - pairing inhibitor

[0162] 3. Light-initiated hybridization probes

[0163] Light - initiated hybridization probes are NA constructs that can specifically recognize 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 as shown in Figure 12A and 12B , and their corresponding example sequences are listed in Table III.

[0164] In Figure 12AIn [reference], a photoactivatable hybridization probe is designed using base pairing inhibitor modification. Initially, the photoactivatable hybridization probe containing the base pairing inhibitor cannot hybridize with its complementary sequence. Applying light can remove the inhibition of hybridization due to the removal of the base pairing inhibitor, and can allow base pairing, so that the transformed photoactivatable hybridization probe can hybridize with its complementary sequence.

[0165] In Figure 12B In [reference], the photoactivatable hybridization probe is designed to contain a photocleavable hairpin monomer structure. Initially, base pairing between the probe and its complementary sequence is thermodynamically unfavorable because of the presence of the hairpin monomer with intramolecular hybridization. Applying light can disrupt the hairpin by cleaving the probe into two separate parts, and make the transformed probe available for base pairing and hybridization with its complementary sequence.

[0166] Table III: Example sequences of photoactivatable hybridization probes

[0167]

[0168] [EI]: Extension inhibitor

[0169] [PC]: Photocleavable modification

[0170] N* : Nucleobase with photocleavable / photoremovable base pairing inhibitor

[0171] 4. Light-blocking hybridization probes

[0172] Photoresist hybridization probes are nucleic acid constructs that can specifically recognize and base pair with their complementary sequences. However, once exposed to light, they can become inactive and can no longer hybridize with their complementary sequences.

[0173] In Figure 13 An example of the photoresist hybridization probe structure is shown in [reference], and the corresponding example sequences are listed in Table IV.

[0174] In Figure 13 In the upper figure of [reference], the photoresist hybridization probe is designed to contain a photocleavable modification that links two segments of the photoresist hybridization probe. Before exposure to light, both segments of the photoresist hybridization probe hybridize with the target NA, thus remaining in the active molecular state. Upon exposure to light, the photoresist hybridization probe can break the photocleavable bond, resulting in two unlinked segments of the photoresist nucleic acid probe, and can make the formation of the probe-template heterodimer thermodynamically unfavorable. For example, at least one segment can be designed to have a sequence non-complementary to the target nucleic acid, and can provide a signal change if it does not hybridize with the target nucleic acid or is separated from the other segment of the photoresist hybridization probe. At least one segment of the transformed photoresist hybridization probe can be in an inactive molecular state.

[0175] InFigure 13 In the following figure, a photoresist hybridization probe is designed to contain one or more base pairing inhibitors. Before exposure to light, the presence of the base pairing inhibitor can prevent self-base pairing within the photoresist hybridization probe from forming a hairpin structure. Instead, the photoresist hybridization probe hybridizes with the target nucleic acid, thus remaining in an active molecular state. When exposed to light, the photoresist hybridization probe can remove the base pairing inhibition and create a stable hairpin structure for at least one segment of the transformed photoresist hybridization probe, thereby making the formation of the probe-template heterodimer thermodynamically unfavorable. The hairpin structure of the photoresist hybridization probe can be in an inactive molecular state.

[0176] Table IV: Example Sequences of Photoresist Hybridization Probes

[0177]

[0178] [EI]: Extension Inhibitor

[0179] [PC]: Photocleavable Modification

[0180] N* : Nucleobase with Photocleavable / Photoremovable Base Pairing Inhibitor

[0181] 5. Light-initiated 5’- Exonuclease probes

[0182] A light-initiated 5'-terminal exonuclease probe is a NA construct containing a 3'-terminal exonuclease protecting agent that can be removed by light. The 5'-terminal exonuclease protecting agent can be a photosensitive system or a photosensitive chemical moiety chemically attached to the 5'-terminal terminal unit of the NA sequence. Due to the presence of the 5'-terminal exonuclease protecting agent, the exonuclease digestion of the 5'-end of the nucleic acid chain can be blocked, protecting the nucleic acid chain from being cleaved or digested. The light-initiated 5'-terminal exonuclease probe is in an inactive molecular state. When exposed to light, the 5'-terminal exonuclease protecting agent can be removed, and the digestion of the 5' to 3' strand can be promoted. For example, Figure 14 such a nucleic acid structure is shown in, where the DNA polymerase 5'-terminal exonuclease is initially blocked, and exposure to light can remove the blocking group and allow the enzyme to digest the strand.

[0183] Generally, the heteroatoms on the nucleobase, 3'-OH, 5'-OH, and phosphate group (at the 3' or 5' position) can be conjugated to a photosensitive chemical moiety, such as any one shown in Figure 1 A photocleavable linker can have one or more photosensitive chemical moieties attached to the ends of the linker, such that when exposed to light, the one or more photosensitive chemical moieties can detach from the nucleic acid fragment to which they are attached. Various photocleavable chemical moieties can be used in various ways.

[0184] Example embodiments using light-triggered NA constructs

[0185] Example 1: Light-initiated PCR

[0186] In this embodiment, as Figure 15 shown, a photoinitiated primer pair is used in the PCR assay. As Figure 15 shown, PCR and extension of the primer begin after light is applied, but cannot begin before light is applied. Prior to exposure to light, neither polymerization nor exponential amplification can occur because 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 is that it can reduce the presence of unwanted products and / or primer dimers that result from non-specific DNA amplification at room temperature (or colder), e.g., during sample introduction into the reaction or other pretreatment steps. Upon exposure to light, the 3'-end extension inhibitor can be removed and the primer can become active in polymerase-catalyzed extension (i.e., extension, elongation of the growing strand).

[0187] This method may hereinafter be referred to as "photoinitiated PCR" and can replace other PCR methods, such as hot start PCR, in which the amplification process is activated by heating at an elevated temperature. 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, photoinitiated PCR may not include reagents and molecules that act as thermolabile switches.

[0188] In some embodiments of the present invention, the photoinitiated PCR method and the hot start PCR method may be used to better ensure that amplification remains inactive at lower temperatures and prior to PCR.

[0189] In some embodiments of the present invention, photoinitiated PCR is included in a quantitative PCR (Q-PCR) system. In some embodiments, the method of using photoinitiated PCR is a Q-PCR method, comprising: (a) nucleic acid amplification of two or more nucleotide sequences in the presence of a photoinitiated primer to generate two or more amplicons in a fluid; (b) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently locatable positions, the array being configured to contact the fluid; and (c) measuring the hybridization of the amplicons with two or more nucleic acid probes when the fluid contacts the array to obtain an amplicon hybridization measurement, wherein the amplicon comprises a quencher. In some embodiments, primers comprising a photo-primer are used to generate the amplicons and the primers comprise 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, a quencher is incorporated into the amplicon upon amplicon formation. In some embodiments, deoxynucleotide triphosphates (d-NTP's) are used to prepare the amplicons, and one or more d-NTP's used to prepare the amplicons comprise a quencher. In some embodiments, the amplicon hybridization measurement is performed by measuring fluorescence from a fluorescent moiety attached to the 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 not covalently attached to the nucleic acid probe. In some embodiments, the amplicon comprises a quencher, and the measurement of hybridization is performed by measuring a decrease in fluorescence due to hybridization of the amplicon with the nucleic acid probe.

[0190] In some embodiments, the method of light-initiated PCR is the 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 at different locatable positions on the surface, each locatable position 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, wherein: (i) the PCR primers for each nucleic acid sequence are light-initiated primers and comprise a quencher; and (ii) the fluid is contacted with the probes such that the amplified molecules can hybridize with the probes, thereby quenching the signal from the fluorescent moieties; (c) detecting over time the signal from the fluorescent moieties at the locatable positions; (d) using the signal detected over time to determine the amount of amplified molecules in the fluid; and (e) using the amount of amplified molecules in the fluid to determine the amount of nucleotide sequences in the sample. In some embodiments, the amount of amplified molecules is determined during or after a plurality of 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 over time the signal from the fluorescent moieties at the locatable positions comprises measuring the hybridization rate of the amplified molecules with the probes. In some embodiments, the sample comprises messenger RNA or nucleotide sequences derived from messenger RNA, and the determination of the amount of nucleic acid sequences in the sample is used to determine the gene expression level in the cells or cell populations from which the sample is derived. In some embodiments, the sample comprises genomic DNA or nucleotide sequences derived from genomic DNA, and the determination of the amount of nucleic acid sequences in the sample is used to determine the genetic constitution in the cells or cell populations 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 locatable positions 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 comprises using light-initiated primers to perform the Q-PCR method on a sample from the individual.

[0191] In some embodiments, the Q-PCR method is a method for determining at least one target nucleic acid molecule, comprising: (a) providing a reaction mixture comprising a nucleic acid sample containing at least one template nucleic acid molecule, a primer pair comprising the photoinitiated 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 produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid molecule and the limiting primer, wherein at least one target nucleic acid molecule comprises the limiting primer; (c) contacting the reaction mixture with a sensor array having (i) a substrate comprising a plurality of probes immobilized at different individually locatable positions on the surface of the substrate, wherein the probes have sequence complementarity with the limiting primer and are capable of capturing the limiting primer, and (ii) a detector array configured to detect at least one signal from a locatable position, wherein the at least one signal indicates binding of the limiting primer to a single probe among the plurality of probes; (d) detecting at least one signal from one or more locatable positions using the detector array at a plurality of time points during the nucleic acid amplification reaction; and (e) detecting the target nucleic acid molecule based on at least one signal indicating binding of the limiting primer to a single probe among the plurality of probes. In some embodiments, at least one signal is generated when the probe binds to the limiting primer. In some embodiments, the reaction mixture comprises a plurality of limiting primers having different nucleic acid sequences, and the probe specifically binds to the plurality of limiting primers. In some embodiments, the reaction mixture is provided in a reaction chamber configured to retain the reaction mixture and allow binding of the probe to the limiting primer. In some embodiments, the method further comprises correlating at least one signal detected at a plurality of time points with the original concentration of at least one template nucleic acid molecule by analyzing the binding rate of the probe to the limiting primer. In some embodiments, the probe is an oligonucleotide. In some embodiments, the target nucleic acid molecule forms a hairpin loop when hybridized to a single probe. 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 indicating an interaction between an energy acceptor and an energy donor. In some embodiments, the energy acceptor is coupled to the excess primer and / or the limiting primer. In some embodiments, the energy acceptor is coupled 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 indicating an interaction between an electrode and a redox tag. In some embodiments, the redox tag is coupled to the excess primer and / or the limiting primer. In some embodiments, the redox tag is coupled to the target nucleic acid molecule. In some embodiments, (d) comprises measuring an increase in the at least one signal relative to the background. In some embodiments, (d) comprises measuring a decrease in the at least one signal relative to the background.In some embodiments, the target nucleic acid molecule is detected with at least about 90% sensitivity. In some embodiments, at least one signal is detected when the reaction mixture containing the target nucleic acid molecule is in fluid contact with the sensor array. In some embodiments, (b) includes generating a plurality of target nucleic acid molecules having sequence complementarity to the template nucleic acid. In some embodiments, the detector array is configured to detect a plurality of signals from locatable positions, wherein each signal of the plurality of signals indicates binding of a restriction primer to a single probe of the plurality of probes. In some embodiments, (d) includes using the detector array to detect a plurality of signals from locatable positions at a plurality of time points, wherein each signal of the plurality of signals indicates binding of a restriction primer to a single probe of the plurality of probes. In some embodiments, (e) includes identifying the restriction primer.

[0192] In some embodiments, the present disclosure provides a system for assaying at least one target nucleic acid molecule, comprising: (a) a reaction chamber containing 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 restriction primer and an excess primer, and wherein the reaction chamber containing the reaction mixture is configured to facilitate a nucleic acid amplification reaction of the reaction mixture to produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid; (b) a sensor array comprising (i) a substrate containing a plurality of probes immobilized at different individually locatable positions on the surface of the substrate, wherein the probes have sequence complementarity to the restriction primer and are capable of capturing the restriction primer, and (ii) a detector array configured to detect at least one signal from the locatable positions, wherein the at least one signal indicates binding of the restriction primer to a single probe of the plurality of probes; and (c) a computer processor coupled to the sensor array and programmed to (i) cause the reaction mixture to undergo a nucleic acid amplification reaction, and (ii) detect at least one signal from one or more locatable positions at a plurality of time points during the nucleic acid amplification reaction.

[0193] In some embodiments, the Q-PCR method is a method for determining at least one template nucleic acid molecule, comprising: (a) activating a sensor array comprising (i) a substrate comprising a plurality of first probes immobilized on a first pixel and a plurality of second probes immobilized on a second pixel, wherein the first probes are configured to capture a single primer of a primer set, and wherein 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 pixel and at least one second signal from the second pixel, wherein the difference over time between the at least one first signal and the at least one second signal indicates the binding of the single primer to a single probe among the plurality of first probes; (b) subjecting a reaction mixture to a nucleic acid amplification reaction under conditions sufficient to produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid molecule, wherein the reaction mixture comprises (i) a nucleic acid sample containing or suspected of containing the template nucleic acid molecule, (ii) a primer set, (iii) a control nucleic acid molecule, and (iv) a polymerase, wherein the single primer of the primer set has sequence complementarity with the template nucleic acid molecule; (c) detecting at least one first signal and at least one second signal using the detector array at a plurality of time points during the nucleic acid amplification reaction; 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, at least one first signal is generated when a single probe binds to a single primer, and wherein 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 comprises a plurality of template nucleic acid molecules, and wherein the first probes specifically bind to a plurality of target nucleic acid molecules that are amplification products of the plurality of template nucleic acid molecules. In some embodiments, the primer set comprises a plurality of single primers having different nucleic acid sequences, and wherein the first probes are configured to specifically bind to the plurality of single primers. In some embodiments, the reaction mixture is provided in a reaction chamber configured to retain the reaction mixture and allow the first and second probes to bind to the single primer and the control nucleic acid molecule. In some embodiments, the method further comprises correlating the at least one first signal detected at a plurality of time points with the initial concentration of at least one template nucleic acid molecule by analyzing the binding rate of the probe to the single primer from 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 indicating a first interaction between a first energy acceptor and a first energy donor associated with the single primer and the single probe, and wherein the at least one second signal is a second optical signal indicating 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 coupled to a single primer, and wherein the second energy acceptor is coupled to a control nucleic acid molecule. In some embodiments, the first energy acceptor is coupled to a target nucleic acid molecule. In some embodiments, the first energy acceptor is a first quencher, and wherein the second energy acceptor is a second quencher. In some embodiments, the first energy donor is a first fluorophore, and wherein the second energy donor is a second fluorophore. In some embodiments, the first energy donor is coupled to a first probe, and wherein the second energy donor is coupled to a second probe. In some embodiments, the target nucleic acid molecule is detected with at least about 90% sensitivity. In some embodiments, at least one first signal is detected when a reaction mixture comprising the target nucleic acid molecule contacts the sensor array in a fluid.

[0194] In some embodiments, a Q-PCR system is used to assay at least one template nucleic acid molecule and includes: (a) a reaction chamber containing a reaction mixture, wherein the reaction mixture contains (i) a nucleic acid sample containing or suspected of containing the template nucleic acid molecule, (ii) a primer set containing a single primer, (iii) a control nucleic acid molecule, and (iv) a polymerase, wherein the single primer of the primer set has sequence complementarity with the template nucleic acid molecule, and wherein the reaction chamber containing the reaction mixture is configured to facilitate a nucleic acid amplification reaction with the reaction mixture under conditions sufficient to produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid molecule, and wherein the nucleic acid amplification reaction does not produce any amplification products of the control nucleic acid; (b) a sensor array that includes (i) a substrate that includes a plurality of first probes immobilized on a first pixel and a plurality of second probes immobilized on a second pixel, wherein the first probes are configured to capture the single primer of the primer set, and wherein the second probes are configured to capture the control nucleic acid molecule, and (ii) a detector array that is configured to detect at least one first signal from the first pixel and at least one second signal from the second pixel, wherein the difference over time between the at least one first signal and the at least one second signal indicates the binding of the single primer to a single probe among the plurality of first probes; and (c) a computer processor that is coupled to the sensor array and programmed to (i) cause the reaction mixture to undergo the nucleic acid amplification reaction and (ii) detect the at least one first signal and the at least one second signal at a plurality of time points during the nucleic acid amplification reaction. In some embodiments, the computer processor is programmed to use the difference between the at least one first signal and the at least one second signal to detect the template nucleic acid molecule. In some embodiments, the reaction mixture contains a plurality of template nucleic acid molecules, and wherein the first probes specifically bind to a plurality of target nucleic acid molecules that are amplification products of the plurality of template nucleic acid molecules. In some embodiments, the primer set contains a plurality of single primers having different nucleic acid sequences, and wherein the first probes are configured to specifically bind to the plurality of single primers. In some embodiments, the detector array includes 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 single primer and the single probe, and wherein 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 includes a complementary metal oxide semiconductor device. In some embodiments, the detector array includes an electrical detector. In some embodiments, the electrical detector includes a complementary metal oxide semiconductor device. In some embodiments, the sensor array includes at least about 100 integrated sensors.

[0195] A variety of processes and techniques can be used for Q-PCR using microarrays or CMOS biochips. For example, many 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.

[0196] In some embodiments of the present invention, photo-removable blocking can be included in an NA affinity-based detection system, such as a DNA microarray. A DNA microarray is essentially a massively parallel affinity-based biosensor, mainly used to measure gene expression levels, that is, to quantify the process of DNA data being transcribed into messenger RNA molecules (mRNA). The information transcribed into mRNA is further translated into proteins, which perform most of the functions in cells. Therefore, by measuring gene expression levels, researchers can infer key information about cell or whole organism function. Thus, perturbations in typical expression levels usually indicate disease; therefore, DNA microarray experiments can provide valuable insights into the genetic causes of diseases. In fact, one ultimate goal of DNA microarray technology is to enable the development of molecular diagnostics and the creation of personalized medicine.

[0197] A DNA microarray is essentially an affinity-based biosensor, where binding is based on hybridization, which is the process by which complementary DNA strands specifically bind to each other to form a structure in a lower energy state. Generally, the surface of a DNA microarray consists of an array of spots (grid), each containing single-stranded DNA oligonucleotide capture molecules as recognition elements, whose positions are fixed during the hybridization and detection processes. The length of each single-stranded DNA capture molecule is typically 25 - 70 bases, depending on the specific platform and application. During DNA microarray detection, the mRNA to be quantified is initially 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 will hybridize, that is, bind to the complementary capture oligonucleotides. However, there is always a significant amount of non-specific binding because the cDNA may non-specifically cross-hybridize to oligonucleotides that are not perfect matches but only partially complementary (with mismatches). In addition, the fluorescence intensity of each spot is measured to obtain an image related to the hybridization process, thereby obtaining gene expression levels.

[0198] 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 biomolecules is affected by non-covalent intermolecular interactions, such as, including hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. In multiplex binding experiments, as contemplated herein, multiple analytes and probes are involved. For example, an experiment may involve detecting binding between multiple different nucleic acid molecules or different proteins. In such an experiment, an analyte will preferentially bind to a probe for which it has a greater affinity. Thus, determining that a particular probe is involved in a binding event indicates the presence in the sample of an analyte that has sufficient affinity for the probe to meet the detection threshold level of the detection system being used. It may be possible to determine the identity of the binding partner based on the specificity and strength of the binding between the probe and the analyte.

[0199] When developing solutions in the context of DNA microarrays, the present invention provides a process in which (i) cross-hybridization is regarded as interference rather than noise (similar to wireless communication interference, cross-hybridization actually has signal content); (ii) hybridization and cross-hybridization are modeled as random processes; (iii) models are constructed using analytical methods (such as melting temperature or Gibbs free energy functions) and fine-tuned using empirical data; (iv) the detection and quantification of gene expression levels are regarded as a random estimation problem; and (v) optimal estimates are constructed. The present invention uses statistical signal processing techniques to optimally detect and quantify targets in microarrays by considering and exploiting the above uncertainties.

[0200] A variety of techniques and processes can be used to synthesize arrays of biological materials on or in a substrate or carrier. For example, many 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.

[0201] In some embodiments of the present invention, the photo-removable blocker is included in a CMOS biochip system. In some embodiments, the present disclosure provides a fully integrated biosensor array that sequentially includes a molecular recognition layer containing an NA construct, an optical layer, and a sensor layer integrated in a sandwich configuration or in series with additional layers, e.g., the additional layer having another layer inserted between any of the molecular recognition layer, the optical layer, and the sensor layer. The molecular recognition layer includes an open surface and a plurality of different probes attached to the open surface at different independently locatable positions. The molecular recognition layer can also transmit light to the optical layer. The optical layer includes a filter layer, wherein the optical layer 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. Additionally, there can be a fluid volume containing an analyte in fluid contact with the molecular recognition layer. The fluid volume can include an NA construct.

[0202] The integrated biosensor array of the present disclosure can measure the binding of an analyte in real time. An integrated biosensor microarray capable of detecting assay binding kinetics can be brought into contact with an affinity-based assay. The biosensor array includes a molecular recognition layer containing binding probes in optical communication, and a sensor for real-time detection of binding to the probes.

[0203] A fluorescence-based integrated microarray system for real-time measurement of the binding of an analyte to a plurality of probes can be constructed using standard complementary metal oxide semiconductor (CMOS) processes, and the system includes a capture probe layer, a fluorescence emission filter, and an image sensor.

[0204] In an embodiment 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 an organic semiconductor or an inorganic semiconductor. In some embodiments, the semiconductor device is a silicon-based sensor. Examples of sensors that can be used 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).

[0205] 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.

[0206] The sensor layer can be created using CMOS processes. The semiconductor detection platform can be a component of an integrated system capable of measuring real-time microarray (RT-microarray) binding events. In some embodiments, the integrated device system involves a sensor array in contact with or near an RT-microarray.

[0207] The semiconductor detection platform of the RT-microarray can include an independent sensor array to receive and / or analyze signals from target and probe binding events of the RT-microarray platform. Multiple sensors can work together to measure multiple binding events at any single microarray spot. For example, sensors dedicated to one spot can add and / or average their respective measurement signals.

[0208] The detection circuit connected to the optical sensor array can be embedded in the sensor layer. The signal processing circuit can also be connected to the optical sensor array and embedded in the sensor layer. In some embodiments, the sensors and / or detection circuits and / or analysis systems are implemented using electrical components fabricated and / or embedded in a semiconductor substrate. Exemplary 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.

[0209] The sensor array can be an image sensor array. Examples of such image arrays include but are not limited to CMOS image sensor arrays, CMOS linear optical sensors, CCD image sensors, and CCD linear optical sensors. The image sensor can be used to detect the activity of probe / analyte interactions within the integrated biosensor array platform.

[0210] Various processes and technologies can be used to fabricate and / or use CMOS biochip systems. For example, many such technologies are described in U.S. Patent Nos. 8,637,436 and 8,969,781.

[0211] Example 2: Photo-induced nested PCR

[0212] In this embodiment, as Figure 16 shown, two pairs of primers are used. One pair is a light-initiated primer, while the other pair is a light-terminated primer. At a specific time within the PCR cycle, light is applied to inactivate the light-terminated primer pair and activate the light-initiated primer pair.

[0213] In some embodiments, the light-terminated primer pair is located beside the light-initiated primer pair (see Figure 16 ), such that the amplicon generated 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. The advantage of this system is that it can improve the specificity and sensitivity of amplification by reducing non-specific amplicons and products that may result from the amplification of non-intended primer binding sites on the template.

[0214] This method may hereinafter be referred to as "photo-nested PCR" and can replace the traditional nested PCR method, in which two PCR amplifications are performed in series in two different reaction chambers. See G. Bein, R. &H. Kirchner, "Rapid HLA-DRB1 genotyping by nested PCR amplification. Tissue antigens," 1992, 39(2): 68-73; M. Pfeffer, B. Linssen, M. D. Parker and R. M Kinney, "Specific detection of Chikungunya virus using a RT-PCR / nested PCR combination". Journal of Veterinary Medicine, Series B, 2002, 49(1): 49-54. However, the advantage of photo-induced nested PCR is that both amplifications can be carried out in a closed-tube manner in the same reaction.

[0215] In some embodiments of the present invention, photo-induced nested PCR is included in a Q-PCR system. The devices, systems and methods disclosed in Example 1 can be modified and applied by using a suitable NA construct as the photo-initiation primer pair and / or photo-termination primer pair in photo-induced nested PCR and irradiating the reaction mixture during the operation of photo-induced nested PCR to initiate or terminate a specific PCR process.

[0216] In some embodiments of the present invention, photo-removable blocking is included in a detection system based on NA affinity (such as a DNA microarray).

[0217] In some embodiments of the present invention, photo-removable blocking is included in a CMOS biochip system.

[0218] Example 3: Photo-removable blocking

[0219] In this embodiment, the photo-terminating hybridization probe is used as a sequence-selective blocker in polymerase chain reaction or other primer-initiated molecular amplification reactions. See P.L. Dominguez, and M.S. Kolodney, “Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens,” Oncogene, 2005, 24(45):6830-6834. J.F. 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).

[0220] In some embodiments, the photo-terminating hybridization probe inhibits the PCR amplification of wild-type sequences while allowing the synthesis of mutant sequences. By doing so, the ratio of wild-type amplicons to mutant amplicons decreases as amplification proceeds. This aids in better detection of mutants at the end of PCR. The presence of the photo-resist construct type further allows for the removal of the blocker by light to produce a clean PCR product without interfering hybridization probes.

[0221] In some embodiments of the present invention, the photo-removable blocking is included in a Q-PCR system. The devices, systems, and methods disclosed in Example 1 can be modified and applied by using a suitable NA construct as a photo-removable blocking probe in tandem with a light-initiated PCR process and irradiating the reaction mixture during the running of a light-induced nested PCR to initiate or terminate the light-initiated PCR.

[0222] In some embodiments of the present invention, the photo-removable blocking is included in a NA affinity-based detection system (such as a DNA microarray). The devices, systems, and methods disclosed in Example 1 can be modified and applied by using an appropriate NA construct as a photo-removable blocking probe in a NA affinity-based detection system (such as a DNA microarray). For example, when using a NA affinity-based detection system to detect a target nucleic acid, the photo-removable blocking probe can interact with the target nucleic acid, a fixed probe, or a solution-based probe or a combination thereof. By irradiating the reaction mixture during the running of the NA affinity-based detection system, different amplicons and / or different hybridization events can be detected by the NA affinity-based detection system.

[0223] In some embodiments of the present invention, the photo-removable blocker is included in the CMOS biochip system. The devices, systems, and methods disclosed in Example 1 can be modified and applied by using a suitable NA construct as the photo-removable blocker probe in the CMOS biochip system. For example, when using the CMOS biochip system to detect a target nucleic acid, the photo-removable blocker probe can interact with the target nucleic acid, the immobilized probe, or the solution-based probe or a combination thereof. By irradiating the reaction mixture during the operation of the CMOS biochip system, different amplicons can be generated and / or different hybridization events can be detected by the CMOS biochip system.

[0224] Example 4: Light-anchored primers

[0225] In this example, a photo-terminator primer is used to change the effective length of the primer during PCR.

[0226] In some embodiments, after a specific number of PCR cycles, the photo-resistant primer is cleaved 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, which can continue PCR after photolysis. This allows the design of an anchor primer with a high melting temperature (T M ) in the initial cycles of PCR. When exposed to light, the length of the primer is shortened to reduce the T M of the primer and the length of the resulting amplicon. Applications of this method include designing high-T M primers to accommodate mismatches within the template in the early cycles of PCR and / or to overcome secondary structures in RNA or DNA templates.

[0227] In some embodiments of the present invention, the photo-anchor primer is included in the Q-PCR system. The devices, systems, and methods disclosed in Example 1 can be modified and applied by using a suitable NA construct as the photo-anchor primer in the photo-anchor PCR process. Before exposure to light, the generated amplicon can contain the full length of the photo-anchor primer. Irradiating the reaction mixture can generate new primer pairs. The length of each new primer is shorter than that of the corresponding full-length photo-anchor primer. Therefore, the amplicon generated using the new primer pair can have a shorter length than before exposure to light. Two sets of amplicons with different lengths can be generated using the same template nucleic acid molecule.

[0228] In some embodiments of the present invention, the photo-anchor primer is included in a detection system based on NA affinity (such as a DNA microarray).

[0229] In some embodiments of the present invention, the photo-anchor primer is included in the CMOS chip system.

[0230] Other terms used in this disclosure

[0231] As used herein, the term "quantitative PCR" or "Q-PCR" generally refers to the polymerase chain reaction (PCR) process that can be used for the qualitative and quantitative determination of nucleic acid sequences. In some cases, Q-PCR is synonymous with real-time PCR. Q-PCR can involve measuring the amount of amplification product (or amplicon) as a function of the amplification cycle, and using this information to determine the amount of nucleic acid sequence corresponding to the amplicon present in the original sample.

[0232] As used herein, the term "reverse transcription polymerase chain reaction" or "RT-PCR" generally refers to a variant of the polymerase chain reaction (PCR) in which a ribonucleic acid (RNA) strand is first reverse transcribed into its DNA complement (complementary DNA cDNA) using reverse transcriptase. The resulting cDNA is then amplified using conventional PCR. RT-PCR uses a pair of primers that are complementary to defined sequences on each of the two strands of the cDNA. These primers are then extended by DNA polymerase and the strand is replicated after each PCR cycle, resulting in exponential amplification. As used herein, the term "quantitative reverse transcription polymerase chain reaction" or "qRT-PCR" refers to the real-time detection of an RT-PCR reaction, similar to the real-time detection in a Q-PCR reaction.

[0233] In the present disclosure, after making the corresponding changes known to those skilled in the art, all methods or systems disclosed for QPCR can be adapted for qRT-PCR.

[0234] As used herein, the term "probe" 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 contain molecules and can be bound directly or via a linker molecule to a substrate or other solid surface.

[0235] As used herein, the term "detector" generally refers to a device that typically includes optical and / or electrical components that can detect a signal.

[0236] As used herein, the term "mutation" generally refers to a gene mutation or sequence variation, such as a point mutation, single nucleotide polymorphism (SNP), insertion, deletion, substitution, transposition, translocation, copy number variation, or another gene mutation, alteration, or sequence variation.

[0237] As used herein, the term "about" or "substantially" generally refers to within + / - 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the specified amount.

[0238] As used herein, the term "marker" refers to a specific molecular structure that can be attached to a target molecule to make the target molecule distinguishable and traceable by providing a unique characteristic not inherent to the target molecule.

[0239] As used herein, the term "limiting" in the context of a chemical or biological reaction generally refers to a species present in a limiting amount (e.g., stoichiometrically limiting) in a given reaction volume such that, at the completion of the chemical or biological reaction (e.g., PCR), the species may no longer be present in the reaction volume.

[0240] As used herein, the term "excess" in the context of a chemical or biological reaction generally refers to a species present in an excess amount (e.g., stoichiometrically limiting) in a given reaction volume such that, at the completion of the chemical or biological reaction (e.g., PCR), the species may still be present in the reaction volume.

[0241] As used herein, the term "nucleotide" generally refers to a molecule that can serve as a monomer or subunit of a nucleic acid (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). Nucleotides can be deoxynucleotide triphosphates (dNTPs) or analogs thereof, such as molecules having multiple phosphates (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphates) in the phosphate chain. Nucleotides generally can include adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) or variants thereof. Nucleotides can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits can be A, C, G, T, or U, or any other subunit specific for or complementary to one or more complementary A, C, G, T, or U, or purines (i.e., A or G, or variants thereof) or pyrimidines (i.e., C, T, or U, or variants thereof). Subunits can enable single nucleic acid bases or base pairs (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or their uracil counterparts) to be resolved. Nucleotides can be labeled or unlabeled. Labeled nucleotides can produce a detectable signal, such as an optical, electrostatic, or electrochemical signal.

[0242] The Q-PCR process can be described in the following non-limiting examples. A PCR reaction is carried out using a primer pair designed to amplify a given nucleic acid sequence in a sample. A suitable enzyme and nucleotides such as deoxynucleotide triphosphates (dNTPs) are added to the reaction, and the reaction is carried out for multiple amplification cycles. The amount of amplicon generated from each cycle is detected, but in the early cycles, the amount of amplicon may be below the detection threshold. Amplification can occur in two phases, namely the exponential phase and a subsequent non-exponential stationary phase. In the exponential phase, the amount of PCR product approximately doubles in each cycle. However, as the reaction proceeds, the reaction components are consumed, and eventually one or more components become limiting. At this point, the reaction slows down and enters the stationary phase. Initially, the amount of amplicon remains at or below the background level, and an increase in amount cannot be detected (even though the amplicon product accumulates exponentially). Eventually, the amplified product accumulates to an amount sufficient to produce a detectable signal. The cycle at which a detectable signal is produced is called the cycle threshold or C t Because C tThe value is measured during the exponential phase when the reagent is not restricted, so Q-PCR can be used to reliably and accurately calculate the initial amount of template present in the reaction. The C of the reaction t can be determined mainly by the amount of nucleic acid sequence corresponding to the amplicons present at the start of the amplification reaction. If a large amount of template is present at the start of the reaction, relatively few amplification cycles may be required to accumulate sufficient product to obtain a signal above background. Thus, the reaction may have a low or early C t . Conversely, if a small amount of template is present at the start of the reaction, more amplification cycles may be required to generate a fluorescent signal above background. Thus, the reaction may have a high or late C t . The methods and systems provided herein allow the accumulation of multiple amplicons in a single fluid to be measured in a single amplification reaction, thus allowing the amount of multiple nucleic acid sequences in the same sample to be determined using the Q-PCR method described above.

[0243] As used herein, the term "real-time" generally refers to measuring the state of a reaction during its transient phase or in biochemical equilibrium while the reaction is occurring. As opposed to measurements made after the reaction is fixed, real-time measurements occur simultaneously with the ongoing event being monitored, measured, or observed. Thus, a "real-time" determination or measurement generally contains not only the quantitative result of the measurement, such as fluorescence, but also represents it at different time points, i.e., in nanoseconds, microseconds, milliseconds, seconds, minutes, hours, etc. "Real-time" can include detecting the dynamic generation of a signal, including acquiring multiple readings to characterize the signal over a period of time. For example, real-time measurement can include determining the rate of increase or decrease in the amount of an analyte. Although real-time measurement of a signal can be used to determine a rate by measuring the change in the signal, in some cases, it may also be useful to measure no change in the signal. For example, a lack of change in the signal over time may indicate that the reaction (e.g., binding, hybridization) has reached a steady state.

[0244] 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), which can be ribonucleotides (RNA) or deoxyribonucleotides (DNA). The term can refer only to the primary structure of the molecule. Thus, the term can include triple-stranded, double-stranded, and single-stranded DNA, as well as triple-stranded, double-stranded, and single-stranded RNA. The term can also include modifications, such as modifications by methylation and / or by capping, as well as unmodified forms of polynucleotides.

[0245] Nucleic acids can include phosphodiester bonds (i.e., natural nucleic acids). Nucleic acids can include nucleic acid analogs that can have alternative backbones, such as phosphoramidates (see, e.g., Beaucage et al., Tetrahedron 49(10):1925 (1993) and U.S. Patent No. 5,644,048), phosphorothioates (see, e.g., Briu et al., J. Am. Chem. Soc. 111:2321 (1989)), methylphosphonates (see, e.g., Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid (PNA) backbones and linkages (see, e.g., Carlsson et al., Nature 380:207 (1996)). Nucleic acids can include other nucleic acid analogs, including those with positively charged backbones (see, e.g., Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)); nonionic backbones (see, e.g., U.S. Patent 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, “Carbohydrate Modifications in Antisense Research”, Y.S. Sanghui and P.D. Cook, eds.; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones (see, e.g., U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Y.S. Sanghui and P.D. Cook, eds.).The nucleic acid may comprise one or more carbocyclic sugars (see, e.g., Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). These modifications of the ribose-phosphate backbone can facilitate the addition of labels, or increase the stability and half-life of such molecules in a physiological environment.

[0246] As used herein, the term "amplicon" generally refers to a molecular species generated by the amplification of a nucleotide sequence, such as by PCR. An amplicon can be a polynucleotide such as RNA or DNA or a mixture thereof, wherein the sequence of nucleotides in the amplicon can be related to (i.e., corresponding or complementary to) the sequence of the nucleotide sequence from which the amplicon is generated. An amplicon can be single-stranded or double-stranded. In some cases, an amplicon can be generated by using one or more primers incorporated into the amplicon. In some cases, an amplicon can be generated in a polymerase chain reaction or PCR amplification, where two primers can be used to produce a pair of complementary single-stranded amplicons or double-stranded amplicons.

[0247] 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 comprise a molecule and can be directly or via a linker molecule bound to a substrate or surface.

[0248] As used herein, the singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise.

[0249] While the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The present invention is not intended to be limited to the specific examples provided in the specification. While the invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not to be construed as limiting. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. Accordingly, it is contemplated that the present invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the present invention and thereby cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A reaction mixture comprising a pair of phototerminator primers and a pair of photoinitiator primers for nested polymerase chain reaction (PCR), wherein each member of the pair of phototerminator primers and the pair of photoinitiator primers is independently a nucleic acid construct comprising: a) a plurality of nucleotides; and b) one or more photocleavable moieties; wherein each of the nucleic acid constructs of the pair of phototerminator primers is active in the nested PCR, and wherein each of the nucleic acid constructs of the pair of phototerminator primers is configured to be inactive in the nested PCR in the absence of the one or more photocleavable moieties on the pair of phototerminator primers, wherein the nucleic acid constructs of the pair of photoinitiator primers are inactive in the nested PCR, and wherein the nucleic acid constructs of the pair of photoinitiator primers are configured to be active in the nested PCR in the absence of the one or more photocleavable moieties on the pair of photoinitiator primers, wherein each of the one or more photocleavable moieties is independently located at: a) the 3'-end of the nucleic acid construct; b) the 5'-end of the nucleic acid construct; c) between the 3'-end and the 5'-end; d) on or attached to the nucleobase of a nucleotide among the plurality of nucleotides; e) on or attached to the ribose of the nucleotide; f) between two consecutive members of the plurality of nucleotides and attached to the two consecutive members of the plurality of nucleotides; or g) a combination thereof, wherein the pair of phototerminator primers is located on both sides of the pair of photoinitiator primers such that the amplicon generated by the active form of the pair of phototerminator primers is used as a template for the active form of the pair of photoinitiator primers.

2. The reaction mixture according to claim 1, wherein the nucleic acid constructs of the pair of phototerminator primers are configured to form a hairpin structure in the absence of the one or more photocleavable moieties on the pair of phototerminator primers.

3. The reaction mixture according to claim 1, wherein the one or more photocleavable moieties of the pair of phototerminator primers are located at the 5'-end of the pair of phototerminator primers.

4. The reaction mixture according to claim 1, wherein the nucleic acid constructs of the pair of phototerminator primers are configured to form nucleic acid molecules after photolysis of the one or more photocleavable moieties on the pair of phototerminator primers, and wherein the nucleic acid molecules are inactive in the nested PCR.

5. The reaction mixture according to claim 1, wherein the nucleic acid constructs of the pair of photoinitiator primers are configured to form nucleic acid molecules after photolysis of the one or more photocleavable moieties on the pair of photoinitiator primers, and wherein the nucleic acid molecules are configured to be active in the nested PCR.

6. The reaction mixture according to claim 5, wherein the one or more photocleavable moieties of the pair of photoinitiator primers are located at the 3'-end.

7. The reaction mixture according to claim 5, wherein each of the one or more photocleavable moieties on the photoinitiation primer pair is independently located between the 3'-end and the 5'-end and on a nucleobase.

8. The reaction mixture according to claim 5, wherein each of the one or more photocleavable moieties of the photoinitiation primer pair is independently located between the 3'-end and the 5'-end and between two consecutive members of the plurality of nucleotides of the nucleic acid construct on the photoinitiation primer pair.

9. The reaction mixture according to claim 7, wherein each nucleic acid construct of the photoinitiation primer pair comprises a first nucleic acid portion and a second nucleic acid portion complementary to the first nucleic acid portion, and wherein the nucleic acid construct forms another hairpin structure.

10. The reaction mixture according to claim 9, wherein the first nucleic acid portion and the second nucleic acid portion of the photoinitiation primer pair do not comprise the one or more photocleavable moieties.

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

12. The reaction mixture according to claim 11, wherein each member of the photoinitiation primer pair has a second sequence complementary to the template nucleic acid molecule, and wherein the first sequence is flanked by the second sequence with respect to the template nucleic acid molecule.

13. A method of performing the nested PCR using the reaction mixture according to any one of claims 1-12, the method comprising: a) providing the reaction mixture comprising the phototermination primer pair, the photoinitiation primer pair, at least one template nucleic acid molecule comprising an internal nucleic acid sequence, and a polymerase; b) using the phototermination primer pair to subject the reaction mixture to conditions for first strand extension 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; and c) irradiating the reaction mixture with photons of light, thereby inactivating the phototermination primer pair and terminating the first strand extension, activating the photoinitiation primer pair and using the activated second primer pair to initiate second strand extension, and forming 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 manner.

14. The method according to claim 13, further comprising: 1) performing the nested PCR on two or more nucleotide sequences in the presence of the phototermination primer pair and the photoinitiation primer pair to generate two or more amplicons in a fluid; 2) providing an array comprising a solid surface having a plurality of nucleic acid probes at independently locatable positions, the array being configured to contact the fluid; and 3) When the fluid contacts the array, measure the hybridization of the two or more amplicons with two or more of the plurality of nucleic acid probes to obtain an amplicon hybridization measurement, wherein the amplicon contains a quencher.

15. The method of claim 13, wherein the nested PCR is quantitative polymerase chain reaction (Q-PCR), and the method further comprises: 1) Provide an array comprising a solid support having a surface and a plurality of different probes, The plurality of different probes are immobilized on the surface at different locatable positions, and each locatable position contains a fluorescent moiety; 2) Perform PCR amplification on a sample comprising a plurality of nucleotide sequences; The PCR amplification is carried out in a fluid, wherein: (i) each of the phototerminator primer pair and the photoinitiator primer pair for each nucleic acid sequence contains a quencher; and (ii) the fluid contacts the plurality of different probes, wherein the amplicons generated in the PCR amplification hybridize with the plurality of probes, thereby quenching the signal from the fluorescent moiety; 3) Detect the signal from the fluorescent moiety at each of the locatable positions over time; 4) Use the signal detected over time and determine the amount of the amplicon in the fluid; and 5) Use the amount of the amplicon in the fluid to determine the amount of the nucleotide sequence in the sample.

16. The method of claim 13, wherein the nested PCR is quantitative polymerase chain reaction (Q-PCR), wherein the photoinitiator primer pair comprises a limiting primer and an excess primer, and wherein the method further comprises: 1) Subject the reaction mixture to the Q-PCR under conditions sufficient to produce at least one target nucleic acid molecule that is an amplification product of the template nucleic acid molecule and the limiting primer, wherein the at least one target nucleic acid molecule contains the limiting primer; 2) Contact the reaction mixture with a sensor array having (i) a substrate comprising a plurality of probes immobilized at different individually locatable positions on the surface of the substrate, wherein the probes are sequence complementary to the limiting primer and capable of capturing the limiting primer, and (ii) a detector array configured to detect at least one signal from the locatable positions, wherein the at least one signal indicates the binding of the limiting primer to a single probe among the plurality of probes; 3) At multiple time points during the nucleic acid amplification reaction, use the detector array to detect the at least one signal from one or more of the locatable positions; and 4) Detect the target nucleic acid molecule based on the at least one signal indicating the binding of the limiting primer to the single probe among the plurality of probes.

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