Detection of target nucleic acids by solid-phase molecular imaging

Abstract

The present invention describes methods for performing real-time PCR for detection and quantification of target nucleic acids using tagged oligonucleotide probes and solid phase molecular imaging.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 782,867, filed on December 20, 2018, which is incorporated herein by reference in its entirety.

[0003] Reference sequence list

[0004] This application contains a sequence listing submitted as an electronic text file, entitled “P34923-WO PCT fling sequence listing”, which is 46kb in size and was created on November 17, 2019. Technical Field

[0005] The present invention relates to a method of polymerase chain reaction (PCR), and more particularly to a method for performing multiplex PCR and detecting a target nucleic acid by a solid phase biosensor. Background Art

[0006] The polymerase chain reaction (PCR) has become a ubiquitous tool in biomedical research, disease monitoring, and diagnosis. Amplification of nucleic acid sequences by PCR is described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the art and has been extensively described in the scientific literature. See PCR Applications, ((1999) Innis et al., ed., Academic Press, San Diego), PCR Strategies, ((1995) Innis et al., ed., Academic Press, San Diego); PCR Protocols, ((1990) Innis et al., ed., Academic Press, San Diego) and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). "Real-time" PCR assays are able to simultaneously amplify and detect and quantify the starting amount of a target sequence. The basic principle of the 5′ to 3′ nuclease activity of a DNA polymerase is to use a 5′ to 3′ nuclease activity of a DNA polymerase to generate a PCR product. Real-time PCR assays are described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Pat. No. 5,210,015. Real-time PCR without nuclease activity (nuclease-free assay) has been described in U.S. Application Serial No. 12 / 330,694 filed December 9, 2008. The use of fluorescent probes in real-time PCR is described in U.S. Pat. No. 5,538,848.

[0007] In most current approaches, the ability to multiplex is limited by the detection instrument. Specifically, the use of multiple probes in the same reaction requires the use of different fluorescent labels. In order to detect multiple probes simultaneously, the instrument must be able to distinguish the light signals emitted by each probe. Most current technologies on the market do not allow more than four to seven different wavelengths to be detected in the same reaction vessel. Therefore, each target uses a uniquely labeled probe, and at most only four to seven different targets can be detected in the same container. In practice, at least one target is usually a control nucleic acid. Therefore, in practice, at most only three to six experimental targets can be detected in the same tube. The use of fluorescent dyes is also limited due to the limitation of spectral width, of which only about six or seven dyes can be applied without significant overlapping interference in the visible spectrum. Therefore, unless fundamental changes are made in amplification and detection strategies, the ability of multiplexing will not keep up with clinical needs.

[0008] There are many methods known for detecting target nucleic acids. Currently available homogeneous assays for nucleic acid detection include Dye-binding, allele-selective kinetic PCR and Primer assay. As mentioned above, since each target nucleic acid to be detected requires different dyes, these assay procedures are not easy to multiplex, and therefore their potential for improvement is limited. In order to overcome such limitations, some recent studies have disclosed the use of oligonucleotide probes containing a "tag" portion of cleavage that is easy to separate and detect (e.g., see Chenna et al., U.S. Patent Application Publication No. 2005 / 0053939; Van Den Boom, U.S. Patent No. 8,133,701). Recently, improved methods for performing multiple nucleic acid target identification by using structure-based oligonucleotide probe cleavage have been described in U.S. Patent Application Publication No. 2014 / 0272955, U.S. 2015 / 0176075 and U.S. 2015 / 0376681, all of which are incorporated herein by reference. Other methods for detecting target nucleic acid sequences from DNA or nucleic acid mixtures by using a combination of "probing and labeling oligonucleotides" (PTO) and "capture and templating oligonucleotides" (CTO) in so-called PTO cleavage and extension assays have been described by Chun et al. in U.S. Pat. No. 8,809,239. However, there remains a need for accurate methods to perform high-throughput multiplexed detection of target nucleic acids, particularly one that is not limited by the use of fluorescent dyes or labels (i.e., label-free multiplexed detection of target nucleic acids).

[0009] Focal molography has recently been described as an analytical method for real-time visualization of specific biomolecular interactions (see Fattinger C., Physical Review X 4, 031024, 2014; Fattinger C., US2016 / 0139115; and Gatterdam, V. et al., Nature Nanotechnology 12, 1089-1095, 2017, the disclosures of which are incorporated herein by reference in their entirety). Focal molography converts affinity modulation on the sensor surface into refractive index modulation caused by target molecules that bind to a precisely assembled nanopattern of molecular recognition sites, called a "mologram". The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal at the focus of the mologram via constructive interference, while scattering at non-specifically bound molecules does not contribute. The mologram can be presented on a chip by near-field reactive immersion lithography on a photosensitive monolithic graft copolymer layer. Selective, sensitive and label-free detection of biomolecules binding to the recognition site of the mologram could be demonstrated. Summary of the invention

[0010] The present invention provides a novel method for nucleic acid sequence detection, in particular, amplification and detection of multiple target nucleic acids using real-time PCR assays and solid phase focused molecular imaging. The method is performed by using oligonucleotide probes, each of which comprises an annealing portion and a label portion, referred to as a "labeled probe", wherein the label portion comprises a nucleotide sequence that is not complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence, and hybridizes with a region of the target nucleic acid subsequence. The method is also performed by using oligonucleotides attached to a molecular imaging chip called a "moligo", each moligo comprising an annealing portion and an extension portion, the annealing portion comprising a nucleotide sequence complementary to the label portion of one of the oligonucleotide probes, and the extension portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence or to the label portion.

[0011] Therefore, in one embodiment, the present invention relates to a method for amplifying and detecting a target nucleic acid in a sample, the method comprising the following steps: contacting the sample containing the target nucleic acid in a single reaction container with a pair of oligonucleotide primers, each oligonucleotide primer being capable of hybridizing to the opposite strand of a subsequence of the target nucleic acid; a labeled probe comprising an oligonucleotide sequence, the oligonucleotide sequence comprising an annealing portion and a label portion, wherein the label portion comprises a nucleotide sequence that is not complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid, the subsequence of the target nucleic acid being bounded by the oligonucleotide primer pair; amplifying the target nucleic acid by PCR using a nucleic acid polymerase having 5′ to 3′ nuclease activity, such that during the extension step of each PCR cycle, the polymerase The nuclease activity of the synthase can cleave and separate the tag portion from the annealing portion of the tagged probe; hybridize the cleaved tag portion from the tagged probe with a moligo template comprising an oligonucleotide sequence, the oligonucleotide sequence comprising an annealing portion and an extension portion, the annealing portion comprising a nucleotide sequence complementary to the tag portion of the tagged probe, the extension portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence or the tag portion of the tagged probe to form a partial duplex molecule, wherein the moligo template is attached to a solid phase chip; react the partial duplex molecule with a nucleic acid polymerase to extend the cleaved tag portion into the extension portion of the moligo template; measure the extension by molecular imaging, wherein an increase in the molecular imaging signal between the hybridization step and the extension step indicates the detection of the presence of the target nucleic acid; wherein all steps are performed in the absence of a label. In a specific embodiment, the length of the moligo template is at least 10 nucleotides longer than the length of the tagged probe. In other embodiments, the length of the moligo is at least 20 nucleotides, 30 nucleotides, 40 nucleotides, or 50 nucleotides longer than the length of the tagged probe. In certain embodiments, the length of the moligo template is at least 10 nucleotides longer than the length of the tag portion of the tagged probe. In other embodiments, the length of the moligo is at least 20 nucleotides, 30 nucleotides, 40 nucleotides, or 50 nucleotides longer than the length of the tag portion of the tagged probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a graphical depiction of the method of the present invention.

[0013] Figure 2 An HPLC chromatogram of a PCR experiment performed as described in Example 1 is shown.

[0014] Figure 3 Molecular imaging signals generated experimentally as described in Example 2 are shown.

[0015] Figure 4 Shown is a bar graph representing the molecular imaging signal by hybridization between an oligonucleotide consisting of a 22 bp primer sequence and an oligonucleotide consisting of a 100 bp sequence complementary to the moligo sequence on a 100 bp moligo. DETAILED DESCRIPTION

[0016] definition

[0017] The term "sample" as used herein includes specimens or cultures (e.g., microbiological cultures) containing nucleic acids. The term "sample" is also intended to include both biological samples and environmental samples. Samples may include specimens of synthetic origin. Biological samples include whole blood, serum, plasma, cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, stomach, peritoneum, catheter, ear, arthroscopy), biopsy samples, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells. In a preferred embodiment, the biological sample is blood, and more preferably plasma. As used herein, the term "blood" encompasses any fraction of whole blood or blood, such as serum and plasma as conventionally defined. Plasma refers to the whole blood fraction produced by centrifugation of blood treated with an anticoagulant. Serum refers to the watery portion of the fluid remaining after the blood sample coagulates. Environmental samples include environmental materials, such as surface materials, soil, water and industrial samples, and samples obtained from food and dairy processing instruments, equipment, equipment, utensils, disposable and non-disposable items. These examples should not be interpreted as limiting the same sample types applicable to the present invention.

[0018] The term "target" or "target nucleic acid" as used herein refers to any molecule whose presence is to be detected or measured or whose function, interaction or property is to be studied. For example, a target can be a biological molecule, such as a nucleic acid molecule, that can be bound to or otherwise contacted by an oligonucleotide probe.

[0019] Throughout this disclosure, the terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably. The term refers to oligonucleotides, oligos, polynucleotides, deoxyribonucleotides (DNA), genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones, plasmids, M13, P1, cosmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), amplified nucleic acids, amplicons, PCR products and other types of amplified nucleic acids, RNA / DNA hybrids and polyamide nucleic acids (PNA), all of which can be in single-stranded or double-stranded form, and unless otherwise limited, known analogs of natural nucleotides that can function in a similar manner will be covered as naturally occurring nucleotides and combinations and / or mixtures thereof. Therefore, the term "nucleotide" refers to naturally occurring and modified / non-naturally occurring nucleotides, including nucleoside triphosphates, nucleoside diphosphates and nucleoside monophosphates and monophosphate monomers present in polynucleic acids or oligonucleotides. Nucleotides can also be ribose; 2'-deoxy; 2', 3'-deoxy and a large number of other nucleotide mimetics well known in the art. Mimetics include chain-terminating nucleotides, such as 3'-O-methyl, halobase or sugar substitutions; alternative sugar structures, including non-sugar, alkyl ring structures; alternative bases, including inosine; deaza modified; chi and psi, modified by linkers; modified by a large number of labels; phosphodiester modifications or substitutions, including thiophosphates, methylphosphonates, boranophosphates, amides, esters, ethers; and substantial or complete internucleotide substitutions, including cleavable bonds such as photolyzable nitrophenyl moieties.

[0020] The presence or absence of a target can be detected quantitatively or qualitatively. The target can appear in a variety of different forms, including, for example, simple or complex mixtures, or in a substantially purified form. For example, the target can be a part of a sample containing other components, or it can be the only or main component of a sample. Thus, the target can be a component of a whole cell or tissue, a cell or tissue extract, a fractionated lysate thereof, or a substantially purified molecule. The target can also have a known or unknown sequence or structure.

[0021] The term "amplification reaction" refers to any in vitro means for amplifying copies of a nucleic acid target sequence.

[0022] "Amplification" refers to the step of submitting a solution to conditions sufficient to allow amplification. The components of the amplification reaction may include, but are not limited to, for example, primers, polynucleotide templates, polymerases, nucleotides, dNTPs, etc. The term "amplification" generally refers to an "exponential" increase in a target nucleic acid. However, "amplification" as used herein may also refer to a linear increase in the number of selective target sequences of a nucleic acid, but is distinct from a one-time, single primer extension step.

[0023] "Polymerase chain reaction" or "PCR" refers to a method by which a specific segment or subsequence of a target double-stranded DNA is amplified exponentially. PCR is well known to those skilled in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., 1990.

[0024] As used herein, "oligonucleotide" refers to a linear oligomer of natural or modified nucleoside monomers connected by a phosphodiester bond or its analogs. Oligonucleotides include deoxyribonucleosides, ribonucleosides, their end group isomer forms, peptide nucleic acids (PNA), etc. that can specifically bind to target nucleic acids. Typically, monomers are connected by phosphodiester bonds or their analogs to form oligonucleotides, and their size ranges from several monomer units, such as 3-4, to tens of monomer units, such as 40-60. Whenever an oligonucleotide is represented by a letter sequence, such as "ATGCCTG", it should be understood that the nucleotides are in 5'-3' order from left to right, and unless otherwise specified, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents ribonucleoside uridine. Typically, oligonucleotides contain four natural deoxynucleotides; however, they can also contain ribonucleosides or non-natural nucleotide analogs. When the enzyme has a specific oligonucleotide or polynucleotide substrate requirement for activity, such as single-stranded DNA, RNA / DNA duplex, etc., then the selection of an appropriate composition for the oligonucleotide or polynucleotide substrate is well within the skill of the art.

[0025] As used herein, "oligonucleotide primer" or simply "primer" refers to a polynucleotide sequence that hybridizes with a sequence on a target nucleic acid template and promotes the detection of an oligonucleotide probe. In an amplification embodiment of the present invention, an oligonucleotide primer is used as a starting point for nucleic acid synthesis. In a non-amplification embodiment, an oligonucleotide primer can be used to generate a structure that can be cracked by a cleavage agent. Primers can have various lengths, and generally have a length of less than 50 nucleotides, such as a length of 12-25 nucleotides. The length and sequence of primers used for PCR can be designed based on principles known to those skilled in the art.

[0026] As used herein, the term "oligonucleotide probe" refers to a polynucleotide sequence that is capable of hybridizing or annealing to a target nucleic acid of interest and allows for specific detection of the target nucleic acid.

[0027] A "label" or "label molecule" is a molecule that confers a detectable signal. For example, a detectable signal can be a colorimetric, fluorescent, or luminescent signal. In contrast, "label-free" refers to the absence of a molecule that confers a detectable signal. For purposes of the present invention, an affinity tag that does not itself confer a detectable signal is not a label, and an assay that employs an affinity tag that does not itself confer a detectable signal is considered a label-free assay.

[0028] "Mismatched nucleotide" or "mismatch" refers to a nucleotide that is not complementary to the target sequence at one or more positions. An oligonucleotide probe may have at least one mismatch, but may also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides.

[0029] As used herein, the term "polymorphism" refers to allelic variants. Polymorphisms can include single nucleotide polymorphisms (SNPs) and simple sequence length polymorphisms. Polymorphisms can be attributed to one or more nucleotide substitutions of an allele compared to another allele, or can be attributed to insertions or deletions, repetitions, inversions and other changes known in the art.

[0030] The term "modification" as used herein refers to changes in the oligonucleotide probe at the molecular level (e.g., base moieties, sugar moieties, or phosphate backbones). Nucleoside modifications include, but are not limited to, the introduction of cleavage blockers or cleavage inducers, the introduction of minor groove binders, isotope enrichment, isotope depletion, the introduction of deuterium, and halogen modifications. Nucleoside modifications may also include parts that increase hybridization stringency or increase the melting temperature of the oligonucleotide probe. For example, nucleotide molecules may be modified with an additional bridge connecting 2' and 4' carbons to produce locked nucleic acid (LNA) nucleotides that are resistant to nuclease cleavage (such as Imanishi et al., U.S. Patent No. 6,268,490 and in U.S. Patent No. 6,794,499 of Wengel et al., the entire contents of which are incorporated herein by reference in their entirety). The composition of the tag portion of the oligonucleotide probe and the quencher oligonucleotide molecule is limited only by their ability to form a stable duplex. Thus, these oligonucleotides may comprise DNA, L-DNA, RNA, L-RNA, LNA, L-LNA, PNA (peptide nucleic acid, as described in Nielsen et al., U.S. Pat. No. 5,539,082), BNA (bridged nucleic acid, e.g., 2′,4′-BNA (NC) [2′-O,4′-C-aminomethylene bridged nucleic acid], as described in Rahman et al., J. Am. Chem. Soc. 2008; 130(14): 4886-96)), L-BNA, etc. (wherein "L-XXX" refers to the L enantiomer of the nucleic acid sugar unit) or any other known variations and modifications on the nucleotide bases, sugars, or phosphodiester backbones.

[0031] Other examples of nucleoside modification include various 2' substitutions introduced in the sugar moiety of oligonucleotides, such as halo, alkoxy and allyloxy groups. Existing evidence shows that 2'-substituted-2'-deoxyadenosine polynucleotides are similar to double-stranded RNA rather than DNA. Ikehara et al., (Nucleic Acids Res., 1978, 5, 3315) have shown that the 2'-fluorine substituents duplexed with their complementary sequences in poly-A, poly-I or poly-C are significantly more stable than ribonucleotide or deoxyribonucleotide polyduplexes, which are determined by standard melting assays. Inoue et al., (Nucleic Acids Res., 1987, 15, 6131) have described the synthesis of mixed oligonucleotide sequences containing 2'-OMe (O-methyl) substituents on the nucleotides of each core. Mixed 2'-OMe substituted oligonucleotides hybridize as strongly to their RNA complements as RNA-RNA duplexes, which are significantly stronger than RNA-DNA heteroduplexes of the same sequence. Thus, examples of substitutions at the 2' position of the sugar include F, CN, CF3, OCF3, OMe, OCN, O-alkyl, S-alkyl, SMe, SO2Me, ONO2, NO2, NH3, NH2, NH-alkyl, OCH3=CH2, and OCCH.

[0032] The term "specific" or "specificity" with respect to the binding of one molecule to another molecule, such as a probe of a target polynucleotide, refers to the recognition, contact, and formation of a stable complex between the two molecules, and substantially less recognition, contact, or formation of a complex with the other molecule. As used herein, the term "annealing" refers to the formation of a stable complex between two molecules.

[0033] A probe is "capable of annealing" to a nucleic acid sequence if at least one region of the probe has substantial sequence identity to at least one region of the complement of the nucleic acid sequence. "Substantial sequence identity" is at least about 80%, preferably at least about 85%, more preferably at least about 90%, 95% or 99%, and most preferably 100% sequence identity. For purposes of determining sequence identity of DNA and RNA sequences, U and T are generally considered to be identical nucleotides. For example, a probe comprising the sequence ATCAGC is capable of hybridizing to a target RNA sequence comprising the sequence GCUGAU.

[0034] As used herein, the term "cleaving agent" refers to any means capable of cleaving an oligonucleotide probe to produce a fragment, including but not limited to an enzyme. For methods in which amplification does not occur, the cleaving agent may be used only to cleave, degrade or otherwise separate the second portion of the oligonucleotide probe or a fragment thereof. The cleaving agent may be an enzyme. The cleaving agent may be natural, synthetic, unmodified or modified.

[0035] For methods in which amplification occurs, the lysing agent is preferably an enzyme having both synthetic (or polymerizing) activity and nuclease activity. Such enzymes are typically nucleic acid amplification enzymes. Examples of nucleic acid amplification enzymes are nucleic acid polymerases, such as Thermus aquaticus (Taq) DNA polymerase. or E. coli DNA polymerase I. The enzyme may be naturally occurring, unmodified or modified.

[0036] "Nucleic acid polymerase" refers to an enzyme that catalyzes the incorporation of nucleotides into nucleic acids. Exemplary nucleic acid polymerases include DNA polymerase, RNA polymerase, terminal transferase, reverse transcriptase, telomerase, and the like.

[0037] "Thermostable DNA polymerase" refers to a DNA polymerase that is stable (i.e., resists decomposition or denaturation) and retains sufficient catalytic activity when the elevated temperature reaches the selected time period. For example, when the time required for denaturing double-stranded nucleic acids is reached at an elevated temperature, the thermostable DNA polymerase retains sufficient activity to carry out subsequent primer extension reactions. The heating conditions required for nucleic acid denaturation are well known in the art and are illustrated in U.S. Patent Nos. 4,683,202 and 4,683,195. As used herein, thermostable polymerases are generally suitable for temperature cycling reactions, such as polymerase chain reaction ("PCR"). Examples of thermostable nucleic acid polymerases include Thermus aquaticus Taq DNA polymerase, Thermus species Z05 polymerase, Thermus flavus polymerase, Thermotogamaritima polymerase, such as TMA-25 and TMA-30 polymerases, Tth DNA polymerases, etc.

[0038] A "modified" polymerase refers to a polymerase in which at least one monomer is different from a reference sequence, such as a native or wild-type form of the polymerase or another modified form of the polymerase. Exemplary modifications include monomer insertions, deletions, and substitutions. Modified polymerases also include chimeric polymerases having identifiable component sequences (e.g., structural or functional domains, etc.) derived from two or more parents. Also included within the definition of modified polymerases are those that contain chemical modifications of the reference sequence. Examples of modified polymerases include G46E E678G CS5 DNA polymerase, G46EL329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46E L329AD640G S671F E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase, ΔZ05-Gold polymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, E678G TMA-30 polymerase, and the like.

[0039] The term "5' to 3' nuclease activity" or "5'-3' nuclease activity" refers to the activity of a nucleic acid polymerase, usually associated with nucleic acid chain synthesis, thereby removing nucleotides from the 5' end of the nucleic acid chain, for example, E. coli DNA polymerase I has this activity, while Klenow fragment does not. Some enzymes with 5' to 3' nuclease activity are 5' to 3' exonucleases. Examples of such 5' to 3' exonucleases include: exonucleases from B. subtilis, phosphodiesterase from spleen, lambda exonuclease, exonuclease II from yeast, exonuclease V from yeast, and exonuclease from Neurospora crassa.

[0040] The term "propylene glycol" or "propylene glycol spacer" refers to 1,3-propylene glycol and is synonymous with propane-1,3-diol, 1,3-dihydroxypropane and trimethylene glycol. The term "HEG" or "HEG spacer" refers to hexaethylene glycol, which is synonymous with 3,6,9,12,15-pentaoxaheptadecane-1,17-diol.

[0041] Various aspects of the present invention are based on the special properties of nucleic acid polymerases. Nucleic acid polymerases can have multiple activities, including 5' to 3' nuclease activity, whereby nucleic acid polymerases can cleave single nucleotides or small oligonucleotides from oligonucleotides annealed to their larger, complementary polynucleotides. In order for cleavage to be effective, the upstream oligonucleotide must also anneal to the same larger polynucleotide.

[0042] Can be Detection of target nucleic acids utilizing 5' to 3' nuclease activity may be performed using a "5'-nuclease assay" as described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280, all of which are incorporated herein by reference. In the assay, there is a labeled detection probe that hybridizes within the amplification region during the amplification reaction. The probe is modified to prevent the probe from acting as a primer for DNA synthesis. Amplification is performed using a DNA polymerase with 5' to 3' exonuclease activity. During each synthesis step of the amplification, any probe that hybridizes with the target nucleic acid downstream from the extended primer is degraded by the 5' to 3' exonuclease activity of the DNA polymerase. Therefore, the synthesis of the new target chain also leads to the degradation of the probe, and the accumulation of degradation products provides a measure of the synthesis of the target sequence.

[0043] The 5' nuclease assay for detecting a target nucleic acid can employ any polymerase having 5' to 3' exonuclease activity. Thus, in some embodiments, the polymerase having 5'-nuclease activity is a thermostable and thermoactive nucleic acid polymerase. Such thermostable polymerases include, but are not limited to, natural and recombinant forms of polymerases from multiple species of the eubacterial genera Thermus, Thermatoga, and Thermosipho, as well as chimeric forms thereof. For example, Thermus species polymerases that can be used in the methods of the invention include Thermus aquaticus (Taq) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus species Z05 (Z05) DNA polymerase, Thermus species sps17 (sps17), and Thermus species Z05 (e.g., as described in U.S. Pat. Nos. 5,405,774; 5,352,600; 5,079,352; 4,889,818; 5,466,591; 5,618,711; 5,674,738 and 5,795,762). Thermatoga polymerases that can be used in the methods of the present invention include, for example, Thermotoga marmma DNA polymerase and Thermotoga neapolitana DNA polymerase, while an example of a Thermosipho polymerase that can be used is Thermosipho africanus DNA polymerase. The sequences of Thermosipho marmma and Thermosipho africanus DNA polymerases are disclosed in International Patent Application No. PCT / US91 / 07035, having Publication No. WO 92 / 06200. The sequence of Thermotoga neapolitana can be found in International Patent Publication No. WO 97 / 09451.

[0044] In a 5' nuclease assay, amplification detection is typically performed simultaneously with amplification (i.e., "real time"). In some embodiments, amplification detection is quantitative, and amplification detection is real time. In some embodiments, amplification detection is qualitative (e.g., endpoint detection of the presence or absence of a target nucleic acid). In some embodiments, amplification detection is after amplification. In some embodiments, amplification detection is qualitative, and amplification detection is after amplification.

[0045] In the present invention, real-time PCR amplification and detection of two or more target nucleic acids are performed in a single reaction vessel (e.g., tube, well) using oligonucleotide probes called tagged probes, each of which comprises two different parts. The first part is the annealing part and comprises a sequence that is at least partially complementary to the target nucleic acid sequence, so that it can hybridize with the target sequence. The second part of the oligonucleotide probe (i.e., the tagged probe) is called the tag part. In one embodiment, the tag part is attached to the 5′ end of the annealing part. In another embodiment, the tag part is attached to the 3′ end of the annealing part. In another embodiment, the tag part is attached to any position between the 5′ end and the 3′ end of the annealing part via a linker. The tag part may comprise a nucleotide sequence that is not complementary to the target nucleic acid sequence and forms a “wing” region that is not capable of binding to the target nucleic acid (see for a graphical representation of a 5′ wing probe). Figure 1 ). The annealing portion and the tag portion of the tagged probe can optionally be separated by a non-nucleotide "linker". The linker can be composed of carbon, carbon and oxygen, carbon and nitrogen, or any combination thereof and can have any length. In addition, the linker can be composed of a linear portion or a cyclic portion. The linker can be derived from a single unit or from multiple identical or different units separated by phosphate bonds. The purpose of the linker is to create a region at the junction of the annealing portion and the tag portion of the tagged probe. When the tag portion is separated from the annealing portion, the linker can also prevent the tag portion from being extended by a nucleic acid polymerase. Alternatively, another modification of the separated tag portion makes it unable to be extended by a nucleic acid polymerase.

[0046] The general principles of performing real-time PCR amplification and target nucleic acid detection using labeled probes in a 5' nuclease assay are described below. First, a sample suspected of containing a target nucleic acid is provided. The sample is then contacted with a PCR reagent containing both an oligonucleotide primer and a novel oligonucleotide probe capable of generating a target nucleic acid amplicon in a single reaction vessel (e.g., a single test tube or a single well in a multi-well microplate). PCR amplification is initiated by using a nucleic acid polymerase having 5' to 3' nuclease activity, so that during the extension step of each PCR cycle, the nuclease activity of the polymerase can cleave and separate the label portion from the annealed portion of the labeled probe. The separated label portion may optionally contain a modification (such as a non-nucleotide linker) so that it cannot be extended by a nucleic acid polymerase.

[0047] The separated label portion is then hybridized with an oligonucleotide attached to a molecular imaging chip. The attached oligonucleotide is called a "moligo" and comprises two parts, an annealing portion and an extension portion, the annealing portion comprising a nucleotide sequence complementary to the label portion of the tagged probe, and the extension portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence or to the label portion of the tagged probe. Moligo acts as a template for the extension of the separated label portion, which acts as a primer because it hybridizes with the annealing portion of moligo and extends along the extension portion of moligo to form an extended duplex molecule. Focused molecular imaging is then used to detect the generation of the extended duplex on the chip.

[0048] Focused molecular imaging has recently been described as a new analytical method for detecting molecular interactions in biological samples (see Fattinger, C., Physical Review X, 4, 031024, 2014; Gatterdam, V. et al., Nature Nanotechnology 12, 1089-1095, 2017). In short, in focused molecular imaging, coherent laser light is diffracted at a coherent assembly of interaction sites. The coherent assembly includes a precise submicron pattern of the same interaction site, which needs to be phase-matched with the incident coherent light over the entire pattern. The light diffracted at the assembly constructively interferes at a diffraction-limited focal spot (Airy disc). Molecular assemblies with these characteristics are called "molograms". In essence, a mologram is a synthetic hologram composed of molecules that scatters light into a diffraction-limited focus and is fine-tuned to allow molecular interaction analysis. The acquisition of molecular imaging signals is achieved by detecting the diffracted coherent light at the focus of the mologram. The light intensity at the focus of molecular imaging is modulated quadratically with the surface mass density of the mologram. Background scattering from molecules randomly attached to the assay surface is essentially separated from the coherent molecular imaging signal by focused molecular imaging. Molecular imaging does not require the presence of labels. Therefore, label-free molecular imaging visualizes biomolecular recognition events by detecting tiny phase difference changes in the nanoenvironment of the recognition site in the mologram. This opens up new perspectives and possibilities for analyzing non-covalent interactions.

[0049] In the measurement of biological interactions, most bioanalytical methods face severe problems with background signals or involve laborious referencing procedures. Although some methods, such as fluorescence-based techniques (e.g., Forster resonance energy transfer, or FRET), circumvent some of these issues, they may interfere with the molecular interactions being studied due to the use of fluorescent dyes or other interfering labels. In contrast, focused molecular imaging converts the invisible affinity modulation of the mologram, which is the difference in the density of target molecule recognition sites between ridges and grooves, into a refractive index modulation, resulting in a focused beam. Discrimination between specific and nonspecific binding can be achieved by minimizing the separation of sensing and reference channels to a minimum (less than the wavelength of light) while embedding hundreds of reference and signal regions into a single sensing spot.

[0050] In order to perform meaningful molecular imaging measurements, several instrumental requirements need to be met. Focused molecular imaging requires appropriate dark-field illumination of the mologram, which can be achieved by guiding the pattern of high-refractive-index thin-film optical waveguides. This ensures a high signal-to-noise ratio. Molecular imaging also requires submicron-scale coherent patterns of identification sites, which can be created by various existing soft lithography methods developed for biochips. One specific embodiment involves the use of a photoactivatable graft copolymer, similar to that described by Serrano et al. (Macromol. Rapid Commun. 37, 622-629, 2016), to coat tantalum pentoxide (Ta2O5) thin-film optical waveguides. The copolymer has four key properties. (1) Prior to photolithographic exposure, the copolymer layer on the assay surface is monolithic and homogeneous in terms of both its chemical and optical properties. The monolithic layer does not contain coherently arranged molecules, and therefore it does not show any molecular imaging signal prior to the photolithography step. (2) The copolymer layer is resistant to nonspecific molecular adsorption. Although the copolymer layer exhibits defects and irregularities at the nanoscale, these defects are not coherently arranged and do not contribute to the molecular imaging signal. (3) The copolymer layer contains photoactivatable functional groups for forming recognition sites upon exposure to light. (4) The formed copolymer adsorption layer can undergo necessary chemical and biochemical modifications after initial surface coating and is sufficiently stable to undergo temperature cycling during PCR reactions.

[0051] Embodiments of the present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0052] Examples

[0053] Example 1 Feasibility study: cleavage and extension of HIV-LTR probe in solution

[0054] Real-time PCR experiments were performed to verify that: the tagged probe contained an annealing portion that, in the presence of the target HIV-1 LTR gene, could hybridize with a portion of the HIV-1 LTR gene and the unique tag portion would be cleaved; and the released tag portion would be able to anneal to and extend from the "moligo" template, which contained a sequence complementary to the tag portion. Table 1 shows the nucleotide sequences of the tagged probe and moligo.

[0055] Table 1

[0056]

[0057] PCR assays were performed in individual wells in the presence (FL HIV(+)) or absence (FL HIV(-)) of the target HIV-1 LTR gene and in the presence or absence of the Moligo tag 3 template. At the end of the assay, aliquots were removed from the wells and subjected to HPLC chromatography to analyze the resulting products. The experimental results are shown in Figure 2 In the PCR reaction in the presence of the target gene and in the absence of the tag 3moligo, peaks representing cleavage fragments of the HIV-1 LTR labeled probe were observed (see FL HIV(+)No Moligo figure). Close inspection of these cleavage product peaks showed that they represented fragments containing intact tag portions with one (+1), two (+2), three (+3), four (+4), or five (+5) nucleotides from the annealed portion (see Figure 2 Interestingly, in the reaction where both the target gene and the tag 3moligo were present, a small peak was observed, the size of which corresponded to the full-length labeled Moligo complementary sequence (see FL (HIV (+) with Moligo map). This result demonstrates that the cleaved tag portion is able to hybridize with and extend the Moligo template in solution, and provides the feasibility of performing this type of assay on a Moligo template immobilized on a solid phase.

[0058] Example 2: Feasibility study of nucleic acid hybridization detection by molecular imaging

[0059] A feasibility study of detecting nucleic acid hybridization and extension by molecular imaging was performed on a Zeptochip microarray chip (Zeptosens AG, Witterswil, Switzerland) and has been described in Pawlak M. et al., Proteomics 2002; 2(4): 383-93, which is incorporated herein by reference in its entirety. A running DNA buffer consisting of 20 mM Tris, 5 mM MgCl2, 50 mM KCl, pH 8.0 was applied to the flow chamber within the chip flow system at a constant flow rate of 15 μL / min. First, streptavidin (SAv) was injected as a normalized signal for focused molecular imaging into a surface mass density modulated to 520 pg / mm 2 Negative controls between cDNA without affinity tag and immobilized SAv did not produce any changes in the molecular imaging signal (see Figure 3 (i)), indicating that no unwanted interactions occurred. Next, immobilization of biotinylated 100 bp HIV-LTR moligo oligonucleotide (SEQ ID NO: 4) was achieved using 100 nM SAv and biotinylated HIV-LTR moligo-100 as a pre-complex at a ratio of 1:1. Figure 3 (i) and Figure 3 (ii) The formation of this pre-complex leads to an increase in the molecular imaging signal as shown in the comparison between the left and right sides. The hybridization between the immobilized HIV-LTR moligo-100 and the following items were then compared: a 22 bp primer (AT-MOLO-3 (SEQ ID NO: 3)) that can bind to the annealed portion of the moligo-100 template ( Figure 3 (iii)); or a 100 bp sequence that is completely complementary to the moligo-100 template ( Figure 3 (ii) Left side). The increase in molecular imaging signal between the hybridization of the 22 bp primer and the 100 bp complementary sequence is Figure 4 This is shown graphically in Figure 2. This demonstrates that molecular imaging is able to distinguish between extended cleaved tags and non-extended cleaved tags.

[0060] Informal sequence listing

[0061] SEQ ID NO 1: HIV-1 LTR tag 3 probe sequence

[0062]

[0063] SEQ ID NO 2: Moligo tag 3 oligonucleotide sequence

[0064]

[0065] SEQ ID NO 3: AT-MOLO-3 oligonucleotide sequence

[0066]

[0067] SEQ ID NO 4: HIV-LTR-moligo-100 oligonucleotide sequence

[0068]

Claims

1. A method for amplifying and detecting a target nucleic acid in a sample, the method comprising the following steps: (a) contacting the sample containing the target nucleic acid in a single reaction vessel with (i) a pair of oligonucleotide primers, each oligonucleotide primer being capable of hybridizing to opposite strands of a subsequence of the target nucleic acid; (ii) a tagged probe comprising an oligonucleotide sequence comprising an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence that is not complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid that is bounded by the oligonucleotide primer pair, and wherein the tag portion is attached to the 5′ end or the 3′ end of the annealing portion, or wherein the tag portion is attached to any position between the 5′ end and the 3′ end of the annealing portion via a linker; (b) amplifying the target nucleic acid by PCR using a nucleic acid polymerase having 5' to 3' nuclease activity, such that during the extension step of each PCR cycle, the nuclease activity of the polymerase can cleave and separate the tag portion from the annealed portion of the tagged probe; (c) hybridizing the cleaved tag portion from the tagged probe with a moligo template, the moligo template comprising an oligonucleotide sequence, the oligonucleotide sequence comprising an annealing portion and an extension portion, the annealing portion comprising a nucleotide sequence complementary to the tag portion of the tagged probe, and the extension portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence or to the tag portion of the tagged probe, so that the cleaved tag portion hybridizes with the annealing portion of the moligo template to form a partial duplex molecule, wherein the moligo template is attached to a solid phase chip; (d) reacting the partial duplex molecule with a nucleic acid polymerase to extend the cleaved tag portion into the extended portion of the moligo template; (e) measuring the extension in step (d) by molecular imaging, wherein an increase in the molecular imaging signal between step (c) and step (d) indicates the detection of the presence of the target nucleic acid; wherein all steps (a) to (e) are performed in the absence of a marker, and The methods described herein are not intended for diagnostic purposes.

2. The method of claim 1, wherein the length of the moligo template is at least 10 nucleotides longer than the length of the tagged probe.

3. The method of claim 1, wherein the length of the moligo template is at least 20 nucleotides longer than the length of the tagged probe.

4. The method of claim 1, wherein the length of the moligo template is at least 30 nucleotides longer than the length of the tagged probe.

5. The method of claim 1, wherein the length of the moligo template is at least 40 nucleotides longer than the length of the tagged probe.

6. The method of claim 1, wherein the length of the moligo template is at least 50 nucleotides longer than the length of the tagged probe.

7. The method of any one of claims 1 to 6, wherein the tagged probe comprises SEQ ID NO: 1, and the moligo template comprises SEQ ID NO:

4.

8. The method according to any one of claims 1 to 7, wherein the annealing portion and the tag portion of the tagged probe are separated by a non-nucleotide linker.

9. The method of claim 8, wherein the linker is a linear moiety or a cyclic moiety.

10. The method according to any one of claims 8 or 9, wherein the linker is derived from a single unit or from a plurality of identical or different units separated by phosphate bonds.

11. The method according to any one of claims 1 to 10, wherein the extension is detected using focused molecular imaging.

12. Use of a labeled probe in the preparation of a kit for a method for amplifying and detecting a target nucleic acid in a sample, the method comprising the following steps: (a) contacting the sample containing the target nucleic acid in a single reaction vessel with (i) a pair of oligonucleotide primers, each oligonucleotide primer being capable of hybridizing to opposite strands of a subsequence of the target nucleic acid; (ii) a tagged probe comprising an oligonucleotide sequence comprising an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence that is not complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid that is bounded by the oligonucleotide primer pair, and wherein the tag portion is attached to the 5′ end or the 3′ end of the annealing portion, or wherein the tag portion is attached to any position between the 5′ end and the 3′ end of the annealing portion via a linker; (b) amplifying the target nucleic acid by PCR using a nucleic acid polymerase having 5' to 3' nuclease activity, such that during the extension step of each PCR cycle, the nuclease activity of the polymerase can cleave and separate the tag portion from the annealed portion of the tagged probe; (c) hybridizing the cleaved tag portion from the tagged probe with a moligo template, the moligo template comprising an oligonucleotide sequence, the oligonucleotide sequence comprising an annealing portion and an extension portion, the annealing portion comprising a nucleotide sequence complementary to the tag portion of the tagged probe, and the extension portion comprising a nucleotide sequence that is not complementary to the target nucleic acid sequence or to the tag portion of the tagged probe, so that the cleaved tag portion hybridizes with the annealing portion of the moligo template to form a partial duplex molecule, wherein the moligo template is attached to a solid phase chip; (d) reacting the partial duplex molecule with a nucleic acid polymerase to extend the cleaved tag portion into the extended portion of the moligo template; (e) measuring the extension in step (d) by molecular imaging, wherein an increase in the molecular imaging signal between step (c) and step (d) indicates the detection of the presence of the target nucleic acid; wherein all steps (a) to (e) are performed in the absence of a label.

13. The method of claim 12, wherein the length of the moligo template is at least 10 nucleotides longer than the length of the tagged probe.

14. The method of claim 12, wherein the length of the moligo template is at least 20 nucleotides longer than the length of the tagged probe.

15. The method of claim 12, wherein the length of the moligo template is at least 30 nucleotides longer than the length of the tagged probe.

16. The method of claim 12, wherein the length of the moligo template is at least 40 nucleotides longer than the length of the tagged probe.

17. The method of claim 12, wherein the length of the moligo template is at least 50 nucleotides longer than the length of the tagged probe.

18. The method of any one of claims 12 to 17, wherein the tagged probe comprises SEQ ID NO: 1, and the moligo template comprises SEQ ID NO:

4.

19. The method of any one of claims 12 to 18, wherein the annealing portion and the tag portion of the tagged probe are separated by a non-nucleotide linker.

20. The method of claim 19, wherein the linker is a linear moiety or a cyclic moiety.

21. The method according to any one of claims 19 or 20, wherein the linker is derived from a single unit or from a plurality of identical or different units separated by phosphate bonds.

22. The method of any one of claims 12 to 21, wherein the extension is detected using focused molecular imaging.

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