WO2019231287A1 - Procédé de détection d'un acide nucléique cible à l'aide d'une amplification par cercle roulant et composition pour la détection d'un acide nucléique cible - Google Patents

Procédé de détection d'un acide nucléique cible à l'aide d'une amplification par cercle roulant et composition pour la détection d'un acide nucléique cible Download PDF

Info

Publication number
WO2019231287A1
WO2019231287A1 PCT/KR2019/006602 KR2019006602W WO2019231287A1 WO 2019231287 A1 WO2019231287 A1 WO 2019231287A1 KR 2019006602 W KR2019006602 W KR 2019006602W WO 2019231287 A1 WO2019231287 A1 WO 2019231287A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
target nucleic
barcode
sequence
template
Prior art date
Application number
PCT/KR2019/006602
Other languages
English (en)
Korean (ko)
Inventor
신세현
나원휘
이호윤
김황수
오예은
Original Assignee
고려대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 고려대학교 산학협력단 filed Critical 고려대학교 산학협력단
Priority to EP19811578.4A priority Critical patent/EP3805408B1/fr
Priority to US15/733,965 priority patent/US11608522B2/en
Priority claimed from KR1020190064750A external-priority patent/KR102293402B1/ko
Publication of WO2019231287A1 publication Critical patent/WO2019231287A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for detecting a target nucleic acid based on rolling circle amplification (RCA), and more particularly, when the target nucleic acid is present, the target nucleic acid and the template form an annular template to perform an amplification reaction.
  • RCA rolling circle amplification
  • NGS Next Generation Sequencing
  • ddPCR Droplet Digital PCR
  • NGS requires a few days of inspection time and the cost of analysis is quite high.
  • ddPCR is difficult to detect multiplely, so only one mutation can be identified in one reagent reaction tube. Therefore, there are limitations to the high cost of many reagent consumptions to test all the major point mutation genes.
  • the general detection method using DNA complementary binding has a limitation in that it is difficult to distinguish minute differences such as point mutations, and thus, a normal gene and a mutant gene cannot be distinguished and recognized as the same signal.
  • CRISPR cas9 is a technology that can detect specific mutant genes by the action of enzyme without such equipment, but the limitation of recognizable mutant sequence is the biggest limitation, and the price is too expensive to use for diagnostic purposes. There are disadvantages.
  • RNA fragments are used together, and there is a limitation that RNA is not stable and can be applied only at the laboratory environment level.
  • a target nucleic acid binding region which is disposed at both ends and complementarily binds to the target nucleic acid sequence ; ii) primer binding regions; iii) restriction enzyme binding region; And iv) a barcode generating region; amplified products of a predefined barcode region can be generated and detected according to the presence or absence of a target nucleic acid (a nucleic acid including a target nucleic acid sequence) using a linear template and a restriction enzyme.
  • a target nucleic acid a nucleic acid including a target nucleic acid sequence
  • Another object of the present invention to provide a composition for amplifying a ring for the detection of target nucleic acids with high sensitivity.
  • the present invention comprises the steps of (a) preparing a single stranded nucleic acid prepared from a sample sample; (b) i) a target nucleic acid binding region disposed at both ends and binding complementarily with the target nucleic acid sequence; ii) primer binding regions; iii) restriction enzyme binding region; And iv) a barcode generation region; and ligation is performed after combining the linear template with the single-stranded nucleic acid to form a cyclic template-target nucleic acid conjugate; (c) rotating single ring amplification in the presence of a primer binding to the primer binding region and a nucleic acid for cleavage having the same sequence as the sequence of the restriction enzyme binding region to generate a single-stranded amplification product, and a restriction enzyme (restriction enzyme) Cutting the region in which the single-stranded amplification product and the cleavage nucleic acid are complementarily bound to each other to include a first
  • RCA Rolling Circle Amplification
  • the present invention also provides a kit comprising: (a) i) a target nucleic acid binding region disposed at both ends and complementarily binding to a target nucleic acid sequence; ii) primer binding regions; iii) restriction enzyme binding region; And iv) a barcode generation region; (b) a primer that binds to the primer binding region; (c) a cleavage nucleic acid having the same sequence as the sequence of the restriction enzyme binding region; (d) restriction enzymes; It provides a composition for amplifying the rotation ring for the target nucleic acid detection, and (e) ligase.
  • Target nucleic acid detection method can detect the presence of the target nucleic acid at the same time multiple times without sequencing by detecting a predefined barcode sequence according to the type of target nucleic acid, do not use expensive enzymes such as CRISPR Therefore, it is not only inexpensive, but also a variety of existing nucleic acid detection systems can be used as a method for detecting barcode sequences, which is useful for detecting genetic variations.
  • the RCA amplification composition for detecting target nucleic acids according to the present invention can not only simultaneously detect multiple targets in a single reaction using a template prepared for each target nucleic acid, but also detect a base unit variation with high sensitivity. It is useful for various molecular diagnosis.
  • Figure 1 shows a method for confirming the generation mechanism of the barcode sequence according to the presence or absence of the target nucleic acid sequence of the present invention by fluorescence detection.
  • Figure 2 is a schematic diagram showing the structure of the enzyme and DNA required for the method of the present invention.
  • Figure 3 is a schematic diagram showing the stage in which the rotation ring amplification according to the method of the present invention.
  • Figure 4 (A) is a schematic diagram showing the product produced by the action of the restriction enzyme in the RCA amplification reaction according to the method of the present invention, (B) is a schematic diagram showing the role of the product.
  • Figure 5 (A) is a result of confirming the detection of wild type and mutant PIK3CA E545K mutation detection according to an embodiment of the present invention by fluorescence signal detection, (B) is the result confirmed by the electrophoresis.
  • Figure 6 confirms the results of the restriction enzyme treatment of the RCA amplification products of wild type and mutant PIK3CA gene according to an embodiment of the present invention (Example 1) by electrophoresis.
  • Figure 7 confirms the results of the restriction enzyme treatment of the RCA amplification products of wild type and mutant PIK3CA gene according to an embodiment of the present invention (Example 2) by electrophoresis.
  • Figure 8 shows the result of detecting the barcode amplification product according to an embodiment of the present invention using a surface plasmon resonance (Surface plasmon resonance) sensor.
  • the present invention to determine whether the target nucleic acid can be detected through the rotation ring amplification reaction containing a restriction enzyme using a template specifically designed in the rotation ring amplification reaction.
  • a target nucleic acid binding region that complementarily binds to a target nucleic acid present at both ends; ii) primer binding regions; iii) restriction enzyme binding region; And iv) a barcode region; and a template comprising a barcode strand binding to a single-stranded target nucleic acid, followed by ligation to produce a cyclic template, and then performing a spin ring amplification reaction, while processing a restriction enzyme to amplify the barcode region. After separation, the barcode region was detected to confirm that various types of target nucleic acids can be detected simultaneously (FIGS. 1 to 4).
  • the present invention in one aspect (a) preparing a single stranded nucleic acid prepared from a sample sample; (b) i) a target nucleic acid binding region disposed at both ends and binding complementarily with the target nucleic acid sequence; ii) primer binding regions; iii) restriction enzyme binding region; And iv) a barcode generation region; and ligation is performed after combining the linear template with the single-stranded nucleic acid to form a cyclic template-target nucleic acid conjugate; (c) rotating single ring amplification in the presence of a primer binding to the primer binding region and a nucleic acid for cleavage having the same sequence as the sequence of the restriction enzyme binding region to generate a single-stranded amplification product, and a restriction enzyme (restriction enzyme) Cutting the region in which the single-stranded amplification product and the cleavage nucleic acid are complementarily bound to each other to include a first amplification product including the bar
  • the present invention relates to a target nucleic acid detection method using Rolling Circle Amplification (RCA), wherein the barcode is prepared.
  • RCA Rolling Circle Amplification
  • target nucleic acid means all kinds of nucleic acids to be detected and includes gene sequences from different species, subspecies, or variants, or gene mutations within the same species. do. It may be characterized by, but not limited to, all types of DNA including genomic DNA, mitochondrial DNA, viral DNA, or any type of RNA including mRNA, ribosomal RNA, non-coding RNA, tRNA, viral RNA, and the like.
  • the target nucleic acid is not limited thereto, but may be characterized by a mutant nucleotide sequence including a mutated nucleotide sequence, wherein the mutant is Single Nucleotide Polymorphism (SNP), insertion, and deletion.
  • SNP Single Nucleotide Polymorphism
  • LHM loss of heterozygosity
  • nucleoside refers to a glycosylamine compound in which a nucleic acid base (nucleobase) is linked to a sugar moiety.
  • Nucleotide means nucleoside phosphate. Nucleotides can be represented using alphabetic letters (letter names) corresponding to their nucleosides, as described in Table 1. For example, A refers to adenosine (nucleosides containing adenine nucleobases), C refers to cytidine, G refers to guanosine, U refers to uridine, and T refers to thymidine (5- Methyl uridine). W refers to A or T / U and S refers to G or C. N represents a random nucleoside and dNTP means deoxyribonucleoside triphosphate. N can be any of A, C, G, or T / U.
  • oligonucleotide refers to an oligomer of nucleotides.
  • nucleic acid means a polymer of nucleotides.
  • sequence refers to the nucleotide sequence of an oligonucleotide or nucleic acid. Throughout the specification, whenever an oligonucleotide or nucleic acid is represented by a sequence of letters, the nucleotides are from 5 ' ⁇ 3' order from left to right. Oligonucleotides or nucleic acids may be DNA, RNA, or analogs thereof (eg, phosphorothioate analogs).
  • Oligonucleotides or nucleic acids may also include modified bases and / or backbones (eg, modified phosphate linkages or modified sugar moieties).
  • modified backbones eg, modified phosphate linkages or modified sugar moieties.
  • synthetic backbones that confer stability and / or other advantages to nucleic acids may include phosphorothioate linkages, peptide nucleic acids, locked nucleic acids, xylose nucleic acids, or analogs thereof.
  • nucleic acid in the present invention refers to a nucleotide polymer and includes known analogs of natural nucleotides that can act in a similar manner (eg, hybridization) to naturally occurring nucleotides unless otherwise defined.
  • nucleic acid is for example genomic DNA; Complementary DNA (cDNA), which is usually the DNA representation of mRNA obtained by reverse transcription or amplification of messenger RNA (mRNA); DNA molecules produced synthetically or by amplification; And any form of DNA or RNA, including mRNA.
  • cDNA Complementary DNA
  • mRNA messenger RNA
  • nucleic acid includes single stranded molecules as well as double or triple stranded nucleic acids.
  • the nucleic acid strands need not be coextensive (ie, the double stranded nucleic acid need not be double stranded along the entire length of both strands).
  • nucleic acid also includes any chemical modification thereof, such as by methylation and / or capping.
  • Nucleic acid modifications may include the addition of chemical groups including additional charge, polarization, hydrogen bonding, electrostatic interaction, and functionality to individual nucleic acid bases or to the nucleic acid as a whole. Such modifications include 2 'sugar modification, 5 position pyrimidine modification, 8 position purine modification, modification in cytosine exocyclic amines, substitution of 5-bromo-uracil, backbone modification, isobasic isocytidine and isoguanidine Base modifications, such as combinations of specific base pairs, and the like.
  • Nucleic acid may be derived from a complete chemical synthesis process, such as solid phase-mediated chemical synthesis, from a biological source, such as through separation from any species that produces nucleic acids, or from DNA replication, PCR amplification, reverse transcription. From processes associated with the handling of nucleic acids by molecular biological tools such as, or from combination of these processes.
  • complement in the present invention refers to the ability for precise pairing between two nucleotides. That is, if a nucleotide can hydrogen bond with a nucleotide of another nucleic acid at a given position of the nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules with only a portion of the nucleotides bound may be “partial” or complementarity may be complete when total complementarity is present between single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.
  • the term “primer” refers to a short linear oligonucleotide that hybridizes to a target nucleic acid sequence (eg, a DNA template to be amplified) for priming a nucleic acid synthesis reaction.
  • the primer may be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence.
  • Primers can contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the primer length are determined experimentally. The lower limit of the primer length is the minimum length required to form a stable duplex after hybridization with a target nucleic acid under nucleic acid amplification reaction conditions.
  • Very short primers do not form a thermothermally stable duplex with the target nucleic acid under these hybridization conditions.
  • the upper limit is usually determined by the possibility of having duplex formation in a region other than the predetermined nucleic acid sequence in the target nucleic acid.
  • suitable primer lengths range from about 3 nucleotides to about 40 nucleotides in length.
  • Rolling Circle Amplification refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (eg, a single stranded DNA ring) via a rolling circle mechanism.
  • Rotating amplification reactions are initiated by hybridization of primers to circular, often single-stranded nucleic acid templates.
  • the nucleic acid polymerase then proceeds around the circular nucleic acid template to expand the primers hybridized to the circular nucleic acid template to replicate the sequence of the nucleic acid template repeatedly (rolling circle mechanism).
  • Rotating amplification typically produces a concatemer comprising a tandem repeat unit of a circular nucleic acid template sequence.
  • Rotating amplification can be linear RCA (LRCA) exhibiting linear amplification kinetics (eg, RCA using single specific primers) or exponential RCA (ERCA) exhibiting exponential amplification kinetics.
  • Rotating amplification may also be performed using a plurality of primers to produce hyperbranched concatemers (multiple primed rolling circle amplification or MPRCA).
  • MPRCA multiple primed rolling circle amplification
  • one primer may be complementary to the circular nucleic acid template as in linear RCA, while the other primer may be complementary to the tandem repeat unit nucleic acid sequence of the RCA product.
  • double-primed RCAs can undergo a chain reaction with exponential (geometric) amplification kinetics characterized by branching cascades of multi-hybridization, primer-extension, and strand-separation phenomena involving both primers. .
  • Rotating ring amplification can be performed in vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.
  • probe binds to a target nucleic acid of a complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation and thus forms a duplex structure. Nucleic acids that can form. Probes bind or hybridize to “probe binding sites”. In particular, the probe may be labeled with a detectable label to facilitate detection of the probe once the probe hybridizes to the complementary target of the probe. Alternatively, however, the probe may be unlabeled, but may be detected directly or indirectly by specific binding to the labeled ligand. Probes can vary considerably in size. Generally probes are at least 7-15 nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides in length.
  • Another probe is somewhat longer and is at least 50, 60, 70, 80, or 90 nucleotides in length. Another probe is even longer and is at least 100, 150, 200 or more nucleotides in length. The probe may also be of any length that is within any range defined by any value of the above values (eg, 15-20 nucleotides in length).
  • hybridization in the present invention means that the double-stranded nucleic acid is formed by hydrogen bonding between single-stranded nucleic acids having a complementary base sequence, is used in a similar sense to the annealing (annealing). In a slightly broader sense, hybridization includes the case where the sequences between two single strands are perfectly complementary (except when some sequences are not complementary).
  • the template may be characterized as being a single stranded linear nucleic acid.
  • the target nucleic acid binding region of the template may be present at both ends of the template.
  • the barcode is any base sequence predefined for each target nucleic acid, and may be characterized in that it does not bind complementarily with the target nucleic acid.
  • the second amplification product may be provided as a single stranded nucleic acid of the step (a). That is, the second amplification product obtained in step (c) acts as a single-stranded nucleic acid containing the target nucleic acid sequence to repeat steps (b) to (d), thereby cutting in step (c)
  • the target nucleic acid sequence-containing DNA may be characterized by binding to a new template DNA to perform a new RCA.
  • one long amplification product is not formed by the rotation ring amplification, but according to the action of the restriction enzyme, an amplification product including the target nucleic acid sequence (second amplification product) and an amplification product including a barcode region (First amplification products) are generated, respectively, and the amplification products containing the target nucleic acid cleaved by restriction enzymes bind to another template to induce a new rotation ring amplification reaction, thereby amplifying the signal and improving sensitivity. It is to be made.
  • the target nucleic acid may be characterized in that one or more, it may be characterized by detecting by using a barcode prepared by amplifying a barcode generating region of a different template for each of the one or more target nucleic acids.
  • the target nucleic acid to be detected is, for example, a point mutation of PIK3CA, a point mutation of EGFR, a point mutation of p53, and a point mutation of BRCA1 / 2, a different barcode for each point mutation to be detected
  • the generation area can be designed and detected simultaneously.
  • the target nucleic acid binding region of the template may be designed such that the point to be detected of the target nucleic acid is linked by ligase. That is, when the point mutation to be detected in the present invention is a point mutation in which the 545th glutamic acid of the PIK3CA protein is changed to lysine, it is a mutation generated by changing the base G at position 1633 of the PIK3CA gene to A. In this case, ligation occurs when the 5 'upstream 10-20 base of the __1633 base binds to the first target nucleic acid binding region, and the 3' downstream 10-20 base binds to the second target nucleic acid binding region. In other words, it forms a cyclic template-target nucleic acid conjugate.
  • the single-stranded nucleic acid of step (a) can be prepared using any known method for preparing single-stranded nucleic acids from nucleic acids extracted from a sample sample, preferably asymmetric polymerase chain reaction It may be characterized in that the production of) but is not limited thereto.
  • the single-stranded nucleic acid of step (a) denatures the double-stranded DNA (deoxyribonucleic acid) extracted from the sample, and then binds the blocker DNA that complementarily binds to the single-stranded single strand. It may be characterized by the production of a nucleic acid.
  • the method for detecting a barcode (barcode generated by amplification of the barcode generation region) of the first amplification product obtained in step (c) comprises a surface measurement sensor comprising a probe complementary to the barcode. It may be characterized by using, but is not limited thereto. Therefore, it will be apparent to those skilled in the art that the sequence complementary to the barcode in the probe complementary to the barcode in the present invention is the same sequence as the barcode generation region of the template of the present invention.
  • the surface measuring sensor is characterized in that it uses a method selected from the group consisting of fluorescence, Surface Plasmon Resonance (SPR), quartz crystal microbalance (QCM) and cantilever It may be, but is not limited thereto.
  • the probe complementary to the barcode region may be selected from the group consisting of oligonucleotides, locked nucleic acid (LNA), peptide nucleic acid (PNA), and mixtures thereof, but is not limited thereto. no.
  • PNA Protein nucleic acid
  • PNA has very high affinity and selectivity, and has high stability against nucleases, so that it is not degraded by existing restriction enzymes.
  • PNAs form double strands through hybridization with native nucleic acids of complementary base sequences.
  • PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands.
  • PNA has a greater ability to detect single nucleotide polymorphism (SNP) than natural nucleic acid because the double strand becomes unstable due to single base mismatch.
  • SNP single nucleotide polymorphism
  • the probe complementary to the barcode may be connected to a reporter and a quencher at both ends.
  • the signal generation is suppressed, and the signal strength increases as the distance between the reporter and the quencher becomes far.
  • the probe hybridizes with the complementary nucleotide sequence, the distance between the reporter and the quencher becomes the longest, and thus the specific nucleotide sequence can be detected through signal generation or signal intensity increase.
  • the reporter is fluorescein (fluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and cadicarbocyanin (thiadicarbocyanine) may be characterized in that at least one fluorescent material selected from the group consisting of dyes.
  • the quencher is Dabcyl, Tamra, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black )
  • FQ, Iowa black RQ and IRDye QC-1 may be characterized in that at least one selected from the group consisting of.
  • the ligation of step (b) may be characterized by using T7 DNA ligase, but is not limited thereto.
  • the phi29 DNA polymerase may be used for DNA polymerization in the rotary ring amplification of step (c), but is not limited thereto.
  • the template may further include a spacer region and a fluorescent probe binding region.
  • the restriction enzyme binding region of the template may be characterized in that at least one.
  • the nucleic acid for cleavage may further include any sequence that does not bind to the template at the 5 'and 3' end.
  • the restriction enzyme binding region sequence of the single-stranded amplification product produced by RCA is complementary to the restriction enzyme binding region of the template, the truncating nucleic acid having the same sequence as the sequence of the restriction enzyme binding region of the template is generated by RCA.
  • restriction enzyme binds to the restriction enzyme binding region of the single-stranded amplification product to form a double strand, and the restriction enzyme is cleaved, whereby the cleaved strand can act as a primer to induce further polymerization, 5 'terminal and 3 It further includes any sequence that does not bind to the template at the end.
  • the cleavage nucleic acid may further comprise a functional group or a base at which the nucleic acid polymerization is inhibited at the 5 'and 3' ends.
  • the functional group or base that the nucleic acid polymerization is inhibited is an amine group, a phosphate group, an alkyl group, an alkane-diol, phosphorothioate (phophorothioate), biotin, non-nucleotide linker, C3-18 spacer, di-deoxynucleotide triphosphate (ddNTP), reverse deoxynucleotide It may be characterized by one or more selected from the group consisting of triphosphate (inverted deoxynucleotide triphosphate, inverted dNTP) and reverse di-deoxynucleotide triphosphate (inverted ddNTP), but is not limited thereto. .
  • the present invention in another aspect, (a) i) a target nucleic acid binding region that is disposed at both ends and complementary to the target nucleic acid sequence; ii) primer binding regions; iii) restriction enzyme binding region; And iv) a barcode generation region; (b) a primer that binds to the primer binding region; (c) a cleavage nucleic acid having the same sequence as the sequence of the restriction enzyme binding region; (d) restriction enzymes; And (e) ligase (ligase); relates to a composition for amplifying the rotation ring for the detection of the target nucleic acid.
  • sample in the present invention encompasses various samples, and preferably, the biological sample is analyzed using the method of the present invention. More preferably, it may be a sample mixed with a virus species or a sample of an individual infected with the virus (eg, humans, mammals and fish, etc.), and may be a plant, animal, human, fungus, bacterium or organism of viral origin. Samples can be analyzed. When analyzing a sample of mammalian or human origin, the sample may be derived from a specific tissue or organ. Representative examples of tissues include connective, skin, muscle or nerve tissue.
  • organs include eyes, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testes, ovaries, uterus, rectum, nervous system, Glands and internal vessels are included.
  • the biosample to be analyzed includes any cell, tissue, fluid from a biological source, or any other medium that can be well analyzed by the present invention, which is the consumption of humans, animals, humans or animals. Samples obtained from food prepared for use are included.
  • the biological sample to be analyzed includes a bodily fluid sample, which includes blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain grinds), spinal fluid, appendix, spleen And tonsil tissue extracts, but is not limited thereto.
  • a bodily fluid sample which includes blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain grinds), spinal fluid, appendix, spleen And tonsil tissue extracts, but is not limited thereto.
  • the present invention relates to a kit for detecting a target nucleic acid comprising the composition from another aspect.
  • the kit is a target nucleic acid amplification reaction (buffer, DNA polymerase (DNA polymerase), DNA polymerase cofactor (DNA polymerase cofactor) and deoxyribonucleotide-5-triphosphate (dNTP))
  • a target nucleic acid amplification reaction buffer, DNA polymerase (DNA polymerase), DNA polymerase cofactor (DNA polymerase cofactor) and deoxyribonucleotide-5-triphosphate (dNTP)
  • the kits of the present invention may also include various oligonucleotide molecules, reverse transcriptases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • the optimal amount of reagent used in a particular reaction of the kit can be readily determined by one skilled in the art having learned the disclosure herein.
  • the equipment of the present invention can be manufactured in a separate package or compartment containing the aforementioned components.
  • the kit may comprise a compartment comprising a compartmentalized carrier means for holding a sample, a container comprising a reagent, a container comprising a surrogate target and a primer and a probe for detecting the amplification product.
  • the carrier means is suitable for containing one or more containers, such as bottles, tubes, each container containing independent components used in the method of the invention.
  • containers such as bottles, tubes
  • each container containing independent components used in the method of the invention.
  • one of ordinary skill in the art can readily dispense the required formulation in the container.
  • beacons for detecting templates, primers, and amplification products are shown in the following table. Produced.
  • sequence structure of the template is as follows.
  • the RCA amplification product produced in Example 1.2 was injected with cutting DNA (SEQ ID NO: 5), followed by reaction to perform electrophoresis.
  • the 5'-CAG // CTG-3 'sequence of the RCA amplification product is recognized and cleaved by the restriction enzyme.
  • the sequence structure of the template is as follows.
  • the sequence structure of the template is as follows.
  • FIG. 8 shows the results of distinguishing barcodes.
  • # 1 barcode # 1 Encoded DNA
  • # 1 Encoded DNA linker a complementary probe linker
  • # 1 barcode a non-complementary probe linker
  • the detection signal is strongly observed in the channel to which the probe linker complementary to the RCA amplification product is fixed. It was confirmed that no signal appeared. Through these results, it could be confirmed that the amplification products are well generated by the RCA reaction according to the present invention.
  • the target nucleic acid detection method of the present invention and the composition for RCA amplification therefor can detect the variation of the base unit with high sensitivity, and thus may be usefully used in the field of molecular diagnosis.

Abstract

La présente invention concerne un procédé de détection d'un acide nucléique cible sur la base d'une amplification par cercle roulant (RCA), et plus spécifiquement, un procédé de détection d'un acide nucléique cible, le procédé dans lequel un acide nucléique cible (un acide nucléique ayant une séquence d'acide nucléique cible), lorsqu'il est présent, forme une matrice circulaire avec une matrice pour réaliser une réaction d'amplification, où, au cours de la réaction d'amplification, une enzyme de restriction est ajoutée pour induire encore une nouvelle réaction de RCA, augmentant ainsi le taux de réaction et la sensibilité, et une composition de RCA pour la mise en oeuvre du procédé. Le procédé de détection d'un acide nucléique cible selon la présente invention, par détection d'une séquence de code-barres prédéfinie selon le type de l'acide nucléique cible, permet de multiples détections de la présence de l'acide nucléique cible sans séquençage, est peu coûteux pour ne pas utiliser d'enzymes coûteuses, telles que CRISPR, peut détecter des séquences de code-barres, et peut utiliser divers systèmes de détection d'acide nucléique existants, et peut ainsi être utile dans la détection de mutations génétiques.
PCT/KR2019/006602 2018-06-01 2019-05-31 Procédé de détection d'un acide nucléique cible à l'aide d'une amplification par cercle roulant et composition pour la détection d'un acide nucléique cible WO2019231287A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP19811578.4A EP3805408B1 (fr) 2018-06-01 2019-05-31 Procédé de détection d'un acide nucléique cible à l'aide d'une amplification par cercle roulant et composition pour la détection d'un acide nucléique cible
US15/733,965 US11608522B2 (en) 2018-06-01 2019-05-31 Method of detecting target nucleic acid using rolling circle amplification and composition for detecting target nucleic acid

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20180063569 2018-06-01
KR10-2018-0063569 2018-06-01
KR10-2019-0064750 2019-05-31
KR1020190064750A KR102293402B1 (ko) 2018-06-01 2019-05-31 회전환 증폭을 이용한 표적핵산 검출 방법 및 표적핵산 검출용 조성물

Publications (1)

Publication Number Publication Date
WO2019231287A1 true WO2019231287A1 (fr) 2019-12-05

Family

ID=68698350

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/006602 WO2019231287A1 (fr) 2018-06-01 2019-05-31 Procédé de détection d'un acide nucléique cible à l'aide d'une amplification par cercle roulant et composition pour la détection d'un acide nucléique cible

Country Status (1)

Country Link
WO (1) WO2019231287A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022099938A1 (fr) * 2020-11-11 2022-05-19 天津大学 Procédé de génération d'adn circulaire simple brin sur la base d'une technique de sonde de type verrou, et son utilisation

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150126376A1 (en) * 2012-06-14 2015-05-07 Fred Hutchinson Cancer Research Center Compositions and methods for sensitive mutation detection in nucleic acid molecules
WO2015079042A1 (fr) * 2013-11-29 2015-06-04 Q-Linea Ab Procédé d'amplification par cercle roulant
WO2016016450A1 (fr) * 2014-08-01 2016-02-04 Olink Ab Procédé de sélection d'une séquence d'acides nucléiques
WO2017177017A1 (fr) * 2016-04-07 2017-10-12 Omniome, Inc. Procédés de quantification d'acides nucléiques cibles et d'identification de variants de séquences

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150126376A1 (en) * 2012-06-14 2015-05-07 Fred Hutchinson Cancer Research Center Compositions and methods for sensitive mutation detection in nucleic acid molecules
WO2015079042A1 (fr) * 2013-11-29 2015-06-04 Q-Linea Ab Procédé d'amplification par cercle roulant
WO2016016450A1 (fr) * 2014-08-01 2016-02-04 Olink Ab Procédé de sélection d'une séquence d'acides nucléiques
WO2017177017A1 (fr) * 2016-04-07 2017-10-12 Omniome, Inc. Procédés de quantification d'acides nucléiques cibles et d'identification de variants de séquences

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUCK MJ ET AL., CANCER PREV RES (PHILA, vol. 5, no. 7, 2012, pages 887 - 900
JOFFROY, BASTIAN ET AL.: "Rolling circle amplification shows a sinusoidal template length-dependent amplification bias", NUCLEIC ACIDS RESEARCH, vol. 46, no. 2, 9 December 2017 (2017-12-09), pages 538 - 545, XP055659491 *
ZHANG F ET AL., SCIENCE, vol. 28, no. 6336, 2017, pages 438 - 442

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022099938A1 (fr) * 2020-11-11 2022-05-19 天津大学 Procédé de génération d'adn circulaire simple brin sur la base d'une technique de sonde de type verrou, et son utilisation

Similar Documents

Publication Publication Date Title
CN109468384B (zh) 一种同时检测45个y基因座的复合扩增检测试剂盒
CN105934523A (zh) 核酸的多重检测
US20140186830A1 (en) Methods for Analyzing Short Tandem Repeats and Single Nucleotide Polymorphisms
KR102592367B1 (ko) 게놈 및 치료학적 적용을 위한 핵산 분자의 클론 복제 및 증폭을 위한 시스템 및 방법
JP2008520245A (ja) 切断−増幅法による核酸多様性の検出
JPH07143900A (ja) 単一プライマー増幅に使用されるポリヌクレオチド、および核酸増幅におけるプライマーとしてのホスホロチオエート含有オリゴヌクレオチド
JP6876437B2 (ja) 鎖侵入に基づくｄｎａ増幅法
US11008626B2 (en) Compositions and methods for detection of drug resistant Mycobacterium tuberculosis
KR102293402B1 (ko) 회전환 증폭을 이용한 표적핵산 검출 방법 및 표적핵산 검출용 조성물
WO2013122319A1 (fr) Procédé de détection de gène cible ou mutation associée utilisant une réaction de ligase et réaction d'amplification d'enzyme de coupure
EP1992703A1 (fr) Procédé pour détecter un gène mutant
WO2020153764A1 (fr) Procédé de détection de multiples cibles sur la base d'une sonde de détection unique utilisant un psn de séquence d'étiquettes
WO2019231287A1 (fr) Procédé de détection d'un acide nucléique cible à l'aide d'une amplification par cercle roulant et composition pour la détection d'un acide nucléique cible
US20080220415A1 (en) Detection Method of Dna Amplification Using Probe Labeled With Intercalating Dye
AU2005250250A1 (en) Exhaustive gene expression profiling analysis using microsample
WO2021182850A1 (fr) Procédé de détection simultanée de multiples miarn et kit de détection de miarn l'utilisant
WO2018016683A1 (fr) Méthode de détection de séquence d'acide nucléique cible utilisant une amplification nichée de signal de type amplification multiple
US20160340711A1 (en) Detecting single nucleotide polymorphism using overlapping hydrolysis probes
EP1275734A1 (fr) Méthode pour la synthèse aléatoire et l'amplification d'ADNc
JP2003510011A (ja) カップル性ポリメラーゼ連鎖反応−制限エンドヌクレアーゼ消化−リガーゼ検出反応法
WO2022203297A1 (fr) Procédé d'amplification d'acide nucléique cible utilisant une sonde de guidage et sonde de serrage et composition pour amplifier un acide nucléique cible le comprenant
TWI570242B (zh) 用於基因型鑑定晶片之雙重等位基因特異性聚合酶鏈鎖反應的方法
CN109312397A (zh) Penta E基因座多态性人体鉴定
KR102543156B1 (ko) 높은 특이도의 표적핵산 증폭방법 및 이를 이용한 표적핵산 증폭용 조성물
WO2020027482A1 (fr) Procédé se fondant sur une sonde d'élimination pour détecter des anomalies chromosomiques numériques, et composition d'acide nucléique pour détecter des anomalies chromosomiques numériques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19811578

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019811578

Country of ref document: EP

Effective date: 20201209