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WO2025127642A1 - Procédé basé sur une réaction en chaîne par polymérase numérique pour fournir des informations pour diagnostiquer une instabilité de microsatellite, et kit associé - Google Patents

Procédé basé sur une réaction en chaîne par polymérase numérique pour fournir des informations pour diagnostiquer une instabilité de microsatellite, et kit associé Download PDF

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WO2025127642A1
WO2025127642A1 PCT/KR2024/020074 KR2024020074W WO2025127642A1 WO 2025127642 A1 WO2025127642 A1 WO 2025127642A1 KR 2024020074 W KR2024020074 W KR 2024020074W WO 2025127642 A1 WO2025127642 A1 WO 2025127642A1
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group
quencher
msi
reporter
probe
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박희경
황보경
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Seasunbio Materials Inc
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/319—Exonuclease
    • 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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR
    • 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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/101—Taqman

Definitions

  • the present invention relates to a method for providing information for diagnosing microsatellite instability based on digital polymerase chain reaction and a kit therefor.
  • Microsatellites are short DNA sequences of six or fewer that are sequentially repeated throughout the human genome, and are scattered on each chromosome with different numbers of repetitions.
  • Microsatellite instability refers to the variation in the length of short, repetitive base sequences that make up microsatellites, which are increased or decreased. Microsatellite instability was first discovered in patients with hereditary nonpolyposis colorectal cancer syndrome as a result of mutations in DNA mismatch repair genes or replication abnormalities due to promoter methylation, and has been found to exist in other cancers, such as sporadic colorectal cancer and sporadic endometrial cancer.
  • the first is a fragment analysis method using multiplex fluorescence PCR amplification and capillary electrophoresis, which determines whether a gene is deleted by measuring the length of the analyzed product after performing capillary electrophoresis after gene amplification.
  • the second is a deletion gene-preferential amplification method, which adds a probe such as PNA, which binds to a normal gene and prevents gene amplification, to the PCR amplification reaction reagent, thereby suppressing the normal gene and inducing amplification of the deleted gene, and then measuring the occurrence of the amplification product to determine the presence of the deletion mutation.
  • Deletions of specific parts of genes like this are known to be important biomarkers for diagnosing and predicting the prognosis of cancer, and the importance of accurate analysis is increasing.
  • conventional molecular diagnostic methods cannot perform quantitative analysis, have low sensitivity when the deleted gene is short or exists at a low ratio, and can be misinterpreted as a deletion mutation if a substitution mutation, not a deletion, occurs in a part that prevents amplification of the normal gene.
  • the present inventors have completed the present invention by confirming that the shortcomings of the above-described molecular diagnostic method can be solved and gene deletion mutations can be determined with significantly improved sensitivity by utilizing external and internal hydrolysis of polymerase.
  • the present invention aims to provide a method for accurately diagnosing microsatellite instability by utilizing external and internal hydrolysis of a polymerase to accurately determine deletion of a specific region of a gene, and a diagnostic kit using the method.
  • One aspect of the present invention relates to a method for providing information for diagnosing microsatellite instability (MSI) using digital polymerase chain reaction (digital PCR), the method comprising the step of analyzing the number of nucleic acids measured in one or more groups selected from the group consisting of: a first group including a reporter and a quencher that distinguish complete hybridization or incomplete hybridization with a microsatellite base repeat region of a target nucleic acid; and a second group including a reporter and a quencher that completely hybridize with a region of the target nucleic acid in which microsatellite instability does not exist and fluoresces.
  • MSI microsatellite instability
  • digital PCR digital polymerase chain reaction
  • the complete hybridization or insecure hybridization may be distinguished by the action of an endonuclease or an exonuclease.
  • the probe including the first group may be composed of a terminal consisting of a microsatellite base repeat sequence and a terminal consisting of 5 to 24 sequences outside the microsatellite base repeat sequence, and may include the reporter and the quencher in the terminal portion consisting of the microsatellite base repeat sequence.
  • the probe comprising the second group may complementarily bind to a sequence other than a microsatellite repeat sequence, and may include a reporter and a quencher at the end or inside.
  • the reporter and the quencher may be spaced apart by 2 to 5 bp.
  • the reporter and quencher of the first group and the reporter and quencher of the second group may be present in one probe.
  • the reporter and quencher of the first group and the reporter and quencher of the second group may be present in one amplicon.
  • the reporter and quencher of the first group and the reporter and quencher of the second group may be present in two independent amplification products.
  • the reporter may be selected from the group consisting of Cy5 (Cyaninine5), FAM (6-carboxyfluorescein), Texas red, and HEX (2', 4', 5', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein).
  • the quencher may be selected from the group consisting of BHQ1, BHQ2, Dabcyl, and TAMRA (6-carboxytetramethyl-rhodamine).
  • the probe may be selected from the group consisting of sequence numbers 1 to 7, 11 to 13, and 24 to 33.
  • kits for detecting base deletion of a target nucleic acid from a method for providing information for diagnosing microsatellite instability (MSI), comprising: a probe comprising one or more reporters and quenchers; and forward primers and reverse primers for the probe.
  • MSI microsatellite instability
  • the probe may be selected from the group consisting of sequence numbers 1 to 7, 11 to 13, and 24 to 33.
  • the forward primer may be selected from the group consisting of SEQ ID NOs: 14 to 18, and the reverse primer may be selected from the group consisting of SEQ ID NOs: 10 and 19 to 23.
  • the information-providing method for diagnosing microsatellite instability exhibits a fluorescence signal through separation of a quencher and a reporter by external hydrolysis of a polymerase when a probe is completely bound, and when the 5-terminal binding of the probe does not occur due to a deletion mutation, the region between the site where the 5-terminal binding did not occur and the site where it was bound undergoes internal hydrolysis, so that the quencher and reporter are removed together and fluorescence is not exhibited, so that the presence or absence of a deletion mutation can be analyzed with high sensitivity, and quantitative analysis can be performed by comparing the number of groups of reporters and quenchers produced in an area where microsatellite instability does not exist.
  • Figure 1 is a schematic diagram of a method for diagnosing microsatellite instability (MSI) by detecting deletions according to the present invention.
  • MSI microsatellite instability
  • Figure 2 is a schematic diagram of the detection principle of the detection probe according to the length of the deletion. a) When there is no deletion (0 bp deletion), the entire probe binds specifically, and fluorescence is detected by the exonuclease function of the polymerase. b) When the number of deleted bases is less than the number of bases between the reporter and the quencher, the reporter and the quencher are cleaved and separated by the exonuclease function of the polymerase, and fluorescence is detected.
  • Figure 3 shows the results of real-time PCR performed to confirm the possibility of detecting deletion of a synthetic oligomer.
  • Figure 4 shows the results of confirming the change in detection resolution according to the number of deletions in the synthetic oligomer by performing digital polymerase chain reaction (digital PCR) and utilizing the design probe NR24_Taq_09. a) uses Cy5 as a detection probe, and b) uses EvaGreen as a reference fluorescence, and the measurement results for perfect match, 2 bp deletion, 3 bp deletion, 4 bp deletion, and 5 bp deletion are shown from the left, respectively.
  • Figure 5 shows the results of comparing the detection resolution of deletions (0 to 5 bp) of synthesized oligomers according to probe design by performing digital polymerase chain reaction.
  • Figure 6 is a schematic diagram of a probe comprising multiple fluorescence-quencher groups for simultaneously detecting the amount of an amplification product and whether or not a deletion is present using a single probe.
  • a probe and primer set (63) structure is shown, which includes a detection probe (61) for detecting whether or not a deletion is present at a suspected deletion site (62) and a reference probe that binds gene-specifically to a non-deletion site to identify an amplification product.
  • Figure 7 is a schematic diagram of the detection principle according to the deletion length of a probe containing multiple fluorescence-quencher groups.
  • Figure 8 shows the results of confirming MSI for cell lines ((1) HeLa MSS, (2) SNU638 MSI) and clinical samples ((3) MSS normal, (4) MSS tumor, (5) MSI normal, and (6) MSI tumor) using a probe containing multiple fluorescence-quencher groups.
  • Figure 9 shows the results of analyzing the correlation by confirming the change in normal and deletion detection ratios for HeLa MSS, 1% deletion MSI, 5% deletion MSI, 10% deletion MSI, 50% deletion MSI, SNU638 MSI, and negative control groups using a detection probe (Cy5) and reference probe (FAM) to which the present invention is applied.
  • Figure 10 shows the results of confirming whether or not MSI is present in cell lines (HeLa MSS, SNU638 MSI) by constructing a detection probe (Cy5) and reference probe (FAM, HEX, TAMRA) that apply the present invention to five types of MSI markers (BAT25, BAT26, NR21, NR24, NR27).
  • Figure 11 shows the results of confirming whether MSI is present in clinical samples (normal tissue, cancer tissue) of endometrial cancer patients (8 patients) using a probe to which the present invention is applied.
  • the X-axis represents the reference probe positive concentration
  • the Y-axis represents the MSS positive concentration
  • first, second, etc. may be used to describe various components, the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.
  • first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component.
  • units used in the specification of the present invention without special mention are based on weight, and for example, units of % or ratio mean weight% or weight ratio.
  • the expression “comprises” is an open description having a meaning equivalent to expressions such as “includes,” “contains,” “has,” or “is characterized by,” and does not exclude additional elements, materials, or processes that are not listed.
  • the expression “consisting substantially of...” means that other elements, materials, or processes that are not listed can be present together with the specified elements, materials, or processes in an amount that does not unacceptably significantly affect at least one basic and novel technical idea of the invention.
  • the expression “consists of” means that only the listed elements, materials, or processes are present.
  • substantially or “essentially” means almost entirely or completely, for example, greater than 95% of some given amount. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.
  • first,” “second,” “third,” “fourth,” or similar terms in a component name are used to distinguish and identify more than one component that shares a particular identity in the component name.
  • first RNA and “second RNA” are used to distinguish two RNAs.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • a polynucleotide may have any three-dimensional structure and may perform any function, known or unknown.
  • a polynucleotide may include modified nucleotides, such as, for example, methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted either before or after assembly of the polynucleotide.
  • the sequence of nucleotides may be interrupted by non-nucleotide moieties.
  • the polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling moiety.
  • the term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present disclosure that is a polynucleotide includes both a double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute a double-stranded form.
  • RNA refers to its generally accepted meaning in the art.
  • the term RNA refers to a polynucleotide comprising at least one ribofuranoside moiety.
  • the term can include double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as additions, deletions, substitutions, and/or alterations of one or more nucleotides.
  • Such alterations can include, for example, the addition of non-nucleotide material to one or more nucleotides of the RNA.
  • the nucleotides in a nucleic acid molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. Such altered RNA can be referred to as an analog or an analog of a naturally occurring RNA.
  • the RNA can be, but is not limited to, messenger RNA (mRNA) or siRNA.
  • mRNA messenger RNA
  • an mRNA as disclosed herein comprises, consists essentially of, or additionally consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail.
  • One aspect of the present invention relates to a method for providing information for diagnosing microsatellite instability (MSI) using digital polymerase chain reaction (digital PCR), the method comprising the step of analyzing the number of nucleic acids measured in one or more groups selected from the group consisting of: a first group including a reporter and a quencher that distinguish complete hybridization or incomplete hybridization with a microsatellite base repeat region of a target nucleic acid; and a second group including a reporter and a quencher that completely hybridize with a region of the target nucleic acid in which microsatellite instability does not exist and fluoresces.
  • MSI microsatellite instability
  • digital PCR digital polymerase chain reaction
  • MSI is generally divided into two types according to international standards.
  • NCI National Cancer Institute
  • MSI-H high-level MSI
  • RER+ replication error positive
  • MSI-L low-level MSI
  • microsatellite stable (MSS) (Boland et al. , Cancer Res., 58:5248-57, 1998; Kim, Deok-Woo, Journal of Genetic Medicine 7:24-36, 2010).
  • the 'base mutation' of the present invention refers to a mutation in the base sequence of a target nucleic acid or a nucleic acid to be analyzed, and may mean not only a single nucleotide polymorphism (SNP), but also a mutation caused by substitution, deletion or insertion of a base.
  • the probe of the present invention can analyze a mutation caused by deletion of 2 or more, preferably 3 or more, more preferably 5 or more, specifically, 2 to 27, preferably 3 to 27, and even more preferably 5 to 27 bases of a target nucleic acid or a nucleic acid to be analyzed, through a digital polymerase chain reaction measurement result, but is not limited thereto.
  • the difference in fluorescence due to complete or insecure hybridization can be distinguished by the action of endonuclease or exonuclease.
  • the 'hybridization' of the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid through a reaction.
  • Hybridization may occur when the complementarity between two nucleic acid strands is complete, that is, a perfect match, or may occur even when some mismatched bases exist.
  • the degree of complementarity required for hybridization may vary depending on the hybridization conditions, and may be controlled by, for example, temperature, but is not limited thereto.
  • a probe comprising the above first group may be composed of a terminal portion consisting of a microsatellite base repeat sequence and a terminal portion including 5 to 20 sequences outside the microsatellite base repeat sequence, and may include the reporter and quencher in the terminal portion consisting of the microsatellite base repeat sequence.
  • a probe comprising the second group may be one that binds complementarily to a sequence other than a microsatellite repeat sequence and includes a reporter and a quencher at the end or inside.
  • the 'target nucleic acid' of the present invention refers to a nucleic acid sequence (including SNP) of a genotype to be detected/determined, and includes a specific portion of the nucleic acid sequence of a 'target gene' encoding a protein having a physiological or biochemical function, and is annealed or hybridized with a primer or probe under hybridization, annealing, or amplification conditions.
  • the 'target nucleic acid' is not different from the terms 'target nucleic acid', 'synthetic DNA', or 'artificial synthetic oligo' used in this specification, and are used interchangeably in this specification.
  • the target nucleic acid is DNA or RNA, and the molecule may be in a double-stranded or single-stranded form.
  • the nucleic acid as the initial material is double-stranded, it is preferable to make the two strands into a single strand or a partially single-stranded form.
  • Known methods for separating the strands include, but are not limited to, heat, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins.
  • strand separation can be achieved by heat treatment at a temperature of 80 to 105°C.
  • a general method for the above-mentioned treatment is disclosed in Joseph Sambrook et al,, Molecular Cloning, 2001.
  • the first reporter and the first quencher may be spaced apart from each other by 2 to 5 bp.
  • the reporter may be linked to the 5'-end or the 3'-end of the probe, and the quencher may be located within the probe sequence and separated from the reporter.
  • the reporter and quencher of the first group and the reporter and quencher of the second group may be present in one probe.
  • the reporter and quencher of the first group and the reporter and quencher of the second group may be present in one amplicon.
  • the reporter and quencher of the first group and the reporter and quencher of the second group may be present in two independent amplification products.
  • the above reporter may be selected from the group consisting of Cy5 (Cyaninine5), FAM (6-carboxyfluorescein), Texas red, and HEX (2', 4', 5', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein).
  • the above quencher may be selected from the group consisting of BHQ1, BHQ2, Dabcyl and TAMRA (6-carboxytetramethyl-rhodamine).
  • the above probe may be selected from the group consisting of sequence numbers 1 to 7, 11 to 13, and 24 to 33.
  • the method for determining microsatellite instability (MSI) and microsatellite stability (MSS) is not particularly limited, but the replication ratio of the following formula 1 is calculated using a target nucleic acid sample obtained from a patient, etc.
  • ⁇ (replication ratio) replication ratio(sample 1) - replication ratio(sample 2)
  • the sample 1 may be normal tissue, and the sample 2 may be cancer or tumor tissue, but is not limited thereto.
  • the difference in the duplication rate is, for example, 5% or more, preferably 7% or more, more preferably 10% or more, and more specifically, 5 to 50%, preferably 7 to 40%, and even more preferably 10 to 30%
  • it is determined to be a deletion marker and if there are, for example, 2 or more, preferably 3 or more, more preferably 4 or more, and specifically, 2 to 20, preferably 3 to 15, and even more preferably 4 to 10 such deletion markers, it is determined to be microsatellite instability (MSI), and information can be provided to doctors and other relevant persons.
  • MSI microsatellite instability
  • kits for detecting base deletion of a target nucleic acid isolated from a specimen sample comprising: a probe comprising one or more reporters and quenchers; and forward primers and reverse primers for the probe; and from the method for providing information for diagnosing microsatellite instability (MSI) of the above specific example.
  • MSI microsatellite instability
  • the specimen sample may be derived from a specific tissue or organ of an animal, including a human.
  • tissue include connective, skin, muscle, or nervous tissue.
  • organ may include, but are not limited to, the eye, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, ovary, uterus, rectum, nervous system, gland, and internal blood vessels.
  • the above specimen sample includes any cell, tissue, fluid, or other medium from biological source that can be analyzed by the present invention, including samples obtained from humans, animals, or food prepared for human or animal consumption.
  • the sample to be analyzed also includes a body fluid sample, including but not limited to sputum, blood, serum, plasma, lymph, breast milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain grounds), spinal fluid, appendix, spleen, and tonsil tissue extracts.
  • the above probe may be selected from the group consisting of sequence numbers 1 to 7, 11 to 13, and 24 to 33.
  • the forward primer may be selected from the group consisting of SEQ ID NOs: 14 to 18, and the reverse primer may be selected from the group consisting of SEQ ID NOs: 10 and 19 to 23.
  • the kit of the present invention may optionally include reagents necessary for performing a target amplification PCR reaction (e.g., a PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate.
  • a target amplification PCR reaction e.g., a PCR reaction
  • the kit may include various polynucleotide molecules, a reverse transcriptase, a buffer and reagents, and an antibody that inhibits DNA polymerase activity.
  • the optimal amount of the reagents used in a particular reaction of the kit can be easily determined by a person skilled in the art who has learned the disclosure herein.
  • the kit may be manufactured as a separate package or compartment containing the components mentioned above.
  • reagents used in the present invention they were purchased from Sigma-Aldrich (MO, USA) and used, and oligomers, primers, probes, etc. were synthesized directly by requesting domestic or foreign oligomer synthesis institutions or by the applicant.
  • a probe containing both a reporter and a quencher and containing locked nucleic acid (LNA) was designed.
  • sequence number 7 was designed, and it was designed to have a specific sequence (a sequence that does not cause microsatellite instability) only at the 3'-terminal of the microsatellite repeat sequence so that the 5'-terminal of the probe that binds according to the sequence deletion forms a flap (i.e. sequence numbers 1, 3, 4, 5, and 6).
  • sequence numbers 1, 3, 4, 5, and 6 are sequence numbers 1, 3, 4, 5, and 6.
  • sequence numbers 9 and 10 correspond to the forward and reverse primers.
  • the probe NR24_Taq_09 of Table 1 was applied to the digital polymerase chain reaction for the samples of Table 2 below, and the ratio of the concentration (ng/ ⁇ L) measured by the detection probe (Cy5) and the reference fluorescence (EvaGreen) was measured. The results are shown in Table 2 and Fig. 4.
  • a probe containing multiple fluorescence-quencher groups was designed as shown in Table 3 below according to the schematic diagram in Fig. 6.
  • Fig. 7 The detection principle of the detection probe according to the length of the deletion is schematically illustrated in Fig. 7.
  • MSI contents were designed to be 0, 1, 5, 10, 50, and 100%, respectively, as in the sample information in [Table 4] below, and then the number of reaction areas in which detection probe fluorescence and reference probe fluorescence were positive was measured through digital PCR. The results are shown in Table 4 and Fig. 9.
  • probes for five types of microsatellite markers were designed, and instead of EvaGreen used as a reference fluorescence, gene-specific TaqMan probes targeting sites where microsatellite instability does not occur were used based on fluorescence other than Cy5 (HEX, TAMRA, and FAM), and MSI detection according to primer design was analyzed as shown in Table 5 below. The results are shown in Fig. 10.
  • tissue samples cancer tissue, normal tissue
  • MSI MSI molecules
  • Table 6 shows the results of the concentration (ng/ ⁇ L) of genes obtained by analyzing the reaction zones in which the detection probe and reference probe were positive according to digital polymerase chain reaction. Using the results obtained therefrom, the ratio of the Cy5 concentration (detection probe) and the reference probe concentration was derived using Equation 1 below, and the results are shown in Table 7.
  • the difference in the ratio of the Cy5 concentration, which is a detection probe for cancer tissue and normal tissue according to the patient and marker, and the reference probe concentration was derived using Equation 2 below. Markers showing a difference of 5% or more were determined to be deletion markers, and patients with two or more such deletion markers were determined to have microsatellite instability (MSI) (see Table 8).
  • MSI microsatellite instability
  • ⁇ (replication rate) replication rate(normal tissue) - replication rate(cancer tissue)
  • the present invention is a result of the following national research and development project.

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Abstract

La présente invention concerne un procédé basé sur une réaction en chaîne par polymérase numérique pour fournir des informations pour diagnostiquer une instabilité des microsatellites, et un kit associé. Dans le procédé de fourniture d'informations pour diagnostiquer l'instabilité des microsatellites, selon la présente invention, un signal fluorescent est produit par la séparation d'un extincteur et d'un rapporteur dû à une hydrolyse externe par une polymérase lorsqu'une sonde est complètement liée, et, si la liaison 5-terminale de la sonde ne se produit pas en raison d'une mutation de délétion, la région entre le site non lié de l'extrémité 5-terminale et le site lié subit une hydrolyse interne de telle sorte que l'extincteur et le rapporteur sont éliminés ensemble, empêchant ainsi la fluorescence de se produire, et ainsi si des mutations de délétion sont présentes peuvent être analysées avec une sensibilité élevée, et une analyse quantitative peut être effectuée par comparaison du nombre de groupes rapporteur et extincteur produits dans des régions sans instabilité des microsatellites.
PCT/KR2024/020074 2023-12-14 2024-12-09 Procédé basé sur une réaction en chaîne par polymérase numérique pour fournir des informations pour diagnostiquer une instabilité de microsatellite, et kit associé Pending WO2025127642A1 (fr)

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KR1020230181528A KR20250091512A (ko) 2023-12-14 2023-12-14 디지털 중합효소연쇄반응 기반 현미부수체 불안정성 진단을 위한 정보제공 방법 및 이를 위한 키트
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101969971B1 (ko) * 2017-03-24 2019-04-18 주식회사 시선바이오머티리얼스 양기능성 pna 프로브를 이용한 융해곡선 분석방법, 및 이를 이용한 현미부수체 불안정성의 진단방법 및 현미부수체 불안정성 진단용 키트
US20200291466A1 (en) * 2017-07-12 2020-09-17 Institut Curie Method for Detecting a Mutation in a Microsatellite Sequence
KR20220031544A (ko) * 2019-05-13 2022-03-11 펜타베이스 에이피에스 변이체 핵산 검출을 위한 용융 온도 방법, 키트 및 리포터 올리고
KR20220056665A (ko) * 2020-10-28 2022-05-06 사회복지법인 삼성생명공익재단 현미부수체 불안정성 진단용 마커 조성물 및 프라이머 세트
KR20220080682A (ko) * 2020-12-07 2022-06-14 (주)디엑솜 현미부수체 지역의 서열 길이의 변동계수를 이용한 현미부수체 불안정성 진단방법
CN116479128A (zh) * 2023-04-04 2023-07-25 浙江绍兴鼎晶生物医药科技股份有限公司 一种检测癌症中微卫星不稳定性的生物标志物组及其检测试剂盒和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170037095A (ko) 2015-09-25 2017-04-04 주식회사 시선바이오머티리얼스 현미부수체 불안정성의 진단을 위한 pna 프로브를 이용한 융해곡선 분석방법, 및 이를 이용한 현미부수체 불안정성의 진단방법 및 현미부수체 불안정성의 진단용 키트

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101969971B1 (ko) * 2017-03-24 2019-04-18 주식회사 시선바이오머티리얼스 양기능성 pna 프로브를 이용한 융해곡선 분석방법, 및 이를 이용한 현미부수체 불안정성의 진단방법 및 현미부수체 불안정성 진단용 키트
US20200291466A1 (en) * 2017-07-12 2020-09-17 Institut Curie Method for Detecting a Mutation in a Microsatellite Sequence
KR20220031544A (ko) * 2019-05-13 2022-03-11 펜타베이스 에이피에스 변이체 핵산 검출을 위한 용융 온도 방법, 키트 및 리포터 올리고
KR20220056665A (ko) * 2020-10-28 2022-05-06 사회복지법인 삼성생명공익재단 현미부수체 불안정성 진단용 마커 조성물 및 프라이머 세트
KR20220080682A (ko) * 2020-12-07 2022-06-14 (주)디엑솜 현미부수체 지역의 서열 길이의 변동계수를 이용한 현미부수체 불안정성 진단방법
CN116479128A (zh) * 2023-04-04 2023-07-25 浙江绍兴鼎晶生物医药科技股份有限公司 一种检测癌症中微卫星不稳定性的生物标志物组及其检测试剂盒和应用

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