Computer-implemented methods for providing oligonucleotides for use in detecting nucleotide variations of interest

Abstract

The present invention relates to a computer-implemented method for providing oligonucleotides used to detect a nucleotide mutation of interest in a target nucleic acid sequence. The present invention includes inputting information about a wild-type target nucleic acid sequence and a nucleotide mutation of interest, providing wild-type and mutant target nucleic acid sequences using the input information, designing oligonucleotides for the mutant target nucleic acid sequence, analyzing whether the designed oligonucleotides match the wild-type target nucleic acid sequence, and selecting and providing oligonucleotides that meet predetermined selection criteria. This makes it possible to provide oligonucleotides capable of detecting nucleotide mutations based on integrated design rules, without the need to develop detailed design rules and modules taking into account the type of nucleotide mutation, the magnitude of the mutation, whether the target to be detected is a wild-type and / or mutant sequence, and sequence content.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to Korean Patent Application No. 2020-0173648 filed with the Korean Intellectual Property Office on December 11, 2020, the disclosure of which is incorporated herein by reference.

[0002] [Technical field] The present invention relates to a computer-implemented method for providing oligonucleotides for use in detecting nucleotide mutations of interest in a target nucleic acid sequence. [Background technology]

[0003] A variety of methods are known in the art for the detection of target nucleic acid sequences.Examples of the various detection methods include the PTOCE (PTO cleavage and extension) method (WO2012 / 096523), the TaqMan probe method (U.S. Pat. No. 5,210,015), the molecular beacon method (Tyagi et al., Nature Biotechnology 14:303 (1996)), the Scorpion method (Whitcombe et al., Nature Biotechnology 17:804-807 (1999)), the Sunrise (or Amplifluor) method (Nazarenko et al., Nucleic Acids Research, 25(12):2516-2521 (1997) and U.S. Pat. No. 6,117,635), the Lux method (U.S. Pat. No. 7,537,886), the CPT (Duck P, et al., Biotechniques, 9:142-148 (1990)), LNA method (U.S. Pat. No. 6,977,295), Plexor method (Sherrill CB, et al., Journal of the American Chemical Society, 126:4550-4556 (2004)), Hybeacons method (D.J.French, et al., Molecular and Cellular Probes (2001) 13,363-374 and U.S. Pat. No. 7,348,141), dual-labeled, self-quenched probe (U.S. Pat. No. 5,876,930), hybridization probe (Bernard PS, et al., Clin Chem. 2000, 46, 147-148), Invader analysis (U.S. Patent No. 5,691,142), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO2013 / 115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (WO2014 / 104818) and CER method (WO2011 / 037306).

[0004] Oligonucleotides (probes and / or primers) used for detecting a target nucleic acid sequence must have appropriate specificity and sensitivity, be compatible with a specific detection method, and meet the conditions set by the analyst. Therefore, it is very important to design oligonucleotides that are suitable for the analytical purpose.

[0005] Meanwhile, nucleotide variations including substitutions (e.g., single nucleotide polymorphisms (SNPs)), inversions, insertions, deletions, duplications, or combinations thereof (complexes), are important in research and clinical fields. Among them, SNPs are most frequently found in the human genome and serve as markers for disease-related locations and pharmacogenetics. SNPs are found in the human genome at a rate of about 1 per 1,000 bp, and the total number of SNPs is estimated to be about 3 million.

[0006] Conventionally, in order to design an oligonucleotide for detecting a nucleotide mutation, it was necessary to consider the type of nucleotide mutation (e.g., substitution, insertion, etc.), the magnitude of the mutation (e.g., the number of mutated bases), and whether the target to be detected is a wild-type and / or mutant sequence. As a result, there was a problem that at least 20 design rules and modules had to be developed based on such considerations.

[0007] In addition, the conventional method has a problem that it is difficult to consider sequence content. For example, when a wild-type target nucleic acid sequence is 5'-CAGATCTGTTTTAAACGACT-3' (SEQ ID NO: 1), and a mutation occurs between the 11th position and the 12th position from the 5' end of the wild-type target nucleic acid sequence, in which a base T is inserted (i.e., the mutant target nucleic acid sequence is 5'-CAGATCTGTTTTTAAACGACT-3'; SEQ ID NO: 2), and a design rule for an insertion mutation is determined to design an oligonucleotide such that the base of the insertion mutation is located at the 3rd position from the 5' end of the oligonucleotide (i.e., 5'-TTTTAAACGACT-3'; SEQ ID NO: 3), there is a problem that the oligonucleotide designed according to the design rule cannot distinguish between the wild-type target nucleic acid sequence and the mutant target nucleic acid sequence for detection.

[0008] Therefore, the present inventors have endeavored to develop an integrated design method that can design oligonucleotides to be used for distinguishing and detecting wild-type and mutant target nucleic acid sequences, taking into account all of the various nucleotide mutation types.

[0009] Throughout this specification, a number of cited documents and patent documents are referenced and citations are provided, the disclosures of which are hereby incorporated by reference in their entirety in order to more clearly describe the state of the art to which this invention pertains and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have attempted to overcome the problems of the above-mentioned conventional methods and develop a computer-implemented method that can effectively provide an oligonucleotide used to detect a nucleotide mutation of interest in a target nucleic acid sequence. As a result, the present inventors have confirmed that it is possible to provide an oligonucleotide capable of detecting a nucleotide mutation based on an integrated design rule without the need to develop detailed design rules and modules in consideration of the type of nucleotide mutation, the size of the mutation, whether the target to be detected is a wild-type and / or mutant sequence, and sequence content by inputting information on a wild-type target nucleic acid sequence and a nucleotide mutation of interest, providing wild-type and mutant target nucleic acid sequences using the input information, designing an oligonucleotide for the mutant target nucleic acid sequence, analyzing whether the designed oligonucleotide matches the wild-type target nucleic acid sequence, and selecting and providing an oligonucleotide that meets a predetermined selection criterion, thereby completing the present invention.

[0011] It is therefore an object of the present invention to provide a computer-implemented method for providing oligonucleotides for use in detecting nucleotide variations of interest in a target nucleic acid sequence.

[0012] It is another object of the present invention to provide a computer readable medium comprising instructions implementing a processor for carrying out a method for providing oligonucleotides for use in detecting nucleotide variations of interest in a target nucleic acid sequence.

[0013] Other objects and advantages of the present invention will become more apparent from the following detailed description, claims and drawings. [Means for solving the problem]

[0014] According to one aspect of the invention, the invention provides a computer-implemented method for providing oligonucleotides for use in detecting a nucleotide variation of interest in a target nucleic acid sequence, comprising the steps of: (a) inputting information about a wild-type target nucleic acid sequence and a nucleotide variation of interest, the wild-type target nucleic acid sequence being a target nucleic acid sequence that does not contain a nucleotide variation of interest, the information including (i) the wild-type target nucleic acid sequence, (ii) a position of the nucleotide variation of interest occurring within the wild-type target nucleic acid sequence, and (iii) information about a wild-type base and a mutant base at the position of the nucleotide variation of interest, the positions of the nucleotide variation of interest being designated as a start position and an end position; (b) providing the wild-type target nucleic acid sequence and the mutant target nucleic acid sequence using the input information, the mutant target nucleic acid sequence being a target nucleic acid sequence containing a nucleotide mutation of interest, the wild-type and mutant target nucleic acid sequences including forward and reverse wild-type and mutant target nucleic acid sequences, respectively; (c) designing oligonucleotides for use in detecting the nucleotide variation of interest in a predetermined region within the mutant target nucleic acid sequence that contains the nucleotide variation of interest, to provide a first set of candidate oligonucleotides for the mutant target nucleic acid sequence; (d) analyzing whether or not oligonucleotides included in the first group of oligonucleotide candidates for the mutant target nucleic acid sequence match with the wild-type target nucleic acid sequence, and providing oligonucleotides that satisfy the following selection criteria as a second group of oligonucleotide candidates for the mutant target nucleic acid sequence, the selection criteria including: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, the center, or the 3' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is equal to or greater than a predetermined ratio; and (e) selecting oligonucleotides from the second set of oligonucleotide candidates for the mutant target nucleic acid sequence to provide a third set of oligonucleotide candidates, the third set of oligonucleotide candidates being used to detect a nucleotide variation of interest in the target nucleic acid sequence.

[0015] The present inventors have attempted to overcome the problems of the above-mentioned conventional methods and develop a computer-implemented method that can effectively provide an oligonucleotide used to detect a nucleotide mutation of interest in a target nucleic acid sequence. As a result, the present inventors have confirmed that it is possible to provide an oligonucleotide capable of detecting a nucleotide mutation based on an integrated design rule without the need to develop detailed design rules and modules in consideration of the type of nucleotide mutation, the size of the mutation, whether the target to be detected is a wild-type and / or mutant sequence, and sequence content, by inputting information on a wild-type target nucleic acid sequence and a nucleotide mutation of interest, providing wild-type and mutant target nucleic acid sequences using the input information, designing an oligonucleotide for the mutant target nucleic acid sequence, analyzing whether the designed oligonucleotide matches the wild-type target nucleic acid sequence, and selecting and providing an oligonucleotide that meets a predetermined selection criterion.

[0016] As used herein, the term "preparing" in reference to an oligonucleotide includes providing the sequence information for the oligonucleotide and manufacturing the oligonucleotide material.

[0017] As used herein, the term "target nucleic acid sequence" or "target sequence" refers to a specific nucleic acid sequence of a target nucleic acid molecule.

[0018] As used herein, the terms "target nucleic acid molecule," "target molecule," or "target nucleic acid" refer to a nucleotide molecule in an organism that is to be detected. Target nucleic acid molecules are generally given specific names and include entire genomes and all nucleotide molecules that make up a genome (e.g., genes, pseudogenes, non-coding sequence molecules, non-coding regions, and partial regions of a genome). Target nucleic acid molecules include, for example, the nucleic acids of an organism.

[0019] As used herein, the term "organism" refers to an organism belonging to a biological classification system, e.g., a kingdom, phylum, class, order, family, genus, species, subspecies, cultivar, variety, subtype, genotype, serotype, strain, isolate, or cultivar. Organisms include, for example, prokaryotic cells (e.g., Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Haemophilus influenzae, Streptococcus pneumoniae, Bordetella pertussis, Bordetella parapertussis, Neisseria meningitidis, Listeria monocytogenes, Streptococcus agalactiae, Campylobacter, Clostridium difficile, Clostridium perfringens, Salmonella, Escherichia coli, Shigella, Vibrio, Yersinia enterocolitica, Aeromonas, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Ureaplasma parvum, Mycobacterium tuberculosis), eukaryotic cells (e.g., protozoa, parasites, fungi, yeast, higher plants, lower animals, and higher animals including mammals and humans), viruses, or viroids. Among the eukaryotic cells, examples of parasites include Giardia lamblia, Entamoeba histolytica, Cryptosporidium, Blastocystis hominis, Dientamoeba fragilis, and Cyclospora cayetanensis.Examples of such viruses include influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus A (RSV A), respiratory syncytial virus B (RSV B), parainfluenza virus 1 (PIV1), parainfluenza virus 2 (PIV2), parainfluenza virus 3 (PIV3), parainfluenza virus 4 (PIV4), metapneumovirus (MPV), human enterovirus (HEV), human bocavirus (HBoV), human rhinovirus (HRV), coronavirus, and adenovirus, which induce respiratory disease; and norovirus, rotavirus, adenovirus, astrovirus, and sapovirus, which induce gastrointestinal disease. Examples of the virus include human papillomavirus (HPV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), dengue virus, Herpes simplex virus (HSV), human herpes virus (HHV), Epstein-Barr virus (EMV), Varicella zoster virus (VZV), cytomegalovirus (CMV), HIV, hepatitis virus, and poliovirus.

[0020] As used herein, the term "nucleotide mutations of interest" refers to nucleotide mutations in a target nucleic acid sequence that one wishes to amplify and / or detect using an oligonucleotide (eg, a primer or a probe).

[0021] The term "nucleotide variation" as used herein refers to any single or multiple nucleotide substitutions, inversions, insertions, deletions, repetitions, or combinations thereof, in a DNA sequence at a specific position in a contiguous DNA segment or in DNA segments with similar sequences. Such contiguous DNA segments may include different sites in a gene or a chromosome. Such nucleotide variations may be mutations or polymorphic allele variations. Examples include single nucleotide polymorphisms (SNPs), mutations, deletions, insertions, substitutions, and translocations, and include various mutations in the human genome (e.g., mutations in the MTHFR (methylenetetrahydrofolate reductase) gene), mutations associated with drug resistance in pathogens, and mutations associated with cancer development.

[0022] According to one embodiment of the present invention, the nucleotide variation of interest is a substitution, an inversion, an insertion, a deletion, a duplication, or a combination thereof.

[0023] The term "oligonucleotide" as used herein means a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing to a target nucleic acid sequence, naturally occurring or synthetically synthesized. The oligonucleotide is preferably single-stranded for maximum efficiency in hybridization. In particular, the oligonucleotide is an oligodeoxyribonucleotide. In the present invention, the oligonucleotide may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), nucleotide analogs, or derivatives. The oligonucleotide may also include ribonucleotides. For example, in the present invention, oligonucleotides may contain backbone-modified nucleotides, such as peptide nucleic acids (PNA) (M. Egholm et al., Nature, 365:566-568 (1993)), locked nucleic acids (LNA) (WO1999 / 014226), bridged nucleic acids (Bridged Nucleic Acids (BNA)), and the like. Acid:BNA) (WO2005 / 021570), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar-modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents include fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-.), 7-deazapurines with C-7 substituents (wherein the substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkinyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine, and diaminopurine. In particular, the term "oligonucleotide" as used herein refers to a single strand of deoxyribonucleotides. The term "oligonucleotide" includes oligonucleotides that hybridize with cleavage fragments generated depending on the target nucleic acid sequence.

[0024] According to one embodiment of the present invention, the oligonucleotide is a probe and / or a primer.

[0025] As used herein, the term "primer" refers to an oligonucleotide that can act as an initiation point for synthesis under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand (template), i.e., in the presence of nucleotides and a polymerizing agent such as a DNA polymerase, and at appropriate temperature and pH. A primer must be sufficiently long to be capable of priming the synthesis of an extension product in the presence of the polymerizing agent. The appropriate length of a primer is determined by several factors, including, for example, temperature, application, and source of primer.

[0026] As used herein, the term "probe" refers to a single-stranded nucleic acid molecule that contains a portion or portions that are complementary to a target nucleic acid sequence. The probe may also contain a label that can generate a signal for detecting the target.

[0027] The oligonucleotide may have a typical primer and probe structure that is composed of a sequence that hybridizes to a target nucleic acid sequence. Or, the oligonucleotide may have a unique structure obtained by modifying the structure of the oligonucleotide. For example, the oligonucleotide may have the structure of a Scorpion primer, a molecular beacon probe, a sunrise primer, a high beacon probe, a tagging probe, a DPO primer or probe (WO2006 / 095981), and a PTO probe (see WO2012 / 096523).

[0028] The oligonucleotide may be a modified oligonucleotide such as a degenerate base-containing oligonucleotide and / or a universal base-containing oligonucleotide in which a degenerate base and / or a universal base is introduced into a conventional primer or probe. As used herein, the terms "conventional primer", "conventional probe" and "conventional oligonucleotide" refer to a conventional primer, probe and oligonucleotide in which a degenerate base or a non-natural base is not introduced. According to one embodiment of the present invention, the degenerate base-containing oligonucleotide or the universal base-containing oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% non-modified oligonucleotide. According to one embodiment of the present invention, the number of degenerate bases or universal bases introduced into the conventional oligonucleotide is specifically 7 or less, 5 or less, 4 or less, 3 or less or 2 or less. Alternatively, the ratio of degenerate bases and / or universal bases introduced into the conventional oligonucleotide is specifically 25% or less, 20% or less, 18% or less, 16% or less, 14% or less, 12% or less, 10% or less, 8% or less, or 6% or less. The ratio of degenerate bases or universal bases represents the ratio of degenerate bases or universal bases to the total nucleotides of the oligonucleotide into which the degenerate bases or universal bases are introduced. The degenerate bases include various degenerate bases known in the art: R: A or G; Y: C or T; S: G or C; W: A or T; K: G or T; M: A or C; B: C, G or T; D: A, G or T; H: A, C or T; V: A, C or G; N: A, C, G or T.The universal base includes various universal bases known in the art, such as dioxyinosine, inosine, 7-diaza-2'-dioxyinosine, 2-aza-2'-dioxyinosine, 2'-OMe inosine, 2'-F inosine, dioxy 3-nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3-nitropyrrole, 1-(2'-dioxy-beta-D-ribofuranosyl)-3-nitropyrrole, dioxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole, dioxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, dioxynebularine, 2'-F nebularine, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2'-0-methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro-benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole and combinations of said bases. More specifically, the universal base is dioxyinosine, inosine, or a combination thereof.

[0029] 1 is a flow chart of a process for carrying out the method of the present invention according to an embodiment of the present invention. The method of the present invention will be described in detail with reference to FIG. 1 as follows: (Step (a): Input information on the wild-type target nucleic acid sequence and the nucleotide mutation of interest (110)) First, the method of the present invention includes the step of (a) inputting information on a wild-type target nucleic acid sequence and a nucleotide variation of interest, the wild-type target nucleic acid sequence being a target nucleic acid sequence that does not contain a nucleotide variation of interest, the information including (i) the wild-type target nucleic acid sequence, (ii) the position of the nucleotide variation of interest occurring in the wild-type target nucleic acid sequence, and (iii) information on a wild-type base and a mutant base at the position of the nucleotide variation of interest, the positions of the nucleotide variation of interest being indicated as a start position and an end position.

[0030] The method of the present invention is a computer-implemented method, in which input of information regarding the sequence of the wild-type target nucleic acid sequence and the nucleotide variation of interest is performed via a user interface (UI).

[0031] In this specification, the step of inputting information refers to a step of receiving information, for example, the step of inputting information via a UI refers to a step of receiving information via a UI.

[0032] The wild-type target nucleic acid sequence is a reference sequence for identifying the position of a nucleotide mutation that has occurred within the wild-type target nucleic acid sequence.

[0033] Among the above information, the wild-type target nucleic acid sequence can be searched from the Refseq database by identifying a gene in which a nucleotide mutation of interest exists, searching for a sequence by gene in a publicly accessible database (specifically, the NCBI nucleotide database), selecting organism information (e.g., taxonomy name or taxonomy ID) containing the nucleotide mutation of interest, or by identifying rs number information as an identifier for a mutation occurring in the wild-type target nucleic acid sequence, and searching for the wild-type target nucleic acid sequence by rs number in a publicly accessible database (specifically, the NCBI SNP database).

[0034] According to one embodiment of the present invention, the wild-type target nucleic acid sequence in step (a) has a predetermined length that includes the position of the nucleotide variation of interest.

[0035] According to one embodiment of the present invention, the wild-type target nucleic acid sequence in step (a) has a predetermined length from the position of the nucleotide variation of interest.

[0036] Specifically, the wild-type target nucleic acid sequence includes a sequence having a predetermined length upstream (upstream, left, -) and a predetermined length downstream (downstream, right, +) based on the position of the nucleotide mutation of interest occurring in the wild-type target nucleic acid sequence, or more specifically, a sequence having a predetermined length upstream (upstream, left, -) based on the start position of the nucleotide mutation of interest and a predetermined length downstream (downstream, right, +) based on the end position.

[0037] The predetermined length may be, for example, selected from 1500 bp to 100 bp, or may be 1500 bp, 1400 bp, 1300 bp, 1200 bp, 1100 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, or 100 bp.

[0038] As used herein, the term "upstream" when referring to direction may be used synonymously with "left side," "left" or "-" with respect to any position in a nucleic acid sequence, and the term "downstream" may be used synonymously with "right side," "right" or "+" with respect to any position in a nucleic acid sequence.

[0039] As used herein, the term "nucleotide" in reference to a nucleotide length may be used interchangeably with "mer," "base," or "bp."

[0040] Among the information, the position of the nucleotide mutation of interest occurring in the wild-type target nucleic acid sequence is a position in the wild-type target nucleic acid sequence, and is indicated as a start position and an end position in the wild-type target nucleic acid sequence. For example, when a substitution mutation occurs in which a base T is replaced with a base G at position 305 of the wild-type target nucleic acid sequence, the start position and the end position of the mutation are position 305. When an inversion mutation occurs in which a base CGTAT is replaced with a base TATGC at positions 333 to 337 of the wild-type target nucleic acid sequence, the start position and the end position of the mutation are position 333 and position 337. When an insertion mutation of a base TGC occurs between a base G at position 354 and a base T at position 355 of the wild-type target nucleic acid sequence, the start position and the end position of the mutation are position 354. When a deletion mutation occurs in which CAT is deleted from the bases GCAT at positions 324 to 327 of the wild-type target nucleic acid sequence, the start position of the mutation is position 324, and the end position is position 327. When a complex mutation occurs in which CAT is deleted from the bases GCAT at positions 544 to 547 of the wild-type target nucleic acid sequence and ACGACT is inserted, the start position of the mutation is position 544, and the end position is position 547. When the length of the nucleic acid sequence changes due to a mutation, for example, in an insertion and deletion mutation, the position before the insertion and deletion positions is indicated as the start position.

[0041] Among the information, the information on the wild-type base and the mutant base at the position of the nucleotide mutation of interest may be, for example, in the description of the start position and end position, in the case of a substitution mutation, the wild-type base is the base T and the mutant base is the base G, in the case of a reversal mutation, the wild-type base is the base CGTAT and the mutant base is the base TATGC, in the case of an insertion mutation, the wild-type base is the base G and the mutant base is the base GTGC, in the case of a deletion mutation, the wild-type base is the base GCAT and the mutant base is the base G, and in the case of a complex mutation, the wild-type base is the base GCAT and the mutant base is the base GACGACT. When the length of the sequence is changed by the mutation, for example, in the case of an insertion and deletion mutation, the information may be entered including the base at the position before the insertion and deletion positions.

[0042] (Step (b): Providing a wild-type target nucleic acid sequence and a mutant target nucleic acid sequence (120)) The method then includes the step of (b) using the input information to provide the wild-type target nucleic acid sequence and a mutant target nucleic acid sequence, the mutant target nucleic acid sequence being a target nucleic acid sequence comprising a nucleotide mutation of interest, the wild-type and mutant target nucleic acid sequences comprising forward and reverse wild-type target nucleic acid sequences and forward and reverse mutant target nucleic acid sequences, respectively.

[0043] This step provides a template against which oligonucleotides are designed in the steps described below.

[0044] The wild-type and mutant target nucleic acid sequences are provided using the information input in step (a). Specifically, the position information of the nucleotide mutation of interest and the information of the mutant base generated in the wild-type target nucleic acid sequence in step (a) can be applied to the wild-type target nucleic acid sequence to provide the mutant target nucleic acid sequence. If the wild-type target nucleic acid sequence is a forward wild-type target nucleic acid sequence, a reverse wild-type target nucleic acid sequence can be provided, and if the provided mutant target nucleic acid sequence is provided from a forward wild-type target nucleic acid sequence, a reverse mutant target nucleic acid sequence can be provided from the forward mutant target nucleic acid sequence.

[0045] In other words, when information on a forward wild-type target nucleic acid sequence is input in step (a), a forward (5' to 3') wild-type target nucleic acid sequence and a reverse (reverse complementary, 3' to 5') wild-type target nucleic acid sequence are provided as templates. According to information on the position of the nucleotide mutation of interest occurring in the wild-type target nucleic acid sequence input in step (a) and the wild-type base and mutant base at the position of the nucleotide mutation of interest, a forward (5' to 3') mutant target nucleic acid sequence and a reverse (reverse complementary, 3' to 5') mutant target nucleic acid sequence are provided as templates.

[0046] As used herein, the term "forward" in reference to the orientation of a target nucleic acid sequence as a template refers to a 5' to 3' direction, and the term "reverse" is complementary to the forward target nucleic acid sequence and refers to a 3' to 5' direction.

[0047] The term "providing" as used herein with reference to the wild-type and mutant target nucleic acid sequences refers to the presentation of sequence information of a target nucleic acid sequence.

[0048] According to an embodiment of the present invention, the mutant target nucleic acid sequence includes information on other nucleotide variations in addition to the nucleotide variation of interest. For example, when a mutation having a non-conserved base is present at any position of the collected target nucleic acid sequences in addition to the nucleotide variation of interest to be detected, information on the mutation can also be included in the mutant target nucleic acid sequence. The mutation having a non-conserved base may be different or the same for different organisms or continents.

[0049] As used herein, the term "non-conserved" refers to a case where no conservation is shown at the alignment position of multiple target nucleic acid sequences, and refers to a case where the ratio or number of specific bases that differ compared to the total bases at the alignment position of multiple target nucleic acid sequences exceeds a predetermined value. For example, other nucleotide variations besides the nucleotide variation of interest refer to, but are not limited to, a case where the ratio of specific bases that differ compared to the total bases is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, or at least 0.7%.

[0050] (Step (c): Providing a first set of oligonucleotide candidates for a mutated target nucleic acid sequence (130)) The method of the present invention then includes the step of (c) designing oligonucleotides to be used for detecting the nucleotide variation of interest in a predetermined region containing the nucleotide variation of interest within the mutant target nucleic acid sequence, thereby providing a first set of oligonucleotide candidates for the mutant target nucleic acid sequence.

[0051] In this step, oligonucleotides used to detect the mutant target nucleic acid sequence are designed to provide a first group of oligonucleotide candidates for the mutant target nucleic acid sequence.

[0052] Since the position of the nucleotide variation of interest that occurred in the wild-type target nucleic acid sequence in step (a) and the mutated base at the position are known, the design of the oligonucleotide in this step is performed in a predetermined region that includes the nucleotide variation of interest in the mutant target nucleic acid sequence, and the predetermined region that includes the nucleotide variation of interest can be named the design region of the oligonucleotide.

[0053] According to one embodiment of the present invention, the oligonucleotide of step (c) is designed to have a sequence that matches or is complementary to a predetermined region containing the nucleotide mutation of interest in the mutant target nucleic acid sequence.

[0054] According to this embodiment, the designed oligonucleotides are designed to have sequences that match or are complementary to sequences that fall within the design domain of the oligonucleotide.

[0055] As for the design range of the oligonucleotide, the predetermined region containing the nucleotide mutation of interest may be selected from the ranges of 20 to 200 bp, 30 to 180 bp, 40 to 160 bp, 50 to 150 bp, 60 to 130 bp, or 70 to 120 bp.

[0056] As the design range of the oligonucleotide, the predetermined region including the nucleotide mutation of interest is a region including a predetermined length upstream and a predetermined length downstream based on the position of the nucleotide mutation of interest present in the mutation target nucleic acid sequence, and the predetermined length upstream can be selected from 0 bp to 60 bp, and the predetermined length downstream can be selected from 0 bp to 60 bp. The predetermined length upstream and the predetermined length downstream can be the same or different from each other. For example, when the design range of the oligonucleotide is a region including 0 bp upstream and 60 bp downstream based on the position of the nucleotide mutation of interest, it means that the oligonucleotide is designed in a region including 60 bp downstream from the nucleotide mutation of interest in the mutation target nucleic acid sequence. Or, when the design range of the oligonucleotide is a region including 10 bp upstream based on the start position of the nucleotide mutation of interest, 60 bp downstream based on the end position, and the length of the start position and the end position is 10 bp, it means that the oligonucleotide is designed in an 80 bp region in the mutation target nucleic acid sequence.

[0057] According to one embodiment of the present invention, the oligonucleotides of step (c) are designed with different lengths from a predetermined position upstream to an end position based on the start position of the nucleotide mutation of interest in the mutation target nucleic acid sequence. In particular, the oligonucleotides of step (c) are designed with different lengths from a predetermined position upstream to an end position based on the start position of the nucleotide mutation of interest in the mutation target nucleic acid sequence. In particular, the oligonucleotides of step (c) are designed with different lengths from a predetermined position upstream to an end position based on the start position of the nucleotide mutation of interest in the mutation target nucleic acid sequence. Alternatively, the oligonucleotides of step (c) are designed with different lengths from a predetermined position upstream to an end position based on the start position of the nucleotide mutation of interest in the mutation target nucleic acid sequence.

[0058] The position of the nucleotide variation of interest within the mutated target nucleic acid sequence refers to the position of the nucleotide variation of interest present in the mutated target nucleic acid sequence provided in step (b), and the positions of the variation may be expressed as a start position and an end position.

[0059] The predetermined position upstream of the start position of the nucleotide variation of interest may be selected from 1 to 50 bp or 2 to 40 bp, for example, 1 bp, 2 bp, 3 bp, 4 bp, 38 bp, 39 bp, 40 bp, or 41 bp.

[0060] The length of the oligonucleotide designed according to the length (particularly, the predetermined length) is, for example, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 45 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 15 to 60 nucleotides, 15 to 50 nucleotides, 15 to 45 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 20 to 60 nucleotides, 20 to 50 nucleotides, 20 to 45 nucleotides, 20 to 40 nucleotides, or 20 to 35 nucleotides.

[0061] When designing oligonucleotides according to this embodiment, for example, a predetermined position is 2 bp upstream based on the start position of the nucleotide mutation of interest, the length (predetermined length) of the oligonucleotides designed by length ranges from 3 to 4 nucleotides, the mutation target nucleic acid sequence is 5'-ATGCGGTTG-3', and base C at the fourth position of the 5' end of the mutation target nucleic acid sequence is the mutated base, which is a substitution mutation in which base A is replaced with base C. In this case, oligonucleotides are designed by 3 to 4 nucleotides at each position from base T to base C, which is 2 bp upstream based on base C, which is the base at the start and end positions of the mutation in the mutation target nucleic acid sequence (i.e., 5'-TGC-3', 5'-TGCG-3', 5'-GCG-3', 5'-GCGG-3', 5'-CGG-3', and 5'-CGGT-3').

[0062] In the present specification, the expression "oligonucleotides having the same start position at which the oligonucleotides are designed" refers to oligonucleotides having the same start position at which the oligonucleotides are designed according to length. In the above example, the oligonucleotides 5'-TGC-3' and 5'-TGCG-3' are oligonucleotides having the same start position at which the oligonucleotides are designed.

[0063] When designing oligonucleotides according to this embodiment, for example, when a predetermined position is 2 bp upstream from the start position of the nucleotide mutation of interest, the length of the oligonucleotides designed by length ranges from 3 to 5 nucleotides, the mutation target nucleic acid sequence is 5'-ATGCTCTGGC-3', and there is a mutation in which CT, which is the 4th and 5th bases, are inserted from base A at the 5' end of the mutation target nucleic acid sequence, oligonucleotides can be designed by 3 to 5 nucleotides at each position from base T, which is 2 bp upstream from base C, which is at the start position of the nucleotide mutation of interest, to base T, which is at the end position of the nucleotide mutation of interest. Specifically, 5'-TGC-3', 5'-TGCT-3', 5'-TGCTC-3', 5'-GCT-3', 5'-GCTC-3', 5'-GCTCT-3', 5'-CTC-3', 5'-CTCT-3', 5'-CTCTG-3', 5'-TCT-3', 5'-TCTG-3', 5'-TCTGG-3' are designed from the second base T from the 5' end of the mutation target nucleic acid sequence, i.e., the base T located 2 bp upstream from the base C located at the start position of the mutation. An oligonucleotide is also designed in the same manner for the reverse sequence of the mutation target nucleic acid sequence.

[0064] In describing an example of oligonucleotide design for the insertion mutation, when describing a position starting from the 5' end of the mutation target nucleic acid sequence, the 5' end base A is considered to be the first position, and when describing a specific position upstream based on the start position of the nucleotide mutation of interest, the base G upstream of the start position base C is considered to be the first position.

[0065] In other words, in the present specification, when the reference point is the end of a nucleic acid sequence, the base at the end of the nucleic acid sequence is considered to be the first base, including the base at the end of the nucleic acid sequence, and when the base present in the nucleic acid sequence is used as the reference, the base upstream or downstream of the base excluding the base is considered to be the first base. On the other hand, when the base present in the nucleic acid sequence is used as the reference, any difference from the above description will be explained separately.

[0066] According to one embodiment of the present invention, the oligonucleotide designed in step (c) is designed to satisfy at least one of the following conditions: (i) a length of 10 to 60 nucleotides; (ii) a Tm value of 50 to 85°C; (iii) 3 or more G-run sequence exclusions, and (iv) GC content at the 5′-end site is ≥40%.

[0067] More specifically, the oligonucleotide design conditions include at least two of the above-mentioned conditions, more specifically at least three, and even more specifically four of the above-mentioned conditions.

[0068] The length in the design conditions is, for example, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 45 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 15 to 60 nucleotides, 15 to 50 nucleotides, 15 to 45 nucleotides, 15 to 40 nucleotides, or 15 to 35 nucleotides.

[0069] Among the design conditions, the Tm value is, for example, 50 to 80° C., 50 to 75° C., 55 to 80° C., 55 to 75° C., 60 to 80° C., 60 to 75° C., 65 to 80° C., or 60 to 75° C. Specifically, among the design conditions, the Tm value is 55 to 80° C., 60 to 78° C., 63 to 78° C., 65 to 75° C., 67 to 75° C., or 65 to 73° C.

[0070] Among the design conditions, the G-run sequences are, for example, excluding three or more, or four or more, G-run sequences.

[0071] The GC content of the 5'-terminal portion of the oligonucleotide is 40% or more, specifically 40 to 70% or 40 to 60%. The 5'-terminal portion refers to a portion within 10 nucleotides from the 5'-terminal of the oligonucleotide.

[0072] (Step (d): Analyzing whether the first oligonucleotide candidate group matches the wild-type target nucleic acid sequence, and providing oligonucleotides that meet the selection criteria as a second oligonucleotide candidate group (140)) The method of the present invention then includes the step of (d) analyzing whether or not oligonucleotides included in the first group of oligonucleotide candidates for the mutant target nucleic acid sequence match with the wild-type target nucleic acid sequence, and providing oligonucleotides that satisfy the following selection criteria as a second group of oligonucleotide candidates for the mutant target nucleic acid sequence: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, middle, or 3' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is equal to or greater than a predetermined ratio. This step is a process of selecting oligonucleotides that meet a predetermined selection criterion so that oligonucleotides designed for a mutant target nucleic acid sequence can distinguish and detect the mutant target nucleic acid sequence from a wild-type target nucleic acid sequence. This selection process solves the conventional problem that different methods of designing oligonucleotides are required for each type of nucleotide mutation.

[0073] The region of the wild-type target nucleic acid sequence to be analyzed for matching with the oligonucleotides included in the first oligonucleotide candidate group may be the entire or a part of the wild-type target nucleic acid sequence. Specifically, since the position where the nucleotide mutation of interest occurs in the wild-type target nucleic acid sequence is known, the presence or absence of matching can be analyzed for a part of the wild-type target nucleic acid sequence corresponding to the sequence region in the mutant target nucleic acid sequence for which the oligonucleotide used to detect the nucleotide mutation of interest is designed.

[0074] The method of analyzing the presence or absence of the match can be carried out by various methods known in the art. For example, the presence or absence of the match can be analyzed by moving an oligonucleotide designed for a mutant target nucleic acid sequence to a sequence region in a wild-type target nucleic acid sequence corresponding to the design range of the oligonucleotide, or the presence or absence of the match can be analyzed by using the designed oligonucleotide as a query sequence and performing BLAST against the wild-type target nucleic acid sequence.

[0075] Matching analysis may be performed between target nucleic acid sequences and oligonucleotides having the same orientation.

[0076] The selection criteria that enable the method of the present invention to design oligonucleotides incorporating the type of nucleotide mutation include: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, middle or 3' end of the oligonucleotide; or (ii) the presence of a predetermined ratio or more of mismatches between the wild-type target nucleic acid sequence and the oligonucleotide relative to the oligonucleotide sequence.

[0077] The selection criterion (i) is a criterion for distinguishing and detecting a mutant target nucleic acid sequence from a wild-type target nucleic acid sequence based on a mismatch between a specific partial region of an oligonucleotide and a wild-type target nucleic acid sequence, where the region other than the specific partial region of the oligonucleotide contains a sequence that matches with the wild-type target nucleic acid sequence.

[0078] The specific partial region of the oligonucleotide for such differential detection is a predetermined region at the 5' end, center or 3' end of the oligonucleotide, specifically a predetermined region at the 5' end or center of the oligonucleotide, or a predetermined region at the 5' end or 3' end of the oligonucleotide, more specifically a predetermined region at the 5' end of the oligonucleotide. The predetermined region may be, for example, a region within 10 nucleotides, 7 nucleotides, 5 nucleotides, 3 nucleotides, 2 nucleotides, or 1 nucleotide from the 5' end or 3' end of the oligonucleotide. The predetermined region at the center of the oligonucleotide may be a region within 5 nucleotides, 3 nucleotides, or 2 nucleotides upstream and downstream from the central nucleotide, including the central nucleotide, or the central nucleotide.

[0079] An example in which a mutant target nucleic acid sequence can be distinguished from a wild-type target nucleic acid sequence by a mismatch between a predetermined region at the 5' end, the middle or the 3' end of the oligonucleotide and the wild-type target nucleic acid sequence is as follows: When a designed oligonucleotide is designed to include a base corresponding to a nucleotide mutation of interest, a target nucleic acid sequence complementary to a mutant target nucleic acid sequence containing the nucleotide mutation of interest is referred to herein as a matching template, and a target nucleic acid sequence complementary to a wild-type target nucleic acid sequence that does not include a base corresponding to the nucleotide mutation of interest is referred to herein as a mismatching template.

[0080] For example, when an oligonucleotide satisfying the above-mentioned selection criterion (i) is used in the PTOCE method (WO2012 / 096523) or VD-PTOCE method (WO2013 / 133561) developed by the present applicant, if the 3'-targeting portion (the oligonucleotide designed in the present invention) of the PTO (Probing and Tagging Oligonucleotide) has a base complementary to the matching template and a base non-complementary to the non-matching template in a predetermined region (e.g., 5' end) of the 5' end, for example, the cleavage of the PTO hybridized to the matching template under upstream primer-dependent cleavage induction may be induced at a position immediately adjacent in the 3' direction to the 5' end of the 3'-targeting portion of the PTO. The PTO fragment is hybridized to a CTO (Capturing and Templating Oligonucleotide) having a capturing portion containing a sequence corresponding to the nucleotide mutation of interest, and then extended to form an extension duplex to provide a target signal.

[0081] If the same PTO is hybridized to a mismatching template that has the same sequence as the matching template except for the nucleotide mutation of interest, cleavage of the PTO can occur at a position two nucleotides away from the 5' end of the 3'-targeting portion of the PTO in the 3' direction. The 3' end of the PTO fragment has an additional cleaved nucleotide in addition to the nucleotide complementary to the single nucleotide mutation. If the position of the CTO that hybridizes to the additional cleaved nucleotide is designed to have a sequence that is non-complementary to the additional cleaved nucleotide, the 3' end of the PTO fragment will not hybridize to the CTO, and the PTO fragment will not be extended under the controlled conditions. Even if the PTO fragment is extended to form an extension mer, the mer has a different Tm value from the mer resulting from hybridization between the PTO and the mismatching template.

[0082] In this manner, oligonucleotides that satisfy selection criterion (i) above are capable of distinguishing and detecting mutant versus wild-type target nucleic acid sequences.

[0083] In addition, in the case of an oligonucleotide that satisfies the above-mentioned selection criterion (i) relating to a mismatch with a predetermined region at the 3' end of the oligonucleotide, the mutant target nucleic acid sequence can be distinguished and detected by the following method.

[0084] For example, when there is a mismatch at the 3' end of the upstream primer, the 3' end of the upstream primer anneals to the matching template and extends to induce PTO cleavage.As a result, the PTO fragment hybridizes to the CTO to provide target signal.On the contrary, when the 3' end of the upstream primer is mismatched to the nucleotide mutation in the mismatching template, the upstream primer is not extended under the condition that the annealing of the 3' end of the primer is essential for the extension reaction, even if it is hybridized to the mismatching template, so that no target signal is generated.

[0085] In the case of oligonucleotides that satisfy the above-mentioned selection criterion (i) relating to mismatches with a predetermined central region of the oligonucleotide, the mutant target nucleic acid sequence can be distinguished and detected in the following manner.

[0086] For example, when the sequence for the nucleotide mutation of interest is located in the middle of the 3'-targeting portion of the PTO (the oligonucleotide designed in the present invention as described above), under controlled conditions, the PTO is cleaved after hybridizing to a matching template.As a result, the PTO fragment is hybridized to the CTO to provide a target signal.On the other hand, under controlled conditions, the PTO is not hybridized to a mismatching template and is not cleaved.

[0087] According to one embodiment of the present invention, the selection criterion (i) is that there is one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end of the oligonucleotide; most specifically, there is one mismatch between the wild-type target nucleic acid sequence and the 5' end of the oligonucleotide.

[0088] According to one embodiment of the present invention, the mismatch in step (d)(i) is a minimal mismatch between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, the middle or the 3' end of the oligonucleotide.

[0089] For example, if the specified region at the 5' end of the oligonucleotide in the selection criterion (i) is a 2mer, the designed oligonucleotide is 5'-ATGCT-3', and the region of the wild-type target nucleic acid sequence to be analyzed for matching is 5'-AAGCT-3', when the 2mer base AT at the 5' end of the oligonucleotide is analyzed for matching with 5'-AAGCT-3' to be analyzed for matching, at least one mismatch is found, and therefore the designed oligonucleotide 5'-AATGC-3' is selected and provided as the second oligonucleotide candidate group.

[0090] The selection criterion (ii) is a criterion for distinguishing and detecting a mutant target nucleic acid sequence from a wild-type target nucleic acid sequence by hybridizing the oligonucleotide to a mutant target nucleic acid sequence (or a sequence complementary to the mutant target nucleic acid sequence) and not hybridizing to a wild-type target nucleic acid sequence (or a sequence complementary to the wild-type target nucleic acid sequence) due to a predetermined mismatch ratio between the entire sequence of the oligonucleotide and the wild-type target nucleic acid sequence.

[0091] For example, when an oligonucleotide satisfying the above-mentioned selection criterion (ii) is used in the PTOCE method (WO2012 / 096523) developed by the present applicant, if the 3'-targeting site (oligonucleotide designed in the present invention) of the PTO (Probing and Tagging Oligonucleotide) has a base complementary to the mutant target nucleic acid sequence and a predetermined ratio or more of non-complementary bases so as to be non-hybridized to the wild-type target nucleic acid sequence, for example, cleavage of the PTO hybridized to the mutant target nucleic acid sequence under upstream primer-dependent cleavage induction may be induced at any one position of the 5' end of the 3'-targeting site of the PTO. The PTO fragment is hybridized to a CTO (Capturing and Templating Oligonucleotide) having a capturing site containing a sequence complementary to the fragment, and then extended to form an extension duplex to provide a target signal.

[0092] If the same PTO contains a predetermined ratio or more of non-complementary bases to the wild-type target nucleic acid sequence, including the nucleotide mutation of interest, the PTO will not hybridize to the wild-type target nucleic acid sequence and therefore will not be cleaved, and will not be extended even when hybridized to the CTO.

[0093] In this manner, oligonucleotides that satisfy selection criterion (ii) above are capable of distinguishing and detecting mutant versus wild-type target nucleic acid sequences.

[0094] The predetermined percentage of mismatches may be selected such that an oligonucleotide designed for a mutant target nucleic acid sequence does not hybridize to a wild-type target nucleic acid sequence, and may be, for example, selected from 10% to 100%, or may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0095] According to one embodiment of the present invention, the mismatch in step (d)(ii) represents a minimum mismatch between the wild-type target nucleic acid sequence and the oligonucleotide, more specifically, the mismatch in step (d)(ii) represents a minimum number of mismatches between the wild-type target nucleic acid sequence and the oligonucleotide.

[0096] In this embodiment, a mismatch greater than or equal to a certain ratio for an oligonucleotide sequence represents the ratio of the number of mismatches in an oligonucleotide that shows the smallest mismatch between the wild-type target nucleic acid sequence and the oligonucleotide as a result of analyzing whether or not there is a match between the wild-type target nucleic acid sequence and the entire oligonucleotide sequence.

[0097] For example, when the number of mismatches is 20mer, 17mer, 10mer, 18mer, and 21mer as a result of the match analysis of an oligonucleotide designed as 30mer with a wild-type target nucleic acid sequence, the minimum number of mismatches is 10mer, and the oligonucleotide sequence is 30mer, so the mismatch ratio is 33.3%. In this case, if the predetermined ratio is 30%, the designed oligonucleotide satisfies the selection criterion (ii) and is provided as a second oligonucleotide candidate group.

[0098] According to one embodiment of the present invention, the selection criteria include: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, middle or 3' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is greater than or equal to a predetermined ratio.

[0099] According to one embodiment of the present invention, the second group of oligonucleotide candidates includes modified oligonucleotides having increased target coverage, in which at least one base of the designed oligonucleotide is replaced with a degenerate base and / or a universal base, in order to increase target coverage for a mutant target nucleic acid sequence containing other nucleotide mutations in addition to the nucleotide mutation of interest.

[0100] According to an embodiment of the present invention, the method further includes, after step (d), a step of: d-1) aligning the oligonucleotides included in the second group of oligonucleotide candidates by assigning a rank to the oligonucleotides according to at least one of the following alignment criteria: (i) the position of the mismatch in the oligonucleotide as a result of the analysis of the presence or absence of matching in the step (d); the position of the mismatch is higher the closer it is to the 5' end, the 3' end, or the center; (ii) the number of mismatches in the oligonucleotide as a result of the matching analysis in step (d); the greater the number of mismatches, the higher the ranking; (iii) the Tm value of the oligonucleotide; the larger the Tm value, the higher the ranking; (iv) the GC content of a given region at the 5' end, the middle or the 3' end of the oligonucleotide; the higher the GC content, the higher the ranking; (v) the number of consecutive G bases contained in the oligonucleotide; the fewer the number of consecutive G bases, the higher the rank; (vi) the number or ratio of consecutive nucleotides involved in the formation of a homodimer when the oligonucleotide forms a homodimer; the smaller the number or ratio, the higher the rank; (vii) Free energy value for hairpin structure formation (ΔG value); the larger the free energy value, the higher the ranking. (viii) Length: The shorter the length, the higher the ranking.

[0101] In this embodiment, the second set of oligonucleotide candidates is screened and ranked to provide a third set of oligonucleotide candidates in step (e) described below.

[0102] According to this embodiment, the oligonucleotides contained in the second oligonucleotide candidate group are aligned to satisfy an alignment criterion taking into account at least one (specifically, alignment criterion (i)), specifically at least two, more specifically at least three, at least four, at least five, at least six, at least seven, or at least eight, most specifically eight, ranks.

[0103] The method for aligning oligonucleotides contained in the second oligonucleotide candidate group can be broadly classified into two types: According to a first approach, the at least two alignment criteria have different criticalities, and the oligonucleotides can be aligned so as to satisfy the alignment criterion with the highest criticality (eg, alignment criterion number (i)).

[0104] In the unlikely event that more than one oligonucleotide satisfies the most important alignment criterion, the oligonucleotides can be aligned so as to satisfy the next most important alignment criterion.

[0105] For example, if the importance of the alignment criteria is in the order of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii), and there are three oligonucleotides that satisfy alignment criterion (i), then these three oligonucleotides are aligned according to alignment criterion (ii). If there are three oligonucleotides that satisfy alignment criterion (ii), then the oligonucleotides are aligned so as to satisfy alignment criterion (iii).

[0106] According to the second method, different weights are assigned to the alignment criteria, and scores are assigned to the values ​​(or ranges of values) in each alignment criterion. Taking into account the ranking of these, a total score for each oligonucleotide can be obtained, and the oligonucleotides can be aligned taking into account this calculated total score.

[0107] According to one embodiment of the present invention, the at least two alignment criteria have different criticalities, and the method of the present invention further includes a step of aligning oligonucleotides included in a second group of oligonucleotide candidates so as to satisfy the at least two alignment criteria taking into account the criticalities.

[0108] (Step (e): Providing a third group of oligonucleotide candidates (150)) Finally, the method of the invention includes (e) selecting oligonucleotides from the second set of candidate oligonucleotides for the mutant target nucleic acid sequence to provide a third set of candidate oligonucleotides, which are used to detect a nucleotide variation of interest in the target nucleic acid sequence.

[0109] According to one embodiment of the present invention, the third group of oligonucleotide candidates in step (e) is selected by a method comprising the steps of: (e-1) aligning oligonucleotides having the same starting position as that of the second oligonucleotide candidate group by assigning a rank to the oligonucleotides according to at least one of the following alignment criteria: (e-2) selecting a top oligonucleotide from oligonucleotides having the same start position as that at which the aligned oligonucleotides are designed; wherein the alignment criteria include: (i) the position of the mismatch in the oligonucleotide as a result of the analysis of the presence or absence of matching in the step (d); the position of the mismatch is higher the closer it is to the 5' end, the 3' end, or the center; (ii) the number of mismatches in the oligonucleotide as a result of the matching analysis in step (d); the greater the number of mismatches, the higher the ranking; (iii) the Tm value of the oligonucleotide; the larger the Tm value, the higher the ranking; (iv) the GC content of a given region at the 5' end, the middle or the 3' end of the oligonucleotide; the higher the GC content, the higher the ranking; (v) the number of consecutive G bases contained in the oligonucleotide; the fewer the number of consecutive G bases, the higher the rank; (vi) when the oligonucleotide forms a homodimer, the number or ratio of consecutive nucleotides involved in the formation of the homodimer; the smaller the number or ratio, the higher the rank; (vii) free energy value for hairpin structure formation (ΔG value); the larger the free energy value, the higher the ranking; and (viii) Length: The shorter the length, the higher the ranking.

[0110] In this embodiment, the oligonucleotides in the second oligonucleotide candidate group having the same start position of the oligonucleotide design are ranked and aligned, and then the top oligonucleotide is selected from among them. Among the oligonucleotides in the second oligonucleotide candidate group, the oligonucleotides in the second oligonucleotide candidate group having the same start position of the oligonucleotide design should exist for each oligonucleotide design position in the mutation target nucleic acid sequence, and therefore the top oligonucleotide is selected for each design position.

[0111] The oligonucleotides to be ranked and aligned in step d-1) are all oligonucleotides contained in the second group of oligonucleotide candidates, whereas step d-1) differs from this embodiment in that the oligonucleotides to be ranked and aligned in this embodiment are oligonucleotides among the second group of oligonucleotide candidates that have the same starting position at which the oligonucleotides are designed.

[0112] According to this embodiment, in step (e-1), among the oligonucleotides included in the second oligonucleotide candidate group, oligonucleotides having the same starting position at which the oligonucleotides were designed are aligned so as to satisfy an alignment criterion taking into account a rank of at least one (specifically, alignment criterion (i)), specifically at least two, more specifically at least three, at least four, at least five, at least six, at least seven, or at least eight, most specifically eight.

[0113] There are two main methods for aligning oligonucleotides having the same starting position at which oligonucleotides are designed among the oligonucleotides included in the second oligonucleotide candidate group: According to a first approach, the minimum two alignment criteria have different criticalities, and the oligonucleotides can be aligned so as to satisfy the alignment criterion with the highest criticality (eg, alignment criterion number (i)).

[0114] In the unlikely event that more than one oligonucleotide satisfies the most important alignment criterion, the oligonucleotides can be aligned so as to satisfy the next most important alignment criterion.

[0115] For example, when the importance of the alignment criteria is in the order of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii), if there are three oligonucleotides that satisfy the alignment criterion (i), these three oligonucleotides are aligned according to the alignment criterion (ii). If there are three oligonucleotides that satisfy the alignment criterion (ii), the oligonucleotides are aligned so as to satisfy the alignment criterion (iii), and the top oligonucleotide can be selected as the third oligonucleotide candidate group.

[0116] According to the second method, different weights are assigned to the alignment criteria, and scores are assigned to the values ​​(or ranges of values) for each alignment criterion, and a total score for each oligonucleotide can be obtained by taking into account the ranking thereof. The oligonucleotides can be aligned taking into account this calculated total score, and the top ranked oligonucleotides can be selected as the third oligonucleotide candidate group based on the ranking based on the total score.

[0117] According to one embodiment of the present invention, the at least two alignment criteria have different criticalities, and the method of the present invention further includes a step of aligning oligonucleotides having the same starting position at which the oligonucleotides are designed, among the oligonucleotides included in the second oligonucleotide candidate group, so as to satisfy the at least two alignment criteria taking into account the criticalities.

[0118] Such a method can provide the third set of candidate oligonucleotides that can be used to detect nucleotide variations of interest in the target nucleic acid sequence.

[0119] According to an embodiment of the present invention, the first to third oligonucleotide candidate groups are first to third probe candidate groups, respectively, and the method further comprises, after step (e), the following step: (f) designing primers to amplify a predetermined region in the mutant target nucleic acid sequence that contains the nucleotide variation of interest, to provide a set of candidate primers for the mutant target nucleic acid sequence; and (g) combining said third set of candidate probes and candidate primers to provide a probe and primer combination;

[0120] This embodiment illustrates a process of providing first to third candidate probe groups by the above-mentioned method, and then providing a candidate primer group, and combining the third candidate probe group and the candidate primer group to provide a combination of probe and primer.

[0121] (Step (f): Providing a group of candidate primers for the mutated target nucleic acid sequence) In step (f), primers are designed to amplify a predetermined region within the mutant target nucleic acid sequence that contains the nucleotide variation of interest.

[0122] The range in which the primers are designed, i.e., the predetermined region containing the nucleotide of interest to be amplified using the primers, may be different from the region in which the probes included in the first group of candidate probes are designed (i.e., the probe design range) in step (c), but the primer design range may be set to amplify a region containing the predetermined region in which the probes are designed.

[0123] The range for designing the primer, i.e., the predetermined region containing the nucleotide of interest for amplification using the primer, may be selected within a region from 500 bp (specifically, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp) upstream of the start position of the nucleotide variation of interest to 500 bp (specifically, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp) downstream of the end position of the nucleotide variation of interest.

[0124] According to an embodiment of the present invention, the primers designed in step (f) are designed to satisfy at least one of the following conditions: (i) a Tm value of 40 to 70°C; (ii) a length of 15 to 50 bp nucleotides, and (iii) Exclusion of 5 or more G-run sequences.

[0125] Among the design conditions, the Tm value is, for example, 40 to 70° C., 50 to 70° C., 55 to 70° C., 45 to 65° C., 50 to 65° C., 55 to 65° C., 45 to 60° C., or 50 to 65° C. Specifically, among the design conditions, the Tm value is 40 to 70° C., 45 to 65° C., 50 to 65° C., 50 to 60° C., 55 to 65° C., or 55 to 60° C.

[0126] The length of the above design conditions is, for example, 15 to 60 nucleotides, 15 to 50 nucleotides, 15 to 45 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 15 to 30 nucleotides, 15 to 25 nucleotides, 18 to 45 nucleotides, 18 to 40 nucleotides, 18 to 35 nucleotides, 18 to 30 nucleotides, or 18 to 25 nucleotides. Specifically, the length of the design conditions is, for example, 15 to 40 nucleotides, 16 to 40 nucleotides, 17 to 40 nucleotides, 18 to 40 nucleotides, 15 to 35 nucleotides, 16 to 35 nucleotides, 17 to 35 nucleotides, 18 to 35 nucleotides, 15 to 30 nucleotides, 16 to 30 nucleotides, 17 to 30 nucleotides, 18 to 30 nucleotides, 18 to 25 nucleotides, or 17 to 25 nucleotides.

[0127] Among the design conditions, the G-run sequence is, for example, excluding 5 or more G-run sequences.

[0128] When the primer is a DPO primer developed by the applicant (see U.S. Pat. No. 8,092,997), the description regarding the Tm and length of the DPO primer disclosed in the patent document may be presented as the design conditions.

[0129] More specifically, the primer design conditions include at least two, more specifically at least three of the above-mentioned conditions.

[0130] According to an embodiment of the present invention, the method further includes, after step (f), the following step: (f-1) aligning primers having the same starting position among the primers included in the primer candidate group by assigning a rank to the primers according to at least one of the following alignment criteria; and (f-2) selecting a top primer from among primers having the same start position as the aligned primers; wherein the alignment criteria include: (i) the number or ratio of degenerate bases and / or universal bases introduced into the primer; the smaller the number or ratio, the higher the ranking; (ii) the number of primer patterns generated by the introduction of degenerate bases; the fewer the number of patterns, the higher the ranking; (iii)(A) n , (T) n or (C) n the number of mononucleotide run sequences; the fewer the number of such sequences, the higher the ranking; (iv) when the primer forms a homodimer, the number or ratio of consecutive nucleotides involved in the formation of the homodimer; the smaller the number or ratio, the higher the rank; (v) free energy value for hairpin structure formation (ΔG value); the larger the free energy value, the higher the ranking; (vi) Tm value; the larger the Tm value, the higher the ranking; (vii) GC content; the higher the GC content, the higher the ranking; and (viii) Length; the shorter the length, the higher the ranking.

[0131] In this embodiment, the primers in the candidate group of primers having the same design start position are ranked and aligned, and then a top primer is selected from among them. Among the primers in the candidate group of primers, primers having the same design start position should exist for each primer design position in the mutation target nucleic acid sequence, and therefore a top primer is selected for each design position.

[0132] According to this embodiment, in step (f-1), among the primers included in the group of candidate primers, primers having the same starting position at which the primers are designed are aligned so as to satisfy an alignment criterion taking into account the rank of at least one (specifically, alignment criterion (i)), specifically at least two, more specifically at least three, at least four, at least five, at least six, at least seven, or at least eight, most specifically eight.

[0133] There are two main methods for aligning primers having the same designed start position among the primers included in the primer candidate group: According to a first method, the at least two alignment criteria have different criticalities, and the primers can be aligned to satisfy the alignment criterion with the highest criticality (eg, alignment criterion number (i)).

[0134] In the unlikely event that multiple primers satisfy the most important alignment criterion, primers can be aligned to satisfy the next most important alignment criterion.

[0135] For example, when the importance of the alignment criteria is in the order of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii), if there are three primers that satisfy the alignment criterion (i), these three primers are aligned according to the alignment criterion (ii). If there are three primers that satisfy the alignment criterion (ii), the primers are aligned so as to satisfy the alignment criterion (iii), and the top oligonucleotide can be selected as a group of candidate primers.

[0136] According to the second method, different weights are assigned to the alignment criteria, and scores are assigned to the values ​​(or ranges of values) for each alignment criterion. Taking into account the ranking of the scores, a total score for each primer can be obtained. The primers can be aligned taking into account this calculated total score, and the top primers can be selected as a group of candidate primers based on the ranking of the total scores.

[0137] According to one embodiment of the present invention, the at least two alignment criteria have different criticalities, and the method of the present invention further includes a step of aligning primers having the same designed start position among the primers included in the candidate primer group so as to satisfy the at least two alignment criteria taking into account the criticalities.

[0138] (Step (g): Providing a combination of probes and primers) The third set of candidate probes and the third set of candidate primers are combined to provide a probe and primer combination.

[0139] If detection of a nucleotide variation of interest in a target nucleic acid sequence is performed using an appropriate combination of a probe and a primer, such as in the PTOCE, VD-PTOCE and TaqMan probe methods, not only are the excellent properties of the probe and primer themselves important, but the partnership between the probe and the primer in detecting the nucleotide variation of interest is also important.

[0140] For example, the primers must be located upstream and downstream of the selected probe and capable of generating an amplicon of an appropriate size (specifically, 100 to 1000, more specifically, 200 to 800, even more specifically, 300 to 700, particularly specifically, 300 to 500, and especially specifically, 300 to 400 nucleotides).

[0141] In addition, it is preferable that the primer does not interfere with the probe. A typical phenomenon of such interference is dimer formation. Even if a primer and a probe have excellent properties, if the primer forms a heterodimer with the probe, the primer cannot be a suitable primer.

[0142] The primer preferably has a lower Tm value than the probe. For example, the Tm value of the primer relative to the probe is preferably in the range of [55° C. to (Tm of the probe minus 10° C.)° C.].

[0143] According to an embodiment of the present invention, a top probe is selected from the probes included in the third probe candidate group, and a primer that matches the top probe is selected from the primers included in the primer candidate group. The term "match" means that the selected probe has at least one of the following characteristics: the primer and the probe do not form a heterodimer, form an amplicon of a desired size, and have a Tm value of [55°C to (probe Tm minus 10°C)°C].

[0144] For example, when a third group of candidate probes including probes selected by the present invention and a group of candidate primers including primers are provided, a top probe is selected from the third group of candidate probes, and a top primer that matches this probe is selected from the group of candidate primers. If there is no primer that matches the top probe in the group of candidate primers, a primer that matches the next top probe is selected from the group of candidate primers, and the combination of this next top probe and primer is used as an oligonucleotide for detecting a nucleotide mutation of interest in a target nucleic acid sequence.

[0145] In the present invention, a primer may be selected based on the following suitability in relation to a probe: First, if the ratio of consecutive nucleotides involved in the formation of a heterodimer is 65% or less (specifically 60% or less, more specifically 55% or less, even more specifically 50% or less, particularly specifically 40% or less) when the primer forms a heterodimer with the probe, the primer may be judged to satisfy the suitability. Second, if the primer generates an amplicon of an appropriate size (specifically 100 to 1000, more specifically 200 to 800, even more specifically 300 to 700, particularly specifically 300 to 500, particularly specifically 300 to 400 nucleotides), the primer may be judged to satisfy the suitability. Third, if the Tm value of the primer is [55°C to (Tm of the probe minus 10°C)°C], the primer may be judged to satisfy the suitability.

[0146] As used herein, the term "providing" in reference to an oligonucleotide includes providing the sequence information of the oligonucleotide as well as manufacturing the oligonucleotide material.

[0147] According to an embodiment of the present invention, the probe is a tagging probe further comprising a tagging site comprising a non-hybridizing nucleotide sequence in the mutant target nucleic acid sequence. The probe may be a tagging probe further comprising a tagging site.

[0148] Representative examples of such tagging probes may be PTO used in the PTOCE method, or PTO-NV used in the VD-PTOCE method.

[0149] The tagging probe is designed so that the tagging site satisfies design conditions for the tagging site in addition to the design conditions for the probe.

[0150] According to one embodiment of the present invention, the tagging portion of the tagging probe is designed to satisfy at least one of the following conditions: (i) a length of the tagging portion of 6 to 30 nucleotides, (ii) a mismatching sequence of 30% or more relative to the length of the tagging portion, and (iii) a mismatching sequence of 40% or more relative to the length of the 3'-terminal portion of the tagging portion.

[0151] Specifically, the length of the tagging portion is 6 to 20 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 12 to 30 nucleotides, or 12 to 20 nucleotides.

[0152] The tagging portion is sufficiently non-complementary to the specific region of the mutant target nucleic acid sequence to which the tagging probe is hybridized, so that it does not hybridize to the specific region under conditions in which the tagging probe (targeting portion) is hybridized. Specifically, the tagging portion contains a mismatch sequence of 30% or more, 40% or more, or 50% or more relative to its length. Specifically, the 3'-terminal portion of the tagging portion contains a mismatch sequence of 40% or more, or 50% or more relative to its length.

[0153] The length of the 3'-terminal portion of the tagging portion may be, for example, 3 to 8 nucleotides, 3 to 7 nucleotides, 3 to 6 nucleotides, 3 to 5 nucleotides, 3 to 4 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides, 4 to 6 nucleotides, or 4 to 5 nucleotides.

[0154] [Recording medium, device and program] According to another aspect of the present invention, there is provided a computer readable recording medium comprising instructions for implementing a processor to perform a method for providing oligonucleotides for use in detecting a nucleotide variation of interest in a target nucleic acid sequence, the method comprising the steps of: (a) inputting information about a wild-type target nucleic acid sequence and a nucleotide variation of interest; the wild-type target nucleic acid sequence is a target nucleic acid sequence that does not include a nucleotide variation of interest, the information comprising (i) the wild-type target nucleic acid sequence, (ii) a position of the nucleotide variation of interest occurring in the wild-type target nucleic acid sequence, and (iii) information about a wild-type base and a mutant base at the position of the nucleotide variation of interest, the position of the nucleotide variation of interest being designated as a start position and an end position; and (b) providing the wild-type target nucleic acid sequence and a mutant target nucleic acid sequence using the input information; the mutant target nucleic acid sequence is a target nucleic acid sequence that includes a nucleotide variation of interest, the wild-type and mutant target nucleic acid sequences being designated as a forward and reverse wild-type and mutant bases. (c) designing oligonucleotides for detecting the nucleotide variation of interest in a predetermined region containing the nucleotide variation of interest in the mutant target nucleic acid sequence to provide a first group of oligonucleotide candidates for the mutant target nucleic acid sequence; (d) analyzing whether the oligonucleotides in the first group of oligonucleotide candidates for the mutant target nucleic acid sequence match the wild-type target nucleic acid sequence, and providing oligonucleotides that satisfy the following selection criteria as a second group of oligonucleotide candidates for the mutant target nucleic acid sequence; the selection criteria include: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, middle or 3' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is equal to or greater than a predetermined ratio; and (e) selecting oligonucleotides from the second group of oligonucleotide candidates for the mutant target nucleic acid sequence to provide a third group of oligonucleotide candidates;The third set of candidate oligonucleotides is used to detect a nucleotide variation of interest in the target nucleic acid sequence;

[0155] According to yet another aspect of the present invention, there is provided a computer program stored on a computer-readable recording medium embodying a processor for executing a method for providing an oligonucleotide for use in detecting a nucleotide variation of interest in a target nucleic acid sequence, the method comprising the steps of: (a) inputting information about a wild-type target nucleic acid sequence and a nucleotide variation of interest; the wild-type target nucleic acid sequence is a target nucleic acid sequence that does not include a nucleotide variation of interest, the information including (i) a wild-type target nucleic acid sequence, (ii) a position of the nucleotide variation of interest occurring in the wild-type target nucleic acid sequence, and (iii) information about a wild-type base and a mutant base at the position of the nucleotide variation of interest, the position of the nucleotide variation of interest being indicated as a start position and an end position; (b) providing the wild-type target nucleic acid sequence and a mutant target nucleic acid sequence using the input information; the mutant target nucleic acid sequence is a target nucleic acid sequence that includes a nucleotide variation of interest, the wild-type and mutant target nucleic acid sequences being indicated as a forward and reverse position. (c) designing oligonucleotides for detecting the nucleotide variation of interest in a predetermined region containing the nucleotide variation of interest in the mutant target nucleic acid sequence to provide a first set of oligonucleotide candidates for the mutant target nucleic acid sequence; (d) analyzing whether or not oligonucleotides included in the first set of oligonucleotide candidates for the mutant target nucleic acid sequence match with the wild-type target nucleic acid sequence, and providing oligonucleotides that satisfy the following selection criteria as a second set of oligonucleotide candidates for the mutant target nucleic acid sequence; the selection criteria include: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, middle or 3' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is equal to or greater than a predetermined ratio; and (e) selecting oligonucleotides from the second set of oligonucleotide candidates for the mutant target nucleic acid sequence to provide a third set of oligonucleotide candidates;The third set of candidate oligonucleotides is used to detect a nucleotide variation of interest in the target nucleic acid sequence;

[0156] According to another aspect of the present invention, there is provided an apparatus for providing oligonucleotides used to detect a nucleotide variation of interest in a target nucleic acid sequence, comprising: (a) a computer processor; and (b) a computer readable recording medium of the present invention coupled to the computer processor.

[0157] The recording medium, device, and computer program of the present invention enable the above-mentioned method of the present invention to be implemented by a computer, and description of the contents common to them will be omitted in order to avoid excessive complexity of this specification due to repetition.

[0158] The program instructions, when executed by a processor, cause the processor to carry out the method of the present invention described above. The program instructions for carrying out the method for providing an oligonucleotide for detecting a nucleotide variation of interest in a target nucleic acid sequence can include the following instructions: (i) instructions for inputting information on a wild-type target nucleic acid sequence and a nucleotide variation of interest; (ii) instructions for using the input information to provide the wild-type target nucleic acid sequence and a mutant target nucleic acid sequence; (iii) instructions for designing an oligonucleotide for detecting the nucleotide variation of interest in a predetermined region containing the nucleotide variation of interest in the mutant target nucleic acid sequence, and providing a first oligonucleotide candidate group for the mutant target nucleic acid sequence; (iv) instructions for analyzing whether an oligonucleotide included in the first oligonucleotide candidate group for the mutant target nucleic acid sequence matches with the wild-type target nucleic acid sequence, and providing an oligonucleotide that meets a predetermined selection criterion as a second oligonucleotide candidate group for the mutant target nucleic acid sequence; (v) instructions for selecting an oligonucleotide from the second oligonucleotide candidate group for the mutant target nucleic acid sequence to provide (e.g., display on an output device) a third oligonucleotide candidate group.

[0159] The methods of the present invention are carried out on a processor, which may be a stand alone computer, a network attached computer or a processor located in a data acquisition device such as a real-time PCR device.

[0160] The computer-readable recording medium includes various storage media known in the art, such as, but not limited to, CD-R, CD-ROM, DVD, flash memory, floppy disk, hard drive, portable HDD, USB, magnetic tape, MINIDISC, non-volatile memory card, EEPROM, optical disk, optical storage medium, RAM, ROM, system memory, and web server.

[0161] The oligonucleotides used to detect the nucleotide variation of interest in the target nucleic acid sequence may be provided in various ways. For example, the oligonucleotides may be provided to a separate system, such as a desktop computer system, by network connection (e.g., LAN, VPN, Internet, and Intranet) or direct connection (e.g., USB or other direct wired or wireless connection), or may be provided on portable media, such as CD, DVD, floppy disk, and portable HDD. Similarly, the oligonucleotides may be provided to a server system through network connection (e.g., LAN, VPN, Internet, Intranet, and wireless communication network) to a client, such as a laptop or desktop computer system.

[0162] Instructions for implementing a processor to carry out the present invention may be included in a logic system. The instructions may be provided on a software storage medium (e.g., portable HDD, USB, floppy disk, CD, and DVD), downloadable, or stored in a memory module (e.g., a hard drive or other memory such as local or embedded RAM or ROM). Computer code for carrying out the present invention may be implemented in a variety of coding languages, such as C, C++, Java, Visual Basic, VBScript, JavaScript, Perl, and XML. Also, a variety of languages ​​and protocols may be used for the external and internal storage and transfer of signals and instructions according to the present invention.

[0163] A computer processor may be constructed such that one processor is capable of performing all of the above-mentioned functions, or a processor unit may be constructed such that multiple processors perform each of the functions. Effect of the Invention

[0164] The features and advantages of the present invention can be summarized as follows: (a) In order to design an oligonucleotide for detecting a nucleotide mutation of interest, such as a substitution, inversion, insertion, deletion, repeat, or a combination thereof, the conventional method has a problem that it is necessary to consider the type of nucleotide mutation, the magnitude of the mutation, and whether the target to be detected is a wild-type and / or mutant sequence, and develop various design rules and modules according to these considerations. In addition, the conventional method has a problem that when an oligonucleotide is designed without considering the content of the sequence, it is not possible to distinguish between a wild-type target nucleic acid sequence and a mutant target nucleic acid sequence, while the design rules become complicated when the content of the sequence is considered.

[0165] (b) In response to this, the present invention inputs information regarding a wild-type target nucleic acid sequence and a nucleotide mutation of interest, provides wild-type and mutant target nucleic acid sequences using the input information, designs oligonucleotides for the mutant target nucleic acid sequence, analyzes whether the designed oligonucleotides match the wild-type target nucleic acid sequence, and selects and provides oligonucleotides that satisfy a predetermined selection criterion, thereby effectively providing oligonucleotides used to detect a nucleotide mutation of interest in a target nucleic acid sequence.

[0166] (c) The present invention provides oligonucleotides capable of detecting nucleotide variations based on comprehensive design rules, without the need to develop detailed design rules and modules taking into account the type of nucleotide variation, the magnitude of the variation, whether the target to be detected is a wild-type and / or mutant sequence, and sequence content, etc. [Brief description of the drawings]

[0167] [Figure 1] 1 is a flow chart showing a process for providing oligonucleotides for use in detecting a nucleotide variation of interest in a target nucleic acid sequence according to one embodiment of the present invention. [Diagram 2]FIG. 2 illustrates a user interface (UI) for inputting information about a wild-type target nucleic acid sequence and a nucleotide mutation of interest according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0168] The present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. EXAMPLES

[0169] Example 1: Provision of oligonucleotides used to detect a mutation (T112C, T to C substitution) in the human ApoE gene A program (HMOD) was implemented to provide oligonucleotides used to detect nucleotide variations of interest in target nucleic acid sequences, providing oligonucleotides used to detect a substitution mutation (T112C) in the human gene ApoE.

[0170] (Information on the wild-type target nucleic acid sequence of the ApoE gene and the substitution mutation (T112C)) In the User Interface (UI) window of the HMOD program (FIG. 2), information on the wild-type target nucleic acid sequence of the ApoE gene and the substitution mutation (T112C) for running the HMOD program was entered. Specifically, in the UI of FIG. 2, ApoE was entered in Gene Name, ApoE in Gene Abb., T112C in Mutation Name, T112C in Mutation Abb., Mutant in Analyte Type, Substitution in Mutation Type, 501 in Start and 501 in End of Mutation Position Info, T in Wild and C in Mutant of Sequence Variants, NC_018930.2 in Acc.ID, and the sequence shown in Table 1 below was entered in Sequence, and the HMOD program was run. The start and end positions of the nucleotide mutation of interest entered in the Start and End of the Mutation Position Info represent the positions in the wild-type target nucleic acid sequence shown in Table 1 below.

[0171] [Table 1]

[0172] The base T written in bold in Table 1 above is at the 501st position in the sequence, and the substitution mutation T112C represents a mutation in which the base T at the 501st position of the sequence is replaced with a base C.

[0173] The HMOD program was run in the following order: (Providing wild-type and mutant target nucleic acid sequences) Oligonucleotides were designed using the input information to create forward and reverse wild-type and mutant target nucleic acid sequences to be used as templates to verify that oligonucleotides designed against mutant target nucleic acid sequences can be distinguished and detected from the wild-type target nucleic acid sequence.

[0174] The forward and reverse wild-type and mutant target nucleic acid sequences are summarized in Table 2 below:

[0175] [Table 2] JPEG0007636546000003.jpg181169 JPEG0007636546000004.jpg131169

[0176] (Providing the first set of probe candidates) The probes used to detect the nucleotide mutation of interest in the mutant target nucleic acid sequence were designed to meet the following criteria: (i) length of 10-60 nucleotides, (ii) Tm value of 50-85°C, (iii) exclusion of 3 or more G-run sequences, and (iv) GC content of 40% or more at the 5' end.

[0177] Specifically, probes were designed according to length (predetermined length: 17-35mer) at each position from 499, which is 2bp upstream based on the start position 501 of the nucleotide mutation of interest (base C) in the provided forward mutation target nucleic acid sequence, to 501, which is the end position of the nucleotide mutation of interest, and provided as a first probe candidate group. In addition, probes were designed according to length (predetermined length: 17-35mer) at each position from 637, which is 2bp upstream based on the start position 639 of the nucleotide mutation of interest (base G) in the provided reverse mutation target nucleic acid sequence, to 639, which is the end position of the nucleotide mutation of interest, and provided as a first probe candidate group. There were 114 probes designed for the forward and reverse mutation target nucleic acid sequences, and among them, forward probes having 19 and 20mers are summarized in Table 3 below.

[0178] [Table 3]

[0179] (Providing the second set of probe candidates) The presence or absence of a match between the wild-type target nucleic acid sequence and the probes included in the first candidate group of probes designed for the mutant target nucleic acid sequence was analyzed, and the probes that satisfied the following selection criteria were provided as a second candidate group of probes for the mutant target nucleic acid sequence: (i) there is one mismatch between the wild-type target nucleic acid sequence and the probe at the 5' end; or (ii) the minimum number of mismatches between the wild-type target nucleic acid sequence and the probe is 30% or more for the probe sequence.

[0180] Among the probes included in the first probe candidate group, 10 probes met the above selection criteria, and only 5 of them are summarized in Table 4 below.

[0181] [Table 4]

[0182] The 10 probes were aligned by assigning ranks according to the following alignment criteria: (i) the position of the minimum mismatch in the probe as a result of the analysis of the presence or absence of matching; the closer the position of the minimum mismatch is to the 5' end of the probe, the higher the rank; (ii) the number of minimum mismatches in the probe as a result of the analysis of the presence or absence of matching; the higher the number of minimum mismatches, the higher the rank; (iii) the Tm value of the probe; the higher the Tm value, the higher the rank; (iv) the GC content of a specific region at the 5' end of the probe; the higher the GC content, the higher the rank; (v) the number of consecutive G bases contained in the probe; the lower the number of consecutive G bases, the higher the rank; (vi) when the probe forms a homodimer, the number or ratio of consecutive nucleotides involved in the formation of the homodimer; the lower the number or ratio, the higher the rank; (vii) the free energy value of hairpin structure formation (ΔG value); the higher the free energy value, the higher the rank; and (viii) the length: the shorter the length, the higher the rank.

[0183] (Providing the third probe candidate group) Among the probes included in the second probe candidate group, the probes with the same starting position at which the probe was designed were ranked highest according to the alignment criteria and provided as a third probe candidate group. Two third probe candidate groups were selected from the 10 second probe candidate groups, and the results are summarized in Table 5 below:

[0184] [Table 5]

[0185] (Providing primer candidates) Primers were designed to amplify a predetermined region containing the nucleotide mutation of interest in the mutant target nucleic acid sequence, and a group of candidate primers for the mutant target nucleic acid sequence was provided. Specifically, the design range of the primers designed to amplify the predetermined region containing the nucleotide mutation of interest was selected from a region from 300 bp upstream of the start position of the nucleotide mutation of interest to 300 bp downstream of the end position of the nucleotide mutation of interest. The primers were designed to satisfy the following conditions: (i) a Tm value of 40 to 70°C, (ii) a length of 15 to 50 bp nucleotides, and (iii) excluding 5 or more G-run sequences.

[0186] Among the primers included in the group of candidate primers, primers having the same start position for designing the primer were ranked according to the following ranking criteria, and the top oligonucleotides were selected: (i) the number or ratio of degenerate bases and / or universal bases introduced into the primer; the smaller the number or ratio, the higher the ranking; (ii) the number of primer patterns generated by the introduction of degenerate bases; the smaller the number of patterns, the higher the ranking; (iii) (A) n , (T) n or (C) n(iv) the number of mononucleotide run sequences; the fewer the number of sequences, the higher the ranking; (v) the number or ratio of consecutive nucleotides involved in the formation of a homodimer when the primer forms a homodimer; the smaller the number or ratio, the higher the ranking; (v) the free energy value for the formation of a hairpin structure (ΔG value); the larger the free energy value, the higher the ranking; (vi) the Tm value; the larger the Tm value, the higher the ranking; (vii) the GC content; the higher the GC content, the higher the ranking; and (viii) the length; the shorter the length, the higher the ranking.

[0187] As a result, 12,289 primers were designed as candidate primers, and 103 top primers were selected from among the primers with the same starting position for the primer design. Examples of the selected top primers are summarized in Table 6 below:

[0188] [Table 6]

[0189] (Probe and primer combinations) The selected third probe candidate group and primer candidate group were combined to provide combinations of probes and primers. Specifically, the selected two probes and 103 primers were combined to generate an amplicon of 300 to 350 bp, and 10 combinations were generated that would not generate dimers between the probes and primers that could be combined. Examples of three of these combinations are summarized in Table 7 below:

[0190] [Table 7]

[0191] Example 2: Provision of oligonucleotides used to detect a mutation (FS89, insertion of base C) in the human gene HBB The same program (HMOD) as in Example 1 was carried out to provide oligonucleotides used to detect insertion mutations in the human gene HBB.

[0192] (Input information on the wild-type target nucleic acid sequence and insertion mutation of the HBB gene) In the User Interface (UI) window of the HMOD program (FIG. 2), information on the wild-type target nucleic acid sequence of the HBB gene and the insertion mutation for running the HMOD program was entered. Specifically, in the UI of FIG. 2, HBB was entered in Gene Name, HBB in Gene Abb., FS89 in Mutation Name, FS89 in Mutation Abb., Mutant in Analyte Type, insertion in Mutation Type, 396 in Start and 396 in End of Mutation Position Info, C in Wild and CC in Mutant in Sequence Variants, NG_046672.1 in Acc.ID, and the sequence shown in Table 8 below was entered in Sequence, and the HMOD program was run. The start and end positions of the nucleotide mutation of interest entered in the Start and End of the Mutation Position Info represent the positions in the wild-type target nucleic acid sequence shown in Table 8 below.

[0193] [Table 8]

[0194] In Table 8 above, the base C written in bold is at position 396 in the sequence, and the insertion mutation FS89 represents a mutation in which the base C is inserted between positions 396 and 397 of the sequence.

[0195] The HMOD program was run in the following order: (Providing wild-type and mutant target nucleic acid sequences) The forward and reverse wild-type and mutant target nucleic acid sequences were prepared in the same manner as in Example 1, and the results are summarized in Table 9 below:

[0196] [Table 9] JPEG0007636546000012.jpg175169

[0197] (Providing the first set of probe candidates) Using the same method as in Example 1, probes were designed for each length (predetermined length: 17-35mer) at each position from 394, which is located 2bp upstream based on the start position 396 of the nucleotide mutation of interest (base C) in the provided forward mutation target nucleic acid sequence, to 397, which is the end position of the nucleotide mutation of interest, and provided as a first probe candidate group. In addition, probes were designed for each length (predetermined length: 17-35mer) at each position from 310, which is located 2bp upstream based on the start position 312 of the nucleotide mutation of interest (base G) in the provided reverse mutation target nucleic acid sequence, to 313, which is the end position of the nucleotide mutation of interest, and provided as a first probe candidate group. 152 probes were designed for the forward and reverse mutation target nucleic acid sequences, and among them, forward probes having 19 and 20mers are summarized in Table 10 below.

[0198] [Table 10]

[0199] (Providing the second set of probe candidates) A second group of probe candidates for the mutant target nucleic acid sequence was provided using the same method and screening criteria as in Example 1.

[0200] Of the probes included in the first probe candidate group, 43 probes met the above selection criteria, and only 5 of them are summarized in Table 11 below.

[0201] [Table 11]

[0202] The 43 probes were then ranked and aligned according to the alignment criteria of Example 1.

[0203] (Providing the third probe candidate group) In the same manner as in Example 1, the top probes among the probes included in the second probe candidate group, which have the same starting position at which the probes were designed, were provided as a third probe candidate group. Five third probe candidate groups were selected from the 43 second probe candidate groups, and the results are summarized in Table 12 below:

[0204] [Table 12]

[0205] (Providing primer candidates) A group of candidate primers was provided according to the same method, design conditions and alignment criteria as in Example 1.

[0206] As a result, 12,287 primers were designed as a group of candidate primers, and 190 top primers were selected from among the designed primers having the same starting position. Examples of the selected top primers are summarized in Table 13 below:

[0207] [Table 13]

[0208] (Probe and primer combinations) The selected third probe candidate group and primer candidate group were combined to provide combinations of probes and primers in the same manner as in Example 1. Specifically, the selected five probes and 190 primers were combined to generate an amplicon of 300 to 350 bp, and 10 combinations were generated that did not generate dimers between the probes and primers that could be combined. Examples of three of these combinations are summarized in Table 14 below:

[0209] [Table 14]

[0210] Although the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A computer-implemented method for providing oligonucleotides for use in detecting a nucleotide variation of interest in a target nucleic acid sequence comprising the steps of: (a) inputting information about a wild-type target nucleic acid sequence and a nucleotide variation of interest; the wild-type target nucleic acid sequence is a target nucleic acid sequence that does not include a nucleotide variation of interest, the information including (i) the wild-type target nucleic acid sequence, (ii) the position of the nucleotide variation of interest occurring in the wild-type target nucleic acid sequence, and (iii) information about a wild-type base and a mutant base at the position of the nucleotide variation of interest, the positions of the nucleotide variation of interest being designated as a start position and an end position; (b) providing the wild-type target nucleic acid sequence and the mutant target nucleic acid sequence using the input information; the mutant target nucleic acid sequence is a target nucleic acid sequence containing a nucleotide mutation of interest, and the wild-type and mutant target nucleic acid sequences include forward and reverse wild-type and mutant target nucleic acid sequences, respectively; (c) designing oligonucleotides for detecting the nucleotide variation of interest in a predetermined region containing the nucleotide variation of interest in the mutant target nucleic acid sequence to provide a first group of oligonucleotide candidates for the mutant target nucleic acid sequence; the oligonucleotides are designed by length from a predetermined position upstream to an end position based on the start position of the nucleotide variation of interest in the mutant target nucleic acid sequence, the predetermined position being selected from 1 to 50 bp, and the length being 10 to 45 nucleotides; (d) analyzing whether or not oligonucleotides included in the first group of oligonucleotide candidates for the mutant target nucleic acid sequence match with the wild-type target nucleic acid sequence, and providing oligonucleotides that satisfy the following selection criteria as a second group of oligonucleotide candidates for the mutant target nucleic acid sequence; the selection criteria include: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is 20% or more; and (e) selecting oligonucleotides from the second set of oligonucleotide candidates for the mutant target nucleic acid sequence to provide a third set of oligonucleotide candidates; the third set of oligonucleotide candidates is used to detect a nucleotide variation of interest in the target nucleic acid sequence.

2. 2. The method of claim 1, wherein the nucleotide variation of interest is a substitution, an inversion, an insertion, a deletion, a duplication, or a combination thereof.

3. The method of claim 1 , wherein the oligonucleotide is a probe and / or a primer.

4. 2. The method of claim 1, wherein the wild-type target nucleic acid sequence of step (a) has a predetermined length that includes the position of the nucleotide variation of interest.

5. 2. The method of claim 1, wherein the oligonucleotide of step (c) is designed to have a sequence that matches or is complementary to a predetermined region containing the nucleotide variation of interest in the mutant target nucleic acid sequence.

6. The method according to claim 1, wherein the oligonucleotide designed in step (c) is designed to satisfy at least one of the following conditions: (i) a length of 10 to 60 nucleotides; (ii) a Tm value of 50 to 85°C; (iii) 3 or more G-run sequence exclusions; and (iv) The GC content at the 5′ end site is 40% or more.

7. 2. The method of claim 1, wherein the mismatch in step (d)(i) is a minimal mismatch between the wild-type target nucleic acid sequence and a predetermined region at the 5' end, middle or 3' end of the oligonucleotide, and the mismatch in step (d)(ii) is a minimal mismatch between the wild-type target nucleic acid sequence and the oligonucleotide.

8. The method of claim 1, further comprising, after step (d), a step of: (d-1) aligning the oligonucleotides included in the second group of oligonucleotide candidates by assigning a rank to the oligonucleotides according to at least one of the following alignment criteria: (i) the position of the mismatch in the oligonucleotide as a result of the analysis of the presence or absence of matching in the step (d); the position of the mismatch is higher the closer to the 5' end, 3' end, or center; (ii) the number of mismatches in the oligonucleotide as a result of the matching analysis in step (d); the greater the number of mismatches, the higher the ranking; (iii) the Tm value of the oligonucleotide; the larger the Tm value, the higher the ranking; (iv) GC content of a predetermined region at the 5' end, the middle or the 3' end of the oligonucleotide The higher the GC content, the higher the ranking. (v) the number of consecutive G bases contained in the oligonucleotide; the smaller the number of consecutive G bases, the higher the rank; (vi) the number or ratio of consecutive nucleotides involved in the formation of a homodimer when the oligonucleotide forms a homodimer; the smaller the number or ratio, the higher the rank; (vii) free energy value for hairpin structure formation (ΔG value); the larger the free energy value, the higher the ranking; (viii) Length: the shorter the length, the higher the ranking.

9. The method of claim 1, wherein the third group of oligonucleotide candidates in step (e) is selected by a method comprising the steps of: (e-1) aligning oligonucleotides having the same starting position for designing the oligonucleotide among the second oligonucleotide candidate group by assigning a rank to the oligonucleotides according to at least one of the following alignment criteria; and (e-2) selecting a top oligonucleotide from among oligonucleotides having the same starting position as that at which the aligned oligonucleotides are designed; wherein the alignment criteria include: (i) the position of the mismatch in the oligonucleotide as a result of the analysis of the presence or absence of matching in the step (d); the position of the mismatch is higher the closer to the 5' end, 3' end, or center; (ii) the number of mismatches in the oligonucleotide as a result of the matching analysis in step (d); the greater the number of mismatches, the higher the ranking; (iii) the Tm value of the oligonucleotide; the larger the Tm value, the higher the ranking; (iv) the GC content of a given region at the 5' end, the middle or the 3' end of the oligonucleotide; the higher the GC content, the higher the ranking; (v) the number of consecutive G bases contained in the oligonucleotide; the fewer the number of consecutive G bases, the higher the ranking; (vi) when the oligonucleotide forms a homodimer, the number or ratio of consecutive nucleotides involved in the formation of the homodimer; the smaller the number or ratio, the higher the rank; (vii) free energy value for hairpin structure formation (ΔG value); the larger the free energy value, the higher the ranking; and (viii) Length: the shorter the length, the higher the ranking.

10. The method according to claim 1, wherein the first to third oligonucleotide candidate groups are first to third probe candidate groups, respectively, and the method further comprises, after step (e), the following steps: (f) designing primers to amplify a predetermined region in the mutant target nucleic acid sequence that contains the nucleotide variation of interest, to provide a set of candidate primers for the mutant target nucleic acid sequence; and (g) combining the third set of candidate probes and the third set of candidate primers to provide a probe and primer combination.

11. The method of claim 10, wherein in step (f), the primers are designed to satisfy at least one of the following conditions: (i) a Tm value of 40 to 70° C.; (ii) a length of 15 to 50 bp nucleotides, and (iii) Exclusion of 5 or more G-run sequences.

12. 11. The method of claim 10, further comprising, after step (f), the step of: (f-1) aligning primers having the same starting position of the primer design among the primers included in the primer candidate group by assigning a rank to the primers according to at least one of the following alignment criteria; and (f-2) selecting a top primer from among primers having the same start position as the aligned primers; wherein the alignment criteria include: (i) the number or ratio of degenerate bases and / or universal bases introduced into the primer; the smaller the number or ratio, the higher the ranking; (ii) the number of primer patterns generated by the introduction of degenerate bases; the fewer the number of patterns, the higher the ranking; (iii) (A) ｎ , (T) ｎ or (C) ｎ the number of mononucleotide run sequences; the fewer the number of such sequences, the higher the ranking; (iv) the number or ratio of consecutive nucleotides involved in the formation of a homodimer when the primer forms a homodimer; the smaller the number or ratio, the higher the rank; (v) free energy value for hairpin structure formation (ΔG value); the larger the free energy value, the higher the ranking; (vi) Tm value; the larger the Tm value, the higher the ranking; (vii) GC content; the higher the GC content, the higher the ranking; and (viii) length; the shorter the length, the higher the ranking.

13. 1. A computer readable medium comprising instructions embodying a processor for carrying out a method for providing an oligonucleotide for use in detecting a nucleotide variation of interest in a target nucleic acid sequence, the method comprising the steps of: (a) inputting information about a wild-type target nucleic acid sequence and a nucleotide variation of interest; the wild-type target nucleic acid sequence is a target nucleic acid sequence that does not include a nucleotide variation of interest, the information including (i) the wild-type target nucleic acid sequence, (ii) the position of the nucleotide variation of interest occurring in the wild-type target nucleic acid sequence, and (iii) information about a wild-type base and a mutant base at the position of the nucleotide variation of interest, the positions of the nucleotide variation of interest being designated as a start position and an end position; (b) providing the wild-type target nucleic acid sequence and the mutant target nucleic acid sequence using the input information; the mutant target nucleic acid sequence is a target nucleic acid sequence containing a nucleotide mutation of interest, and the wild-type and mutant target nucleic acid sequences include forward and reverse wild-type and mutant target nucleic acid sequences, respectively; (c) designing oligonucleotides for detecting the nucleotide variation of interest in a predetermined region containing the nucleotide variation of interest in the mutant target nucleic acid sequence to provide a first group of oligonucleotide candidates for the mutant target nucleic acid sequence; the oligonucleotides are designed by length from a predetermined position upstream to an end position based on the start position of the nucleotide variation of interest in the mutant target nucleic acid sequence, the predetermined position being selected from 1 to 50 bp, and the length being 10 to 45 nucleotides; (d) analyzing whether or not oligonucleotides included in the first group of oligonucleotide candidates for the mutant target nucleic acid sequence match with the wild-type target nucleic acid sequence, and providing oligonucleotides that satisfy the following selection criteria as a second group of oligonucleotide candidates for the mutant target nucleic acid sequence; the selection criteria include: (i) the presence of one or more mismatches between the wild-type target nucleic acid sequence and a predetermined region at the 5' end of the oligonucleotide; or (ii) the mismatch between the wild-type target nucleic acid sequence and the oligonucleotide is 20% or more; and (e) selecting oligonucleotides from the second set of oligonucleotide candidates for the mutant target nucleic acid sequence to provide a third set of oligonucleotide candidates; the third set of oligonucleotide candidates is used to detect a nucleotide variation of interest in the target nucleic acid sequence.

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