KR100881924B1 - Method for Detecting Nucleotide Variations Using Labelled Primers - Google Patents

Abstract

The present invention relates to a method of simultaneously detecting at least two or more nucleotide variations of a target nucleotide sequence. According to the method of the present invention, two or more nucleotide variations can be accurately and simultaneously detected without errors by a simple amplification reaction and hybridization process without the conventional additional restriction enzyme processing or sequencing process.

Mutations, nucleotides, amplifications, primers, hybridizations, probes

Description

Method for Detecting Nucleotide Variations Using Labeled Primers

1 is a schematic diagram illustrating a process of detecting nucleotide variation of a human methylenetetrahydrofolate reductase (MTHFR) gene according to an embodiment of the present invention.

The present invention relates to a method for detecting nucleotide variations.

Genetic variation is of great significance both academically and medically. Accordingly, many methods have been developed to detect gene mutations accurately and more conveniently.

For example, in order to detect mutations in Hepatitis B virus (HBV) that show drug resistance, gene sequencing is performed directly after PCR, electrophoresis after restriction enzyme treatment, and mass after restriction enzyme treatment. Analysis method (PCR-RFMP method), LightCycler probe hybridization, primer-specific real-time PCR, etc., but these methods have a problem in accuracy or poor convenience.

On the other hand, the detection of one nucleotide is also a problem of accuracy with the current technology, but there is no reliable method for detecting two or more nucleotide variations simultaneously.

For example, a method for detecting two nucleotide variations through an allele specific amplification reaction by the tetra-primer PCR method has been proposed (Shu Ye, et al., Nucleic Acids, Res. , 20 ( 5): 1152 (1992). However, this method does not produce reproducible data.

Therefore, there is a need for a method that can easily, accurately and sensitively detect two or more variations on nucleic acids such as genome DNA.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors of the present invention have made efforts to solve the above-mentioned demands of the art, and have produced primers with unique structures and label them with appropriate labels, perform amplification reactions, and then hybridize with probes. The present invention has been completed by confirming that not only can be accurately detected, but also that at least two kinds of variations can be easily detected at the same time.

Accordingly, it is an object of the present invention to provide a method for simultaneously detecting at least two or more nucleotide variations of a nucleotide target nucleotide sequence.

Other objects and advantages of the present invention will become apparent from the following examples and claims.

In accordance with an aspect of the present invention, the present invention provides a method for simultaneously detecting at least two kinds of nucleotide variations of a target nucleotide sequence in a sample comprising a target nucleotide sequence exhibiting a nucleotide variation comprising:

(a) contacting the primers of the following (i) to (iv) with the target nucleotide sequence under hybridization conditions:

           (i) hybridizes specifically to a mutation-generating site of a target nucleotide sequence having a nucleotide corresponding to a first nucleotide variant, comprising a nucleotide complementary to the nucleotide corresponding to the first nucleotide variant, and having a 5'-end A nucleotide variation specific primer 1: NVS-P1 having a detectable label bound thereto;

          (Ii) target specific primer 1: TSP1 that hybridizes to a specific site of a target nucleotide sequence located upstream of the mutation-occurring site where said NVS-P1 primer hybridizes;

          (Iii) specifically hybridizes to a sequence complementary to a mutation-occurring site of a target nucleotide sequence having a nucleotide corresponding to a second nucleotide variation at the same site or adjacent site where the first nucleotide variation occurs, wherein the second nucleotide A second nucleotide variation specific primer (NVS-P2) comprising a nucleotide corresponding to a mutation and having a detectable label bound to the 5′-end; And

          (Iii) a target sequence specific primer 2: TSP2 that hybridizes to a specific site of a target nucleotide sequence located downstream of the mutation-producing site from which the NVS-P2 primer hybridizes;

(b) performing at least two cycles of primer annealing, primer extension and denaturation to amplify the target nucleotide sequence;

(c) hybridizing the amplification product generated in step (b) with the first probe and the second probe, wherein the first probe is selected from (i) and (ii) among the products that can be amplified in step (b) Is specifically hybridized only to the product amplified by the primer set of a) and the second probe is amplified by the primer sets of (iii) and (iii) among the products that can be amplified in step (b). Specifically hybridize only to the resulting product; And,

(d) detecting the hybridization signal of step (c).

The present invention provides a method for simultaneously detecting various nucleotide variations or single nucleotide polymorphisms (SNPs) in a nucleotide sequence. In particular, the present invention provides a method capable of simultaneously and quantitatively or qualitatively detecting variations in the same base or in the same codon in a nucleotide sequence. In addition, the method of the present invention is a very advantageous method for automating the whole process.

To date, no method has been developed that can simultaneously detect two or more nucleotide variations, in particular two or more variations in the same base or in the same codon. The present invention provides for the first time a practical method which can confirm such simultaneous variation detection by combining amplification reaction and hybridization reaction.

As used herein, the term "nucleotide variation" refers to various alleles occurring in the same gene. That is, the term “nucleotide variant” encompasses both wild and mutants. Accordingly, the "method of detecting nucleotide variation", which is the subject of the present invention, may be expressed as "genotyping method" or "detection method of allele type".

The nucleotide sequence to be detected in the present invention means gDNA, cDNA and RNA.

According to a preferred embodiment of the invention, the NVS-P1 and NVS-P2 primers are represented by the following general formula (I):

5'-XA _p -Y _q -V _r -3 '(I)

Wherein X is a detectable label, A _p is a variation adjacent specificity portion having a hybridization sequence substantially complementary to the target sequence to hybridize, and Y _q comprises at least three universal bases Is a separation portion, V _r is a variation specificity portion comprising a nucleotide corresponding to or complementary to a nucleotide variation and having a hybridization sequence substantially complementary to the target sequence, p, q and r Is the number of nucleotides, X, Y and Z are deoxyribonucleotides or ribonucleotides, the Tm of the variant adjacent specificity region is higher than the Tm of the variant specificity region, the partition having the lowest Tm of the three regions, Split regions have mutation specificity in terms of hybridization specificity This allows the region to be divided from the variant specificity region, which allows the hybridization specificity of the oligonucleotide overall structure to be determined by the variant adjacent specificity region and the variant specificity region, which in turn improves the hybridization specificity of the oligonucleotide overall structure. .

The basic structure of the nucleotide variation specific primer (NVS) used in the present invention is first proposed by the present inventors, and may be referred to as a dual specificity structure, and an oligonucleotide having such a structure It is termed dual specificity oligonucleotide (DSO). DSO is a new concept oligonucleotide developed by the inventors, which is divided into 5'-high Tm specificity portions and 3'-low Tm specificity regions (3'-low) separated by a splitting region. Since hybridization is determined by Tm specificity portion, the hybridization specificity can be greatly improved (PCT / KR2005 / 001206).

NVS primer in the present invention is a modification of the DSO, and has the structure of Formula (I). NVS primers include a variation adjacent specificity portion, a separation portion and a variation specificity portion.

The partitioning region has the function of physically and functionally partitioning the variant adjacent specificity region and the variant specificity region. By such division, the variant adjacent specificity region and the variant specificity region are divided from each other in terms of hybridization specificity. This specificity cleavage allows the hybridization (annealing) specificity of the oligonucleotide overall structure to be determined double by the variant adjacent specificity region and the variant specificity region, which in turn enhances the hybridization specificity of the primer overall structure.

More specifically, when an NVS primer is annealed to a nucleotide variant-containing target sequence, the specificity is not determined by the full length of the primer as with conventional primers, but rather by dual adjacent mutation regions and mutation specific regions divided from one another. Is determined. Thus, when an NVS primer is annealed to a target sequence, it anneals in a different way from conventional primers, which results in greatly improved annealing specificity.

According to a preferred embodiment of the present invention, the universal base located in the division zone is deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'- OMe inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3-nitropyrrole, 1- (2'-di Oxy-beta-D-ribofuranosyl) -3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, dioxy nebulin, 2'-F nebulin, 2'-F 4- Nitrobenzimidazole, PNA-5-introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpho Lino-nebulin, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholine 3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate- 3-nitropyrrole, 2'-0-methoxyethylinosine, 2'-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4 -Nitro-benzimidazole, 2'-0-methoxyethyl 3-nitropyrrole and combinations of said bases, more preferably dioxyinosine, inosine, 1- (2'-di Oxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole, most preferably dioxyinosine.

According to a preferred embodiment of the invention, said partitioning zone comprises a continuous universal base.

Preferably, the length of the variant adjacent specificity region is longer than the variant specificity region. The variant contiguous specificity region preferably has a length of 15-40 nucleotides, more preferably 15-25 nucleotides in length.

The variant specificity region preferably has a length of 3-15 nucleotides, more preferably 5-15 nucleotides, most preferably 6-13 nucleotides in length.

The partitioning zone preferably has a length of 3-10 nucleotides, more preferably 4-8 nucleotides, most preferably 5-7 nucleotides.

According to a preferred embodiment of the invention, said variant adjacent specificity zone has a Tm of 40-80 ° C, preferably having a Tm of 45-65 ° C. Preferably, the variant specificity region has a Tm of 10-40 ° C. The partition zone preferably has a Tm of 3-15 ° C.

Variation specificity regions of the primers of the invention include nucleotide variations that correspond to or complement the nucleotide variations. When the primers of the present invention hybridize with the sense strand of the target nucleotide sequence, the variant specificity region comprises nucleotides complementary to the nucleotide variant, and when hybridized with the antisense strand, include the nucleotide corresponding to the nucleotide variant. .

According to a preferred embodiment of the present invention, the nucleotide corresponding to or complementary to the nucleotide variation is located at a position 1 to 10 bases away from the 3'-end or 3'-end of the mutation specific region, more preferably the mutation. Located 2 to 7 bases from the 3'-terminus of the specificity region, even more preferably 3 to 6 bases from the 3'-terminus. Most preferably, the nucleotide corresponding to or complementary to the nucleotide variant is located at or near the center of the variant specificity region. For example, where the mutation specific region comprises 8 nucleotides, the nucleotide corresponding to or complementary to the nucleotide variation is a position 3 to 6 bases apart, preferably 4-5 bases away from the 3'-end of the variation specificity region More preferably 4 bases apart.

In the mutation specific region of the NVS primers, if the nucleotide corresponding to or complementary to the nucleotide variation is located 3-6 bases apart or centered away from the 3′-end of the mutation specific region, Advantageous effects are produced.

For example, using a common primer, the mutation site is located at the 3'-end when detecting SNPs, in which case the base at the 3'-end is not annealed to the target sequence (i.e. In case of mismatching, Tm value is not very different. Therefore, even when mismatched, the amplification reaction is annealed and a false positive result tends to occur. Conversely, if the mutation site is placed in the center, most of the thermostable polymerizations differ slightly in the Tm value when the mutation site is annealed to the target sequence (ie when mismatched). The enzyme catalyzes the polymerization even in the mismatched part, leading to false positive results.

According to the present invention, the above-mentioned problems of the prior art can be completely overcome. For example, if a nucleotide variant corresponding or complementary nucleotide is located in the center of the variation specificity region, if mismatching occurs at this position, the mismatching occurs in the center of the structure only in the variation specificity region. However, the thermostable polymerase does not catalyze the polymerization reaction because the Tm value of the mutation specific region is greatly reduced compared with the match, and because of mismatching at the 3'-end in terms of the entire primer structure. Thus, if mismatched, no false positive result occurs.

According to a preferred embodiment of the present invention, the NVS primer used in the present invention is represented by the following general formula II:

5'-XA _p- (dI) _q -V _r -3 '(I)

Wherein X is a detectable label, A _p is a variation adjacent specificity portion having a hybridization sequence substantially complementary to the target sequence to hybridize, and (dI) _q is a continuous deoxyinosine residue Is a separation portion comprising: V _r is a variation specificity portion comprising a nucleotide corresponding to or complementary to a nucleotide variation and having a hybridization sequence substantially complementary to the target sequence, p is 15 -25, q is 4-8, r is 6-13, and the nucleotide corresponding to or complementary to the nucleotide variant is located in the center of V _r .

In the NVS primer of Formula II, (dI) is a division region comprising consecutive deoxyinosine residues, and the number of deoxyinosine residues is 4-8. In this case, other bases may be inserted within the range in which (dI) can serve as a division region.

In addition, in the NVS primer of Formula II, the nucleotide corresponding to or complementary to the nucleotide variation is located in the middle of V _r . For example, if V _r consists of 6, 7, 8, 9, 10, 11, 12, and 13 nucleotide residues, the nucleotides corresponding to or complementary to the nucleotide variation are each 3 from the 3'-terminus of V _r . It is preferred to be located at -4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7 and 6-8 nucleotides.

As used herein, the term “primer” refers to a synthetic or natural oligonucleotide. The primer serves as an initiation point for the synthesis under conditions in which the synthesis of the primer extension product complementary to the template is induced, i.e. the presence of polymers such as nucleotides and DNA polymerase, and conditions of suitable temperature and pH. For maximum efficiency of amplification, the primer is preferably single chain. Preferably, the primer is deoxyribonucleotide. Primers of the invention may include naturally occurring dNMPs (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primer may also include ribonucleotides. For example, oligonucleotides of the present invention may be selected from the group consisting of backbone modified nucleotides such as peptide nucleic acids (PNA) (M. Egholm et al., Nature , 365: 566-568 (1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-0-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohex Tall DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, Tityl-, propynyl-, alkynyl-, thiazolyl-, imidazoryl-, pyridyl-, 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro- , Iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazoryl-, pyridyl-), inosine and diaminopurine .

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function.

As used herein, the term “hybridization” means that two single stranded nucleic acids form a duplex structure by pairing of complementary base sequences. Hybridization can occur when perfect complementarity between single stranded nucleic acid sequences occurs or even when some mismatch base is present. The degree of complementarity required for hybridization may vary depending on the hybridization reaction conditions, and in particular, may be controlled by temperature. In general, if the hybridization temperature is high, there is a high probability that hybridization will occur in a perfect match, and if the hybridization temperature is low, hybridization may occur even if some mismatches are present. The lower the hybridization temperature, the higher the degree of mismatch that hybridization can occur.

Methods of amplifying target nucleotide sequences by carrying out primer annealing, primer extension and denaturation steps are well known in the art.

In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash time, buffer components and their pH and ionic strength depend on various factors such as the length and GC amount of the oligonucleotide and the target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc. NY (1999).

According to a preferred embodiment of the present invention, the hybridization (annealing) temperature is 40 ° C. to 70 ° C., more preferably 45 ° C. to 68 ° C., even more preferably 50 ° C. to 65 ° C., most preferably 60 ° C. To 65 ° C.

The method of the present invention for amplifying a nucleic acid sequence comprising a variant allows for detecting any desired variant. Such nucleic acid molecules are DNA or RNA. The nucleic acid molecule may be double stranded or single stranded, preferably double stranded. When the nucleic acid is a double strand as a starting material, it is preferable to make the two strands into a single strand or a partial single strand form. Methods for separating chains include, but are not limited to, heat, alkali, formamide, urea and glycoxal treatments, enzymatic methods (eg helicase action) and binding proteins. For example, chain separation can be accomplished by heat treatment to a temperature of 80-105 ° C. General methods of such treatments are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

If mRNA is used as the starting material, a reverse transcription step is required before amplification, and details of the reverse transcription step can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001). And Noonan, KF et al., Nucleic Acids Res. 16: 10366 (1988). In the reverse transcription step, oligonucleotide dT primers are used that hybridize to the poly A tail of the mRNA. Oligonucleotide dT primers consist of dTMPs, and one or more of the dTMPs can be replaced with other dNMPs, to the extent that the dT primer can act as a primer. The reverse transcription step can be accomplished by reverse transcriptase with RNase H activity. When using enzymes with RNase H activity, careful selection of reaction conditions can avoid individual RNase H cleavage.

Primers used in the present invention are hybridized or annealed to one site of the template to form a double chain structure. Conditions for nucleic acid hybridization suitable for forming such double chain structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

Various DNA polymerases can be used in the amplification step of the present invention and include “Clenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which is Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu). Most of the polymerases can be isolated from the bacteria themselves or can be purchased commercially. In addition, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase.

When carrying out the polymerization reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel. Excess of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is desired.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing or hybridization in the present invention is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer (i.e., the conditions where the partitioning region cannot hydrogen bond to the target sequence). Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables. Preferably, the annealing temperature is 40 ° C to 70 ° C, more preferably 45 ° C to 68 ° C, even more preferably 50 ° C to 65 ° C, and most preferably 60 ° C to 65 ° C.

In the most preferred embodiment of the present invention, the amplification process is carried out according to the PCR disclosed in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.

According to a preferred embodiment of the invention, the detectable label bound to the NVS primer of the invention is a chemical label (eg biotin), an enzyme label (eg alkaline phosphatase, peroxidase, β- Galactosidase and β-glucosidase), radiolabels (e.g. I ¹²⁵ and C ¹⁴ ), fluorescent labels (e.g. fluorine, phycoerythrin, rhodamine, lysamine, TAMRA, Cy5, Cy3, HEX, TET, Dabsyl and FAM), luminescent labels, chemiluminescent labels, fluorescence resonance energy transfer (FRET) labels or heavy metal labels (eg gold). More preferably, the label is a chemical label or metal label, even more preferably a chemical label, most preferably biotin. As the enzyme label, an enzyme catalyzing a color reaction, a fluorescence reaction, a luminescence reaction or an infrared reaction may be used. The labeling of primers by labeling materials can be carried out in various ways conventionally known in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)), and chination methods. (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)).

Since the target sequence specific primer (TSP) used in the present invention is annealed to the outer region where the mutation for analysis purposes occurs, a conventional primer, that is, a primer having a conventional primer structure without a division region, can be used. have. Preferably, the TSP has the structure represented by Formula I above, wherein the variant specificity region has a hybridization sequence that is substantially complementary to the target sequence without the nucleotide corresponding to or complementary to the nucleotide variant.

According to another variant of the present invention, step (a) comprises one or more nucleotide variation specific primers that hybridize to other variants other than the hybridization of the NVS-P1 primer and the NVS-P2 primer. It is additionally used. For example, when applying the method of the present invention to detect lamivudine resistant variants of hepatitis B virus (HBV), it utilizes three or more NVS-P primers that hybridize to YIDD motifs, YVDD motifs and L528M mutations. 3 variations can be detected simultaneously.

After the amplification process described above, the resulting amplification product is hybridized with two probes. One of the two probes is specifically hybridized only to the product amplified by the primer sets of (iii) and (ii) among the products that can be amplified in step (b), and the other probe is Of the products that can be amplified in (b) it is specifically hybridized only to the product amplified by the primer sets of (iii) and (iii). Referring to FIG. 1, probe 1 and probe 2 are the minimum primers used in the hybridization reaction.

According to a preferred embodiment of the invention, the probe is immobilized on a suitable substrate to form a microarray. The probe is used as a hybridizable array element and immobilized on a substrate. Preferred substrates include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The hybridization array element described above is arranged and immobilized on the substrate. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV. According to one specific embodiment of the invention, the hybridization array element can be bonded to the glass surface modified to include an epoxy compound or an aldehyde group and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element may be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine).

Conditions suitable for hybridization of step (c) include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Decisions may be made with reference to those disclosed in Hybridization, A Practical Approach , IRL Press, Washington, DC (1985). Stringent conditions used for hybridization can be determined by adjusting the temperature, ionic strength (buffer concentration) and the presence of compounds such as organic solvents. Such stringent conditions can be determined differently depending on the sequence to be hybridized.

For example, among the stringent conditions, the high stringency conditions were hybridized to 65 ° C. in 0.5 M NaHPO 4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 68 at 0.1 × standard saline citrate / 0.1% SDS. It means washing under the condition of ℃. Alternatively, high stringency conditions mean washing at 48 ° C. in 6 × SSC / 0.05% sodium pyrophosphate. Low stringency means washing at 42 ° C. conditions, for example, at 0.2 × SSC / 0.1% SDS.

After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. Hybridization signals can be carried out in various ways depending on the type of label bound to the NVS primers.

For example, when NVS is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization reaction product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate).

When NVS is labeled with gold particles, it can be detected by silver dyeing using silver nitrate.

According to a preferred embodiment of the invention, step (d) comprises the following sub-steps: (d-1) avidin-horseradish peroxidase (HRP) or strep to the hybridization product of step (c) Contacting tavidin-HRP; (d-2) reacting the substrate of HRP with the product of sub-step (d-1); And (d-3) detecting whether a reaction of the small step (d-1) occurs.

By detecting the hybridization signal, it is possible to finally confirm whether there is any variation in the sample nucleic acid molecule. For example, if only hybridization with the first probe was detected, the sample nucleic acid molecule was analyzed to have a homo genotype for the first nucleotide variant, and conversely, if only hybridization with the second probe was detected, then the sample nucleic acid molecule was identified as the second. It is analyzed to have a homo genotype for nucleotide variations. If hybridization reactions were detected for both the first and second probes, the sample nucleic acid molecule is analyzed as heterogenotype as having both the first nucleotide variant and the second nucleotide variant.

According to a specific embodiment of the present invention, the hybridization signal detection step may be carried out according to a method known as an “Array tube” technique (Stefan Monecke et al, Genome Letters , 2: 106-118 (2003)). . A suitable device for implementing the technique is sold as "ArrayTube" by Clondiag (Germany).

The methods of the present invention can be used for the detection of various nucleotide variations. For example, it can be used to detect various variations in the human genome (eg, variations of the methylenetetrahydrofolate reductase (MTHFR) gene), nucleotide variations of drug resistant pathogens, and mutations of cancer development-related nucleotides.

According to a preferred embodiment of the present invention, the method of the present invention is for detecting drug resistant pathogens, more preferably drug resistant viruses. The drug resistant virus is preferably human immunodeficiency virus (HIV) -1, HIV-2, hepatitis B virus (HBV), hepatitis C virus (HCV) or human herpesvirus (HBV), and most preferably HBV. to be.

According to a preferred embodiment of the present invention, the drug resistant to the virus is zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine , Delavirdine, efavirenz, adefovir, adefovir dipivoxil, FTC, D4FC, BCH-189, F-ddA, tetrahydroimidazo [4 Tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -one, tetrahydroimidazo ,, 5,1-jk [] 1,4] benzodiazepine-2 (1H) -one [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -thione (tetrahydroimidazo [4,5,1-jk [] 1,4] benzodiazepine-2 (1H) -thione), (S ) -4-isopropoxycarbonyl-6-methoxy-3- (methylthiomethyl) -3,4-dihydroquinoxalin-2 (1H) -thione ((S) -4-isopropoxycarbonyl-6-methoxy -3- (methylthiomethyl) -3,4, dihydroquinoxaline-2 (1H) -thione, saquinavir, ritonavir, indinavir, nelphi Nelfinavir amprenavir, entecavir, famciclovir, benzo-1,2,4-thiazine antiviral agent, At least one antiviral agent selected from the group consisting of ribavirin and interferon, most preferably lamivudine.

According to the method of the present invention, an NVS-P primer is prepared by referring to the nucleotide sequence of the pathogen that exhibits drug resistance.

Examples of mutations in HIV-1 reverse transcriptase that result in resistance to antiviral agents (such as zalcitabine, zidovudine, didanosine, lamivudine, etc.) include: Met41Leu, Glu44Asp, Glu44Ala, Ile50Val, Ala62Val, Lys65Arg, Asp67Asn , Ser68Gly, Thr69Asp, Thr69Ser-Ser-Gly, Thr69Ser-Thr-Gly, Thr69Ser-Val-Gly, Lys70Arg, Lys70Glu, Leu74Ile, Leu74Val, Val75Ile, Val75Leu, Val75Thr, Phe77Leu, LeslOlIle, Vall , Ilel35Val, Glnl51Met, Thrl65Ile, Vall79Asp, Tyrl81Ile, Metl84Ala, Metl84Ile, Metl84Val, Tyrl88His, Tyrl88Leu, Glyl90Ala, Glyl90Cys, Glyl90Ser, Glyl90Trr, Lyu210Tr 215

Examples of mutations that occur in HIV-1 proteases that indicate resistance to antiviral agents (saquinavir, ritonavir, indinavir, nlpinavir, amprenavir, etc.) include: Leu10Ile, LeulOVal, LeulOPhe, Val32Ile, Glu35Asp, Met36Ile, Met46Ile, Met45Leu, Ile47Val, Gly48Val, Ile50Val, Ile54Met, Ile54Ser, Ile54Val, Leu63Pro, Ala71Thr, Ala71Val, Val77Ile, Val82AlaTalPheI, Pal As Phe 82 Leu89Pro and Leu90Met.

Examples of variations in HCV RNA-dependent RNA polymerase that result in resistance to antiviral agents (eg, benzo-1,2,4-thiazine antiviral agents) include: Lys50Arg, Met71Val, Asn41 lSer, Met414Thr, Phe415Ty and Val581Ala.

Examples of mutations in the HCV NS5A protein resulting in resistance to antiviral agents (eg, interferon, etc.) include: Leu2190Lys, Val2198Leu, Val2198Met, Val2198Glu, Thr2217Ala, Thr2217Val, Asn2218Asp, Asn2218Lys, Assp2218220Ger, Asp218220Ger, Asp218220Ger, Asp218220 , Glu2225Asp, Glu2228Gln, Glu2236Ala, Asn2248Asp, He2252Val, Ile2268Val, Arg2276Leu, Lys2277Arg, Ser2278Pro, Arg2280Lys, Arg2280Glu, Ala2282Thr, Pro2283Gln, Pro2283Arg, Val2287Ile, Leu2298Val, Leu2298Ile, Thr2300Pro, Thr2300Ala, Lys2302Asn, Lys2303Asn, Asp2305Gly and Pro2315Ala.

Examples in which mutations in HBV polymerase occur to show resistance to antiviral agents are as follows: (iii) Lamivudine: Leu426Ile, Leu426Val, Val173Leu, Met552Ile, Met552Val, Met552Ser, Val555Ile, Leu528Met; (Ii) entecavir: Ile169Thr, Thr184Gly, Ser202Ile, Met250Val; (Iii) famciclovir: Val173 Leu, Met552Ile, Val555Ile; And (iii) adefovir difficile: Asn236Thr.

Nucleotide variant specific NVS-P primers used in the present invention are designed with reference to the mutated nucleotide sequences of the drug resistant pathogens described above.

According to a preferred embodiment of the invention, the invention is directed to a drug resistant nucleotide variant in a polymerase of HBV, more preferably a lamivudine resistant nucleotide variant in a polymerase of HBV, most preferably at 552th in a polymerase of HBV. Applied to detect variations in codons.

The present invention is useful for detecting two or more nucleotide variations simultaneously. As used herein, the term "simultaneously" means to obtain a reaction product that can confirm the presence or absence of two or more mutations in one amplification reaction solution. Therefore, according to the method of the present invention, the multiplex amplification reaction is necessarily accompanied.

When the method of the present invention is used to detect mutations at the same site, it is useful for detecting two SNPs simultaneously. When the method of the present invention is used to detect two or more kinds of mutations occurring at different sites, that is, adjacent or spaced sites, there are largely the following applicability. (Iii) simultaneously detect two nucleotide variations at adjacent sites in the same codon but not at the same site; And (ii) simultaneously detecting two or more nucleotide variations at adjacent sites or spaced apart locations that are not included in the same codon.

In the case of detecting two or more mutations in the same site or in the same codon, the present invention is advantageous for detecting two kinds of nucleotides simultaneously. In this case, the NVS-P1 primer and the NVS-P2 primer to be used generally have overlapping sequences, and may hybridize with each other to form a duplex. Dimer formation between these primers can make the amplification abnormal, resulting in false amplification results. However, the NVS primer of the present invention overcomes the problems caused by the formation of dimers between the primers, and this feature and advantage make it possible to detect two variations simultaneously.

According to one specific embodiment of the present invention for detecting lamivudine resistant variants of hepatitis B virus (HBV), the process of the present invention will be described as follows.

First, the coding sequence of the DNA polymerase of HBV as a target sequence in which nucleotide variations inducing lamivudine resistance occurs is obtained. Such sequences are known in the art and can be found, for example, in GenBank Accession Numbers NC003977, AY167096, AY167095 and AY306136. There is a single base mutation in this sequence that induces lamivudine resistance, and in the wild type it encodes methionine (YMDD motif). However, HBV resistant to antiviral agents such as lamivudine and famcyclovir have mutations in the YMDD motif where methionine is replaced with isoleucine (YIDD motif), valine (YVDD motif) or serine (YSDD motif). In the YIDD motif, g is changed to t, in the YVDD motif, a is converted to g, and in the YSDD motif, tg is changed to gt.

In order to detect variant YVDD motifs and YIDD motifs simultaneously, the YVDD-R primers of SEQ ID NO: 3 and the YIDD-F primers of SEQ ID NO: 4 are used. YVDD-R primers pair with HLT-F primers (SEQ ID NO: 1) to form amplification products, and YIDD-F primers pair with HLT-R primers (SEQ ID NO: 2) to form amplification products do. HLT-F and HLT-R are primers that anneal to the outer region where mutations occur, which may be paired with each other to form amplification products). The primers are selected and designed such that the amplification products produced by them have a predetermined size, and the sizes of the amplification products produced by each primer set are different from each other. Therefore, by checking only the size of the amplification product through simple electrophoresis, it is possible to easily detect what mutation the target nucleotide sequence in the sample has. Although YVDD-R and YIDD-F corresponding to NVS-P primers have sequences that can hybridize with each other, they do not affect each other in the amplification reaction and show accurate amplification results. When the YSDD motif is to be detected, the YSDD-F primer of SEQ ID NO: 4 may be used.

The method of the present invention can be usefully used to simultaneously detect two or more variations in positions spaced from each other. If the method of the present invention is applied to simultaneously detect YIDD-F motifs and L528M mutations at mutually spaced positions on the HBV DNA polymerase gene, the YIDD-F primer and SEQ ID NO: 6 of SEQ ID NO: 4 L528M-R primers of the sequence are used. L528M-R pairs with HLT-F primers to form amplification products, and YIDD-F primers pair with HLT-R primers to form amplification products.

Examples when the method of the present invention detects various variations in the human genome, such as those at the 677th nucleotide in the MTHFR gene, are described in the Examples below.

According to the method of the present invention, through a simple amplification reaction, it is possible to detect two or more nucleotide variations accurately and quantitatively or qualitatively and without error.

In summary, the features and advantages of the present invention are as follows:

(Iii) The method of the present invention is carried out according to a combination of a multiplex amplification reaction and a hybridization reaction using at least two or more primer sets.

(Ii) According to the method of the present invention, two or more nucleotide variations on the target nucleotide sequence can be detected simultaneously with very high specificity.

(Iii) The method of the present invention exhibits very good workability in a multiplex reaction, so that one set of amplification reactions can finally detect two or more nucleotide variations through hybridization reactions.

(Iii) In the NVS-P primer used in the method of the present invention, if a nucleotide variation or complementary nucleotide is located in the center of the variation specificity region, a single base mismatch can be completely distinguished from the amplification reaction. have.

(Iii) According to the method of the present invention, not only nucleotides in the same site or in the same codon, but also two or more nucleotides in spaced positions can be detected simultaneously.

(Iii) In general, when primer pairs having overlapping sequences are used, the primers may form dimers, resulting in an incorrect amplification. According to the method of the present invention, it is possible to overcome this problem of dimer formation. Amplification results can be prevented.

(Iii) The method of the present invention enables quantitative and qualitative analysis of nucleotide variations, and is particularly advantageous for automation.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Example  I: primer  Design and manufacture

Example  I-1: human MTHFR  For gene amplification primer

Based on the nucleotide sequence encoding the human methylenetetrahydrofolate reductase (MTHFR) gene (Ensemble, Vega Havana Gene ID: OTTHUMG00000002277), it is a forward and reverse that can serve as a target specific primer (TSP). ) Primers were designed.

TSP1 5'- TGG GGT CAG AAG CAT ATC AGI III IAG CCC AGC -3 '(SEQ ID NO: 7)

TSP2 5'- GCT TAT CAA GTG GTT CTG ATG III IIC ACC TTT GG -3 '(SEQ ID NO: 8)

I in the sequence represents deoxyinosine. By these two primers, 712 bp of MTHFR gene sequence is amplified.

Example  I-2: human MTHFR  For amplifying gene 677C type primer

Based on the human MTHFR gene 677C type (the 677th sequence of the MTHFR gene, which encodes a functioning MTHFR protein) sequence, it is specifically annealed to 677C to successfully detect SNPs according to the PCR method. Nucleotide nucleotide variation specific primer (NVS) was designed and labeled with biotin at the 5'-end.

NVS-P1 5'- CAC TTG AAG GAG AAG GTG TII III GGA G C C GA -3 '(SEQ ID NO: 9)

I in the sequence represents deoxyinosine. The underlined C in the sequence is the site for detecting 677C.

Nucleotide variant specific primers used in the present invention have a structure of general formula (I), so that the hybridization with the target sequence with a very high specificity. In these primers having a structure of Formula I, the two specificity regions are physically and functionally divided by the cleavage region, and the hybridization specificity of the entire primer structure is dually regulated by the mutation adjacent specificity region and the variation specificity region. In addition, the base corresponding to the nucleotide variation or hybridization to the mutation is located in the central portion of the mutation specific region, so as to increase the Tm value according to the mismatch while preventing the DNA synthesis by Taq polymerase during mismatch.

Example  I-3: human MTHFR  For amplifying gene 677T type primer

Based on the human MTHFR gene 677T type (the 677th sequence of the MTHFR gene is T, coding for a low activity MTHFR protein variant) sequence, it is specifically annealed to 677T to successfully perform SNP detection according to the PCR method. Nucleotide variant specific primers (forward primers) were designed and labeled with biotin at the 5′-end.

NVS-P2 5'- AGA AAA GCT GCG TGA TGI III IAT CG A CTC -3 '(SEQ ID NO: 10)

 I in the sequence represents deoxyinosine. The underlined A in the sequence is the site for detecting 677T.

Example  II: Preparation of Human Genome DNA

Using human blood AccuPrep the genomic DNA ^TM Genomic DNA Extraction Kit (Bioneer, South Korea) was extracted and used as a template for PCR amplification.

Example  Ⅲ: Human MTHFR  Multiplex of genes PCR

Multiplex PCR was performed using the human DNA samples obtained in Example II, respectively, using the primer pairs shown in Table 1 below.

target Primer pair PCR product size (bp) Human MTHFR gene TSP1 / TSP2 712 677C genotype TSP1 / NVS-P1 470 677T genotype NVS-P2 / TSP2 297

2 μl of 10 × PCR reaction buffer (Roche) containing 15 mM MgCl ₂ , 4 μl of 1.25 μM primer pair, 2 μl of dNTP (2 mM dATP, dCTP, dGTP and dTTP, respectively) 20-30 ng and 0.5 of template DNA Multiplex PCR was performed using the final 20 μl of reaction mixture containing μl of Taq polymerase (5 units / μl, Roche). The tube containing the reaction mixture was placed in a preheated (94 ° C.) thermal cycler and denatured at 94 ° C. 15 minutes, followed by 94 ° C. 30 seconds, 65 ° 30 seconds and 72 ° C. 35 The mixture was cycled and then reacted at 72 ° C for 10 minutes.

Example  IV: Microarray  analysis

First, a DNA microarray is produced. The amino-modified oligonucleotide probe is immobilized on an epoxide-coded 3 × 3 mm glass surface. As the probe, two oligonucleotides are designed. One of the two probes specifically binds to a variant 1 specific PCR product, which specifically binds to the product amplified by the TSP1 and NVS-P1 primer pairs, ie the amplification product of the human MTHFR gene 677C type. . Another probe specifically binds to a variant bispecific PCR product, which specifically binds to the product amplified by the TSP2 and NVS-P2 primer pairs, ie the amplification product of the human MTHFR gene 677T type.

DNA microarrays are made and then inserted into ArrayTubes ^™ (Clondiag, Germany).

The multiplex PCR product of Example III is then hybridized with the probes in the ArrayTubes. The PCT product is placed in ArrayTubes, denatured at 95 ° C. for 5 minutes and then reacted at 50 ° C. for 1 hour, followed by washing at 30 ° C. in 2 × SSC, 0.1% SDS for hybridization.

Then, streptavidin-horseradish peroxidase (HRP) is added to the ArrayTube and reacted at 30 ° C. for 15 minutes. Then, 100 μl of a tetramethylbenzidine (TMB) substrate solution was added to the ArrayTube, and then reacted for 10 minutes at room temperature to induce a color reaction, and then the ArrayTube was mounted on an ATR 03 reader (Clondiag, Germany) to read an AT array image. do.

In the AT array image, if a color reaction was observed only in a probe that specifically binds to a variant 1 specific PCR product, the sample is of the MTHFR gene 677C homo genotype and only at a probe that specifically binds to a variant 2 specific PCR product. If a response is observed, the sample is MTHFR gene 677T homo genotype, and if colorimetric reactions are observed in both probes that specifically bind to variant 1 and 2 specific PCR products, then the heterogenotype with MTHFR genes 677C and 677T is observed. You can decide.

In summary, it can be seen that, according to the method of the present invention, analysis (especially genome typing) of DNA in a biological sample can be easily and error-free in a multiplex manner.

According to the method of the present invention, two or more nucleotide variations can be accurately and simultaneously detected without errors by a simple amplification reaction and hybridization process without the conventional additional restriction enzyme processing or sequencing process.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> Seegene, Inc. <120> Method for Detecting Nucleotide Variations Using Labeled Primers <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLT-F <400> 1 ctcgtggtgg acttctctca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HLT-R <400> 2 gtgtaaaagg ggcagcaaag 20 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> YVDD-R <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 3 cttggccccc aataccannn nntccacata 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> YIDD-F <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 4 ccccactgtt tggctttnnn nnatattgat 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> YSDD-F <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 5 ccccactgtt tggctttnnn nnatagtga 29 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> L528M-R <220> <221> misc_feature (222) (19) .. (23) <223> n denotes deoxyinosine <400> 6 aacaaatggc gctagtaann nnngccatga g 31 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TSP1 <220> <221> misc_feature (222) (21) .. (25) <223> n denotes deoxyinosine <400> 7 tggggtcaga agcatatcag nnnnnagccc agc 33 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TSP2 <220> <221> misc_feature (222) (22) .. (26) <223> n denotes deoxyinosine <400> 8 gcttatcaag tggttctgat gnnnnncacc tttgg 35 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> NVS-P1 <220> <221> misc_feature (222) (20) .. (24) <223> n denotes deoxyinosine <400> 9 cacttgaagg agaaggtgtn nnnnggagcc ga 32 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> NVS-P2 <220> <221> misc_feature (222) (18) .. (22) <223> n denotes deoxyinosine <400> 10 agaaaagctg cgtgatgnnn nnatcgactc 30  

Claims (19)

A method for simultaneously detecting at least two kinds of nucleotide variations of a target nucleotide sequence in a sample comprising a target nucleotide sequence exhibiting a nucleotide variation comprising the following steps: (a) contacting the primers of the following (i) to (iv) with the target nucleotide sequence under hybridization conditions:            (i) is specifically hybridized to a mutation-generating site of a target nucleotide sequence having a nucleotide corresponding to a first nucleotide variant, comprises a nucleotide complementary to the nucleotide corresponding to the first nucleotide variant and is 5'-terminal A first nucleotide variation specific primer 1: NVS-P1 to which a detectable label is bound;           (Ii) target specific primer 1: TSP1 that hybridizes to a specific site of a target nucleotide sequence located upstream of the mutation-occurring site where said NVS-P1 primer hybridizes;           (Iii) specifically hybridizes to a sequence complementary to a mutation-occurring site of a target nucleotide sequence having a nucleotide corresponding to a second nucleotide variation at the same site or adjacent site where the first nucleotide variation occurs, wherein the second nucleotide A second nucleotide variation specific primer (NVS-P2) comprising a nucleotide corresponding to a mutation and having a detectable label bound to the 5′-end; And           (Iii) a target sequence specific primer 2: TSP2 that hybridizes to a specific site of a target nucleotide sequence located downstream of the mutation-producing site from which the NVS-P2 primer hybridizes; (b) performing at least two cycles of primer annealing, primer extension and denaturation to amplify the target nucleotide sequence; (c) hybridizing the amplification product generated in step (b) with the first probe and the second probe, wherein the first probe is selected from (i) and (ii) among the products that can be amplified in step (b) Is specifically hybridized only to the product amplified by the primer set of a) and the second probe is amplified by the primer sets of (iii) and (iii) among the products that can be amplified in step (b). Specifically hybridize only to the product; And, (d) detecting the hybridization signal of step (c). The method of claim 1, wherein the NVS-P1 and NVS-P2 primers are represented by the following general formula (I): 5'-XA _p -Y _q -V _r -3 '(I) Wherein X is a detectable label, A _p is a variation adjacent specificity portion having a hybridization sequence substantially complementary to the target sequence to hybridize, and Y _q comprises at least three universal bases Is a separation portion, V _r is a variation specificity portion comprising a nucleotide corresponding to or complementary to a nucleotide variation and having a hybridization sequence substantially complementary to the target sequence, p, q and r Is the number of nucleotides, X, Y and Z are deoxyribonucleotides or ribonucleotides, the Tm of the variant adjacent specificity region is higher than the Tm of the variant specificity region, the partition having the lowest Tm of the three regions, Split regions have mutation specificity in terms of hybridization specificity This allows the region to be divided from the variant specificity region, which allows the hybridization specificity of the oligonucleotide overall structure to be determined by the variant adjacent specificity region and the variant specificity region, which in turn improves the hybridization specificity of the oligonucleotide overall structure. . Claim 3 was abandoned when the setup registration fee was paid. The method of claim 1, wherein the detectable label is a chemical label, an enzyme label, a radiolabel, a fluorescent label, a luminescent label, a chemiluminescent label, a FRET (fluorescence resonance energy transfer) label, or a metal label. . Claim 4 was abandoned when the registration fee was paid. 4. The method of claim 3 wherein the label is biotin. 5. The method of claim 4, wherein step (d) comprises the following sub-steps: (d-1) contacting avidin-horseradish peroxidase (HRP) or streptavidin-HRP with the hybridization product of step (c); (d-2) reacting the substrate of HRP with the product of sub-step (d-1); And (d-3) detecting whether a reaction of the small step (d-1) occurs. Claim 6 was abandoned when the registration fee was paid. 6. The method of claim 5, wherein the substrate of HRP is TMB (tetramethylbenzidine). The method of claim 2, wherein the universal base is deoxyinosine, inosine, 7-diaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine , Deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3-nitropyrrole, 1- (2'-dioxy-beta-D-ribofura Nosyl) -3-nitropyrrole, deoxy 5-nitropyrrole, 5-nitroindole, 2'-OMe 5-nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4 -Nitrobenzimidazole, dioxy 4-aminobenzimidazole, 4-aminobenzimidazole, dioxy nebulin, 2'-F nebulin, 2'-F 4-nitrobenzimidazole, PNA-5 Introindole, PNA-nebulin, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebulinine, morpho Lino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nit Indole, phosphoramidate-nebulin, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethylinosin , 2'-0-methoxyethyl nebulin, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro-benzimidazole, 2'-0-methoxyethyl And 3-nitropyrrole and a combination of said bases. Claim 8 was abandoned when the registration fee was paid. 8. The method of claim 7, wherein the universal base is deoxyinosine, inosine, 1- (2'-deoxy-beta-D-ribofuranosyl) -3-nitropyrrole or 5-nitroindole. . Claim 9 was abandoned upon payment of a set-up fee. 3. The method of claim 2, wherein said variant adjacent specificity region is 15-40 nucleotides in length. Claim 10 was abandoned upon payment of a setup registration fee. The method of claim 2, wherein the variant specificity region has a length of 3-15 nucleotides. Claim 11 was abandoned upon payment of a setup registration fee. The method of claim 2, wherein said partitioning region has a length of 3-10 nucleotides. 3. The method of claim 2, wherein said variant adjacent specificity region has a Tm of 40-80 ° C. The method of claim 2, wherein the variant specificity zone has a Tm of 10-40 ° C. 4. 3. The method of claim 2, wherein the partition zone has a Tm of 3-15 ° C. The method of claim 2, wherein the nucleotide corresponding to or complementary to the nucleotide variant is located at the 3′-end of the variant specificity region or 1 to 10 bases away from the 3′-end. Claim 16 was abandoned upon payment of a setup registration fee. 16. The method of claim 15, wherein the nucleotide corresponding to or complementary to the nucleotide variant is located 2 to 7 bases apart from the 3'-end of the variant specificity region. Claim 17 was abandoned upon payment of a registration fee. The method of claim 16, wherein the nucleotide corresponding to or complementary to the nucleotide variant is located 3 to 6 bases apart from the 3′-end of the variant specificity region. Claim 18 was abandoned upon payment of a set-up fee. The method of claim 15, wherein the nucleotide corresponding to or complementary to the nucleotide variation is located in the central portion of the variation specificity region. Claim 19 was abandoned upon payment of a registration fee. The method of claim 2, wherein the variant specificity regions of the NVS-P1 primer and NVS-P2 primer comprise sequences that partially overlap each other.

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