KR20190071950A - Pepetide nucleic acid probe for genotyping age related hearing loss (presbycusis) and Method using the same - Google Patents

Abstract

The present invention relates to a peptide nucleic acid (PNA) probe for determining a genotype of a gene related to age-related hearing loss, and a method for determining a genotype related to an age-related hearing loss by using the PNA probe. According to the present invention, the PNA probe for determining a genotype related to age-related hearing loss and the method for determining a genotype related to an age-related hearing loss by using the PNA probe rapidly and simply determine the genotype related to an age-related hearing loss, and are useful for predicting a risk degree of age-related hearing losses by determining genotypes of genes of CDH23, OTOF, COCH, ATP1A3, GRHL2, ITGA8, IQGAP2, PCDH15, PCDH20, GRM7, MTHFR, KCNQ1, KCNQ4 and SLC28A3.

Description

TECHNICAL FIELD The present invention relates to a PNA probe for genetic type related to senile hearing loss and a method for discriminating a genotype of senile hearing loss using the same,

More particularly, the present invention relates to a PNA probe for genotype-type determination of senile deafness, and more particularly to at least one PNA probe selected from the group consisting of SEQ ID NOS: 1 to 30 and SEQ ID NOS: 31 to 90 The present invention relates to a method for amplifying a senile deafness-related gene using two or more primers selected from the group consisting of genes and determining the presence or absence of a gene mutation through hybridization of a PNA probe.

The number of geriatric patients is rapidly increasing due to an increase in life expectancy and aging. As a result, the proportion of medical expenses is rapidly increasing, and it is important to prevent and manage disease in advance in order to reduce medical expenses.

The quality of life of the elderly has a close relationship with health. According to the research report of the US Centers for Disease Control and Prevention, the abnormalities of the sensory organs, especially the "hearing problems" are reported as the main indicators for evaluating the quality of life in the old years. More than 33% of elderly people over 70 years of age have hearing problems, and these sensory organs related diseases are considered to be the main causes of lower quality of life in old age. In addition, these sensory organs-related diseases lead to limitations in activities, which can lead to mental problems such as hypertension, heart disease, and social isolation, leading to more social problems.

In order to prevent personalized disease, gene and genotype candidates related to disease are derived, and they are utilized in the diagnosis and treatment of diseases. Individual genome analysis is being carried out to recognize the inherent risk of disease and to control disease by controlling external factors such as lifestyle. Since it is possible to treat before disease worsens, the need for individual genome analysis technology is increasing .

The analysis of individual genome for prevention of such diseases can improve the prediction ability by analyzing a plurality of genes and genotypes, and it is urgently required to develop a technology capable of simultaneously discriminating a plurality of genotypes.

To date, genotyping methods have been used for direct sequencing of polymerase chain reaction (PCR) products, restriction fragment length polymorphism (PCR-RFLP), line probe assay, Enzyme fragmentation mass spectrometry (PCR-RFMP) and multiplex PCR methods and DNA chip detection methods.

DNA chip technology has been constrained to produce a new chip when a new mutation is found. However, DNA chip technology has been limited to DNA chip or sequencing by PCR as a diagnostic method for hereditary hearing loss. Therefore, there is a need for a technique to inexpensively and accurately diagnose a large number of genotypes such as those disclosed in Korean Patent Publication No. 2012-0067384.

Recently, the detection method using a probe has been spotlighted because of its high sensitivity and specificity. However, the detection of a probe of a complicated design method is expensive and inconvenient to use. Therefore, it is urgently required to develop a technology capable of quickly detecting highly accurate signals through a more simple design.

Therefore, the present inventors have made intensive efforts to develop a method for genotyping genes of senile hearing loss genes that are fast and accurate. As a result, the inventors amplified senile hearing-related genes with primers and then hybridized with gene mutation specific probes to analyze the melting curve , The present inventors confirmed that the mutation of the senile hearing loss gene can be determined promptly and accurately and completed the present invention.

An object of the present invention is to provide a probe and a primer pair capable of discriminating senile deafness-associated genotypes.

Another object of the present invention is to provide a method for distinguishing senile deafness-related genotypes using the probe and primer pairs.

In order to achieve the above object, the present invention provides a PNA probe for discriminating senile deafness-related genotypes comprising any one of sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.

The present invention also relates to

A nucleotide sequence of SEQ ID NO: 31 as a forward primer, a nucleotide sequence of SEQ ID NO: 32 as a reverse primer,

SEQ ID NO: 33 as a forward primer, SEQ ID NO: 34 as a reverse primer,

SEQ ID NO: 35 as a forward primer, SEQ ID NO: 36 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 37 as a forward primer, a nucleotide sequence of SEQ ID NO: 38 as a reverse primer,

SEQ ID NO: 39 as a forward primer, SEQ ID NO: 40 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 41 as a forward primer, a nucleotide sequence of SEQ ID NO: 42 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 43 as a forward primer, a nucleotide sequence of SEQ ID NO: 44 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 45 as a forward primer, a nucleotide sequence of SEQ ID NO: 46 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 47 as a forward primer, a nucleotide sequence of SEQ ID NO: 48 as a reverse primer,

SEQ ID NO: 49 as a forward primer, SEQ ID NO: 50 as a reverse primer,

SEQ ID NO: 51 as a forward primer, SEQ ID NO: 52 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 53 as a forward primer, a nucleotide sequence of SEQ ID NO: 54 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 55 as a forward primer, a nucleotide sequence of SEQ ID NO: 56 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 57 as a forward primer, a nucleotide sequence of SEQ ID NO: 58 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 59 as a forward primer, a nucleotide sequence of SEQ ID NO: 60 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 61 as a forward primer, a nucleotide sequence of SEQ ID NO: 62 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 63 as a forward primer, a nucleotide sequence of SEQ ID NO: 64 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 65 as a forward primer, a nucleotide sequence of SEQ ID NO: 66 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 67 as a forward primer, a nucleotide sequence of SEQ ID NO: 68 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 69 as a forward primer, a nucleotide sequence of SEQ ID NO: 70 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 71 as a forward primer, a nucleotide sequence of SEQ ID NO: 72 as a reverse primer,

The nucleotide sequence of SEQ ID NO: 73 as the forward primer, the nucleotide sequence of SEQ ID NO: 74 as the reverse primer;

A nucleotide sequence of SEQ ID NO: 75 as a forward primer, a nucleotide sequence of SEQ ID NO: 76 as a reverse primer,

The nucleotide sequence of SEQ ID NO: 77 as the forward primer, the nucleotide sequence of SEQ ID NO: 78 as the reverse primer;

A nucleotide sequence of SEQ ID NO: 79 as a forward primer, a nucleotide sequence of SEQ ID NO: 80 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 81 as a forward primer, a nucleotide sequence of SEQ ID NO: 82 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 83 as a forward primer, a nucleotide sequence of SEQ ID NO: 84 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 85 as a forward primer, a nucleotide sequence of SEQ ID NO: 86 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 87 as a forward primer, a nucleotide sequence of SEQ ID NO: 88 as a reverse primer, And

A nucleotide sequence of SEQ ID NO: 89 as a forward primer, and a nucleotide sequence of SEQ ID NO: 90 as a reverse primer; and a primer pair for discriminating senile deafness-associated genotypes.

The present invention also provides a composition for senile deafness-associated genotyping comprising the PNA probe and the primer pair.

The present invention also relates to a method for detecting a sample, comprising the steps of: a) extracting gDNA from a specimen sample; b) a pair of forward and reverse primers capable of amplifying a gene or a variant thereof selected from the group consisting of CDH23, OTOF, COCH, ATP1A3, GRHL2, ITGA8, IQGAP2, PCDH15, PCDH20, GRM7, MTHFR, KCNQ1, KCNQ4 and SLC28A3 Amplifying the gene or a mutant thereof, and hybridizing the PNA probe with a PNA probe capable of hybridizing with the gene or a mutant thereof; And c) analyzing the fusion curve of the hybridized reactant in step b) to detect the gene or a mutant thereof.

The kit for predicting the risk of senile hearing loss according to the present invention reduces the social cost by preventing the disease, and the PNA probe and the MeltingArray method are simple, quick, accurate, and cost-effective.

FIG. 1 shows the PCR conditions of the genotype-type discrimination kit for senile hearing loss.
FIG. 2 shows the results of the present invention in comparison with a standard experiment (sanger sequencing).
FIG. 3 shows the result of multiplex analysis using a PNA probe for discriminating the genotypes related to senile hearing loss.
Fig. 4 shows the results of analyzing the reproducibility between PNA probes for genotyping related to senile hearing loss.
FIG. 5 shows the results of analyzing the reproducibility of the PNA probes for discriminating the genotypes related to the senile hearing loss.
FIG. 6 shows the results of analyzing the reproducibility of the PNA probes for discriminating the genotypes related to senile hearing loss according to the test time.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or an mRNA. The oligonucleotide probe, A single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection, but the present invention is not limited thereto.

The term "hybridization" of the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur either in perfect match between two nucleic acid strands, or even in the presence of some mismatching bases. The degree of complementarity required for hybridization can vary depending on the hybridization conditions, and can be controlled, in particular, by temperature.

In the present invention, a target nucleic acid means a nucleic acid sequence to be detected, and is annealed or hybridized with a primer or a probe under hybridization, annealing or amplification conditions.

The present invention aims to develop a fast and accurate method for genotyping the genes associated with senile hearing loss.

In the present invention, the genotype of the senile deafness-related gene was detected by the PNA probe, and it was confirmed that the genotype could be identified only by the analysis of the melting curve without complicated process.

That is, in one embodiment of the present invention, PNAs specifically binding to the SNPs of CDH23, OTOF, COCH, ATP1A3, GRHL2, ITGA8, IQGAP2, PCDH15, PCDH20, GRM7, MTHFR, KCNQ1, KCNQ4 and SLC28A3, When the probe was designed and the melting curve was analyzed using the probe, it was confirmed that the genotype of the gene could be determined only by analysis of the melting curve without further analysis (FIG. 3).

Thus, in one aspect, the present invention relates to a PNA probe for detecting hereditary deafness, which comprises any one of the sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.

PNA (Peptide Nucleic Acid) is a gene recognition material such as LNA (Locked Nucleic Acid) and MNA (Mopholino nucleic acid). It is synthesized artificially and its basic structure is composed of polyamide. PNA has very good affinity and selectivity and is highly stable against nucleic acid degrading enzymes and thus is not degraded into existing restriction enzymes. In addition, it has the advantage of being easy to store due to high thermal / chemical property and stability and not being easily decomposed. In addition, the PNA-DNA binding power is superior to the DNA-DNA binding force, resulting in a melting temperature (Tm) difference of about 10 to 15 ° C for one nucleotide mismatch. Using this difference in binding force, SNP (single nucleotide polymorphism) and InDel (insertion / deletion) nucleic acid changes can be detected.

The Tm value changes depending on the difference between the nucleic acid of the PNA probe and the DNA complementarily binding thereto, and it is easy to develop an application technique using the same. PNA probes are analyzed using a hybridization reaction that is different from the hydrolysis reaction of TaqMan probes. Molecular beacon probes and scorpion probes are similar probes.

Fluorescence Melting Curve Analysis (FMCA) is used for analysis of the hybridization reaction. Fluorescence melting curve analysis is performed by dividing the difference in binding force between the product produced after completion of the PCR reaction and the applied probe into the melting temperature . Unlike other SNP detection probes, the probe design is very simple and is made using 11 to 18 mer nucleotide sequences containing SNPs. Therefore, to design a probe with the desired fusion temperature, the Tm value can be adjusted according to the length of the PNA probe, and even the PNA probe of the same length can be adjusted to change the Tm value by changing the probe. Because PNA has better binding ability than DNA, it has a basic Tm value, so it can be designed shorter than DNA, so it can detect SNPs that are close to each other. In the conventional HRM method, the difference in Tm values is very small, about 0.5 ° C, so that additional analysis programs or detailed temperature changes are required. When two or more SNPs are present, analysis becomes difficult, whereas PNA probes are affected by SNPs other than probe sequences It is possible to analyze quickly and accurately.

In the present invention, the PNA probe is not limited to the CDH23 gene, but may be any of the rs121908354, rs373168635, rs137937502, rs746971522, rs121908349, A1713D, rs80356605, rs772729658, rs368155547 of the OCHF gene, G38D of the COCH gene, rs587777771 of the ATP1A3 gene, rs10955255, ITGA8 gene rs2236579, rs1417664, IQGAP2 gene rs457717, PCDH15 gene rs7081730, PCDH20 gene rs78043697, rd78043697 of the PCDH20 gene, rs11928865, rs779706, rs77901 and rs161927 of the GRM7 gene, rs1801133 and rs1801131 of the MTHFR gene, rs12277647 of the KCNQ1 gene, KCNQ4 gene and hybridizing with any one gene selected from the group consisting of rs727146, rs4660468, rs2149034, rs13374844, rs12143503 and rs7032430 of the SLC28A3 gene.

In the present invention, the PNA probe is not limited but may be characterized in that a reporter or a quencher is bonded thereto. The PNA probe comprising the reporter and the quencher of the present invention hybridizes with the target nucleic acid and generates a fluorescence signal. As the temperature rises, the fluorescence signal is rapidly extinguished with the target nucleic acid at the optimal melting temperature of the probe, The presence or absence of the target nucleic acid can be detected through the analysis of the melting curve of the high resolution obtained from the fluorescence signal according to the present invention.

In the present invention, a "PNA probe" is a probe that binds preferentially to a target nucleic acid in the presence of a target nucleic acid and exhibits a predicted melting temperature (Tm) (Tm), and the difference between the internal control and the target nucleic acid fusion curve can be used to distinguish the false positive and false negative signals. The PNA probes are analyzed using a hybridization method different from the hydrolysis method of the TaqMan probe. The probe having a similar role includes a molecular beacon probe and a scorpion probe (scorpion probe).

The probe of the present invention may include a fluorescent material of a quencher capable of quenching a reporter and a reporter fluorescence at both ends, and may include an intercalating fluorescent material. The reporter may be one or more selected from the group consisting of FAM (6-carboxyfluorescein), HEX, Texas red, JOE, TAMRA, CY5, CY3, and Alexa680. The demagnetizer may be TAMRA (6-carboxytetramethyl- rhodamine) BHQ2 or Dabcyl is preferably used, but not limited thereto. The intercalating fluorescent material may be selected from the group consisting of Acridine homodimer and derivatives thereof, Acridine Orange and derivatives thereof, 7-aminoactinomycin D (7-AAD) and Actinomycin D and derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) and derivatives thereof, DAPI and derivatives thereof, dihydroeti Ethidium bromide and derivatives thereof, Ethidium homodimer-1 (EthD-1) and derivatives thereof, Ethidium homodimer-2 (EthD-2) and derivatives thereof, Ethidium bromide and its derivatives, (Hexidium iodide and derivatives thereof, bisbenzimide (Hoechst 33258) and derivatives thereof, Hoechst 33342 and derivatives thereof, and derivatives thereof, Hoechst 345 < RTI ID = 0.0 > 80) and derivatives thereof, hydroxystilbamidine and derivatives thereof, LDS 751 and derivatives thereof, Propidium Iodide (PI) and derivatives thereof, and Cy-dyes ) Derivatives.

The present invention, in another aspect,

A nucleotide sequence of SEQ ID NO: 31 as a forward primer, a nucleotide sequence of SEQ ID NO: 32 as a reverse primer,

SEQ ID NO: 33 as a forward primer, SEQ ID NO: 34 as a reverse primer,

SEQ ID NO: 35 as a forward primer, SEQ ID NO: 36 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 37 as a forward primer, a nucleotide sequence of SEQ ID NO: 38 as a reverse primer,

SEQ ID NO: 39 as a forward primer, SEQ ID NO: 40 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 41 as a forward primer, a nucleotide sequence of SEQ ID NO: 42 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 43 as a forward primer, a nucleotide sequence of SEQ ID NO: 44 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 45 as a forward primer, a nucleotide sequence of SEQ ID NO: 46 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 47 as a forward primer, a nucleotide sequence of SEQ ID NO: 48 as a reverse primer,

SEQ ID NO: 49 as a forward primer, SEQ ID NO: 50 as a reverse primer,

SEQ ID NO: 51 as a forward primer, SEQ ID NO: 52 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 53 as a forward primer, a nucleotide sequence of SEQ ID NO: 54 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 55 as a forward primer, a nucleotide sequence of SEQ ID NO: 56 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 57 as a forward primer, a nucleotide sequence of SEQ ID NO: 58 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 59 as a forward primer, a nucleotide sequence of SEQ ID NO: 60 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 61 as a forward primer, a nucleotide sequence of SEQ ID NO: 62 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 63 as a forward primer, a nucleotide sequence of SEQ ID NO: 64 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 65 as a forward primer, a nucleotide sequence of SEQ ID NO: 66 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 67 as a forward primer, a nucleotide sequence of SEQ ID NO: 68 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 69 as a forward primer, a nucleotide sequence of SEQ ID NO: 70 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 71 as a forward primer, a nucleotide sequence of SEQ ID NO: 72 as a reverse primer,

The nucleotide sequence of SEQ ID NO: 73 as the forward primer, the nucleotide sequence of SEQ ID NO: 74 as the reverse primer;

A nucleotide sequence of SEQ ID NO: 75 as a forward primer, a nucleotide sequence of SEQ ID NO: 76 as a reverse primer,

The nucleotide sequence of SEQ ID NO: 77 as the forward primer, the nucleotide sequence of SEQ ID NO: 78 as the reverse primer;

A nucleotide sequence of SEQ ID NO: 79 as a forward primer, a nucleotide sequence of SEQ ID NO: 80 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 81 as a forward primer, a nucleotide sequence of SEQ ID NO: 82 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 83 as a forward primer, a nucleotide sequence of SEQ ID NO: 84 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 85 as a forward primer, a nucleotide sequence of SEQ ID NO: 86 as a reverse primer,

A nucleotide sequence of SEQ ID NO: 87 as a forward primer, a nucleotide sequence of SEQ ID NO: 88 as a reverse primer, And

SEQ ID NO: 89 as a forward primer, and SEQ ID NO: 90 as a reverse primer; and a primer pair for detecting hereditary deafness selected from the group consisting of:

In another aspect, the present invention relates to a composition for senile deafness-associated genotyping comprising the PNA probe and the primer pair.

The composition of the present invention may include, but is not limited to, one or more of the primer pairs, the PNA probes, and the FMCA buffer. For example, the PNA probes of SEQ ID NOs: 1 to 4 and SEQ ID NOs: 18 to 19 and the primer pairs of SEQ ID NOs: 31 and 32 can mutate the base mutations rs121908354, rs373168635, rs137937502, rs746971522, rs121908349 and A1713D of CDH23, The PNA probe of SEQ ID NO: 6 and the primer pair of SEQ ID NOs: 41 to 42 correspond to the rs78043697 base mutation of the PCDH20 gene, the PNA probes of SEQ ID NOS: PNA probes and the primer pairs of SEQ ID NOS: 43 to 46 correspond to the base mutations of ITGA8 or rs2236579 and rs1417664, the PNA probes of SEQ ID NO: 9 and the primer pairs of SEQ ID NOS: 47 to 48 correspond to the G38D base mutation of the COCH gene, 10 and the primer pairs of SEQ ID Nos. 49 to 50 are the base mutation of rs587777771 of the gene of ATP1A3, the PNA probe of SEQ ID NO: 11 and the primers of SEQ ID NOS: 51 to 52 A pair of PNA probes of SEQ ID Nos. 12 to 15 and a pair of primers of SEQ ID Nos. 53 to 60, a base mutation of rs11928865, rs779706, rs779701, rs161927 base mutation of the GRM7 gene, a PNA probe of SEQ ID NO: 62, the PNA probe of SEQ ID NO: 17 and the primer pair of SEQ ID NOs: 63 to 64 are the base mutation of rs12277647 of KCNQ1, the PNA probe of SEQ ID NOs: 20 to 21 and the PNA probe of SEQ ID NO: (Rs1801133, rs1801131), the PNA probes of SEQ ID NOS: 22 to 26 and the primer pairs of SEQ ID Nos. 69 to 76 and SEQ ID Nos: 81 to 82 are the KCNQ4 gene or the rs727146, rs4660468, rs2149034, rs13374844, rs12143503 base mutation, the PNA probe of SEQ ID NO: 27 and the primer pair of SEQ ID NO: 83 or 84 are rs10955255 base mutation of GRHL2, the PNA probe of SEQ ID NO: 28 to 30 A pair of primers of SEQ ID NOS: 85-90 may be the detection of the PCR composition OTOF gene or nucleotide mutation (rs80356605, rs772729658, rs368155547).

According to another aspect of the present invention, there is provided a method for producing a sample, comprising the steps of: a) extracting gDNA from a specimen sample; b) a pair of forward and reverse primers capable of amplifying a gene selected from the group consisting of CDH23, OTOF, COCH, ATP1A3, GRHL2, ITGA8, IQGAP2, PCDH15, PCDH20, GRM7, MTHFR, KCNQ1, KCNQ4 and SLC28A3 Amplifying the gene or a mutant thereof, and hybridizing the PNA probe with a PNA probe capable of hybridizing with the gene or a mutant thereof; And c) analyzing the fusion curve of the hybridized reactant in step b) to detect the gene or a mutant thereof.

In the present invention, the sample is DNA or RNA, and the molecule may be a double-stranded or single-stranded form. When the nucleic acid as the starting material is a double strand, it is preferable to make the two strands into a single strand, or a partial single strand form. Methods known to separate strands include, but are not limited to, heat, alkaline, formamide, urea and glycoconjal treatment, enzymatic methods (e.g., helicase action) and binding proteins. For example, the strand separation can be achieved by heat treatment at a temperature of 80 to 105 ° C.

In the present invention, the above-mentioned discrimination method shows a predicted melting temperature (Tm) value by preferentially binding to a target when a target DNA or RNA exists, and when the target is absent, that is, in the case of mutation, , And it is possible to discriminate the senile hearing-related genotype by using this.

The variants of the gene of the present invention include, but are not limited to, rs121908354, rs373168635, rs137937502, rs746971522, rs121908349, A1713D, rs80356605, rs772729658, rs368155547 of the CDH23 gene, G38D of the COCH gene, rs587777771 of the ATP1A3 gene of the OCHF gene, rs10955255, ITGA8 gene rs2236579, rs1417664, IQGAP2 gene rs457717, PCDH15 gene rs7081730, PCDH20 gene rs78043697, rd78043697 of the PCDH20 gene, rs11928865, rs779706, rs77901 and rs161927 of the GRM7 gene, rs1801133 and rs1801131 of the MTHFR gene, rs12277647 of the KCNQ1 gene, KCNQ4 gene rs727146, rs4660468, rs2149034, rs13374844, rs12143503 and rs7032430 of the SLC28A3 gene.

In the present invention, the sample of the sample is not limited, but may be a DNA sample derived from human hair, oral epithelial cells, sputum, blood, saliva, or urine.

In the present invention, a "specimen sample" includes various specimens, and preferably a bio-sample is analyzed using the method of the present invention. Biological samples of plant, animal, human, fungal, bacterial and viral origin can be analyzed. When analyzing a sample of mammalian or human origin, the sample may be from a particular tissue or organ. Representative examples of tissues include binding, skin, muscle or nervous tissue. Representative examples of organs include, but are not limited to, eyes, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testes, Line and inner blood vessels. The biological sample to be analyzed includes any cells, tissues, fluids from the biological source, or any other medium that can be well analyzed by the present invention, including human, animal, human or animal Lt; RTI ID = 0.0 > a < / RTI > In addition, the biological sample to be analyzed includes a body fluid sample, which may be a blood sample, serum, plasma, lymph, milk, urine, feces, eye milk, saliva, semen, brain extract And tonsil tissue extracts.

In the present invention, the melting curve analysis method is not limited thereto, but may be performed by a fluorescence melting curve analysis (FMCA) method.

The fusion curve analysis of the present invention is a method of analyzing the melting temperature of a double-stranded nucleic acid formed with a target DNA or RNA and a probe. Such a method is called a fusion curve analysis because it is performed, for example, by Tm analysis or analysis of the melting curve of the double chain. A hybrid (double-stranded DNA) of the target single-stranded DNA of the detection sample and the probe is formed using a probe complementary to the detection target sequence including the mutation for detection purpose, and then the hybridization product is subjected to heat treatment (Melting) of a hybrid due to a rise in temperature is detected by variation of a signal such as an absorbance and then a Tm value is determined based on the detection result to determine the presence or absence of a point mutation to be.

The Tm value is higher as the homology of the hybrid construct is higher, and lower as the homology is lower. Therefore, a Tm value (evaluation reference value) is previously obtained for a hybrid formed body of a detection target sequence including a point mutation and a probe complementary thereto, and the Tm value of the target single strand DNA of the detection sample and the probe If the measured value is equal to the evaluation reference value, it can be determined that a mutation exists in the match, that is, in the target DNA. If the measured value is lower than the evaluation reference value, a mismatch, that is, It can be judged that it does not exist.

The fluorescence melting curve analysis of the present invention is a method of analyzing a melting curve using a fluorescent substance, and more specifically, a melting curve can be analyzed using a probe containing a fluorescent substance. The fluorescent material may be a reporter and a quencher, and may be an intercalating fluorescent material.

In the present invention, the amplification is not limited, but real-time PCR can be used.

In the real-time PCR method of the present invention, a fluorescent substance is interchelated in a double strand DNA chain in a PCR process, and the DNA product is amplified by amplifying the PCR product, (Tm) at which the DNA is melted (denatured) by analyzing the pattern of the melting curve at which the amount of the fluorescent substance present in the sample is reduced, in particular, the mutation of the base sequence between the normal control and the mutation.

Hereinafter, the present invention will be described in more detail by way of examples. However, these embodiments are provided to aid understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense.

Example 1: Preparation of a risk prediction probe for prevention of senile sensory organs diseases

Studies on base mutations confirmed to have clinical relevance to genes related to senile hearing loss have been actively conducted, and mutations related to Asian and Korean have been obtained. A kit was developed to predict the risk of senile sensory disease using 14 genes and 30 SNPs related to senile hearing loss (Table 1).

The PNA probes that can specifically bind to the base mutation site were designed to be able to distinguish between wild type and mutant type and designed as shown in Table 2. PNA probes were synthesized by HPLC purification method in panagene (Korea) and the purity of all synthesized probes was confirmed by mass spectrometry. In addition, FAM, HEX, Texas Red, and Cy5 fluorescence were selected for multiple analysis, and unnecessary secondary structure was avoided.

In order to perform PCR using human DNA extracted from human samples such as blood, tissue, body fluids, etc., eight kits were constructed and multiple analyzes were performed.

Genetic and genetic information related to senile hearing loss No. Gene Nucleotide change Location REF seq. ALT seq. One CDH23 rs121908354 Chr10: 73330641 C T 2 rs373168635 Chr10: 73405652 C T 3 rs137937502 Chr10: 73501595 C T 4 rs746971522 Chr10: 73545422 G A 5 rs121908349 Chr10: 73553289 G A 6 A1713D Chr10: 73538016 C A 7 OTOF rs80356605 chr2: 26460203 C T 8 rs772729658 Chr2: 26477174 C T 9 rs368155547 Chr2: 26460998 G A 10 COCH G38D (G1113A) chr14: 30877602 G A 11 ATP1A3 rs587777771 chr19: 41970275 C T 12 GRHL2 rs10955255 chr8: 101524177 A G 13 ITGA8 rs2236579 chr10: 15720115 A G 14 rs1417664 chr10: 15696655 T G 15 IQGAP2 rs457717 chr5: 76625147 A G 16 PCDH15 rs7081730 chr10: 54273261 C T 17 PCDH20 rs78043697 chr13: 61892906 T C 18 GRM7 rs11928865 chr3: 7114015 T A 19 rs779706 chr3: 7524042 G C 20 rs779701 chr3: 7518772 A G 21 rs161927 chr3: 7796555 G A 22 MTHFR rs1801133 Chr1: 11796321 G A 23 rs1801131 Chr1: 11794419 T G 24 KCNQ1 rs12277647 Chr11: 2442795 C A 25 KCNQ4 rs727146 Chr1: 40806587 A G 26 rs4660468 Chr1: 40819415 T C 27 rs2149034 Chr1: 40811960 T A 28 rs13374844 Chr1: 40817247 C T 29 rs12143503 Chr1: 40819919 A G 30 SLC28A3 rs7032430 Chr9: 84099087 C A

The mutated portion of the base is usually designed to be positioned in the middle of the PNA probe binding site so that the difference between the target nucleic acid and the melting temperature (Tm) is 5 ° C or more. However, in order to optimize the melting temperature (Tm) (See Table 2). In the PNA probe, quencher used Dabcyl, O means linker, and K means lysine.

PNA probe sequence for discriminating genotypes related to senile hearing loss SEQ ID NO: Probe name Base sequence SEQ ID NO: 1 CDH23_R1916H Dabcyl-CTTGAAAATGAAAATG-O-K (FAM) SEQ ID NO: 2 CDH23_R1588W Dabcyl-TAAACTGACATAACCTG-O-K (HEX) SEQ ID NO: 3 CDH23_P240L Dabcyl-AATCAAGGATTGATAC-O-K (TxR) SEQ ID NO: 4 CDH23_D2202N Dabcyl-GCAAATGAAAAGTAC-O-K (Cy5) SEQ ID NO: 5 PCDH15 Dabcyl-TCACTGCCACAGAC-O-K (FAM) SEQ ID NO: 6 PCDH20 Dabcyl-ATCTCCCCGACCTCC-O-K (HEX) SEQ ID NO: 7 ITGA8_1 Dabcyl-AGACACTTTCTTCACTG-O-K (TxR) SEQ ID NO: 8 ITGA8_2 Dabcyl-GAAATCGGCTCCCT-O-K (Cy5) SEQ ID NO: 9 COCH Dabcyl-TCCTTTGTTGGATGAA-O-K (FAM) SEQ ID NO: 10 ATP1A3 Dabcyl-CTTCTCAGCCAGGTA-O-K (HEX) SEQ ID NO: 11 IQGAP2 Dabcyl-CGCACACATGGCT-O-K (Cy5) SEQ ID NO: 12 GRM7_1 Dabcyl-CCCTCTCCCTCATGTTG-O-K (FAM) SEQ ID NO: 13 GRM7_2 Dabcyl-GGTTTCTGTCCCAC-O-K (HEX) SEQ ID NO: 14 GRM7_779701 Dabcyl-TTAGCATAAAGTGATTC-O-K (TxR) SEQ ID NO: 15 GRM7_4 Dabcyl-CGCACCTCGTCCG-O-K (Cy5) SEQ ID NO: 16 SLC28A3_1 Dabcyl-GCCCCGCGGG-O-K (FAM) SEQ ID NO: 17 KCNQ1 Dabcyl-CAGCCGGCCCG-O-K (HEX) SEQ ID NO: 18 CDH23_A1713D Dabcyl-CTGGACCGCGAG-O-K (TxR) SEQ ID NO: 19 CDH23_P402L Dabcyl-CCACACGGCCTG-O-K (Cy5) SEQ ID NO: 20 1298AC Dabcyl-AACCTGCCTTACAG-O-K (FAM) SEQ ID NO: 21 677CT Dabcyl-TGACCACGACCTCA-O-K (HEX) SEQ ID NO: 22 KCNQ4_3 Dabcyl-GAAGACTACTGAATAC-O-K (TxR) SEQ ID NO: 23 KCNQ4_4 Dabcyl-ATTTAAAAAATAAGACA-O-K (Cy5) SEQ ID NO: 24 KCNQ4_1 Dabcyl-ATACATATAAAATTAGG-O-K (FAM) SEQ ID NO: 25 KCNQ4_2 Dabcyl-TCTATTGCAGTCCCATC-O-K (HEX) SEQ ID NO: 26 KCNQ4 Dabcyl-TCCAAGCCTCTGGT-O-K (TxR) SEQ ID NO: 27 GRHL2 Dabcyl-TCGCTTTCGGCAG-O-K (Cy5) SEQ ID NO: 28 OTOF-2 Dabcyl-CGTTAGTTCAATGTTTC-O-K (FAM) SEQ ID NO: 29 OTOF-1 Dabcyl-AAATTACAGTAAAAGC-O-K (HEX) SEQ ID NO: 30 OTOF Dabcyl-TGATTTCAATTGCCCTA-O-K (TxR)

In addition, bidirectional primers were designed (Table 3, 4) to amplify the mutation sites that can predict the risk of senile organ disease (Table 3, 4) and to avoid unnecessary secondary structure in PNA and multiplex analysis. The gene was synthesized and used as a reference material.

Primer sequences for discriminating genotypes related to senile hearing loss SEQ ID NO: Primer name Base sequence SEQ ID NO: 31 R1916H_F GCCAAACTGAAGAGAGAAATTA SEQ ID NO: 32 R1916H_R TAGCCAATCCTAATCCTGCATAC SEQ ID NO: 33 R1588W_F CAAGGTCTTTCAGGAGACAGG SEQ ID NO: 34 R1588W_R TGACACGGGACTTCAATGACAGAG SEQ ID NO: 35 CDH23e8-F CAGCTGTGTCATGGCAAGTAAAGA SEQ ID NO: 36 CDH23e8-R CACTTCCCTGAAGCTCTTCAGAG SEQ ID NO: 37 D2202N_F CTCTTCTCTCTCAACTGTAGTTGC SEQ ID NO: 38 D2202N_R GTCTCAATCCCATAGACATTAACC SEQ ID NO: 39 PCDH15_F TACCTTAATGGGACATGGTTACC SEQ ID NO: 40 PCDH15_R CAAGGACACCTCAATAATGATGC SEQ ID NO: 41 PCDH20_F ATCCTATCAACTGAAAGTACTAGAG SEQ ID NO: 42 PCDH20_R GGTTCATGACCTTTGCACTTATG SEQ ID NO: 43 ITGA8_2_F CTGCCTCTCCCACAGAGTTGCC SEQ ID NO: 44 ITGA8_2_R GGTGCTGCAGACCTACCACCC SEQ ID NO: 45 ITGA8_1_F GGCTGTGTCCCATGCACCCAC SEQ ID NO: 46 ITGA8_1_R CCCTTCCTGACTGGCTTCTTCTG SEQ ID NO: 47 COCH_F GCCAGTGAGTGGGGCCTTACATC SEQ ID NO: 48 COCH_R CCTGCCTGTAACCTGTTTGTGTCT SEQ ID NO: 49 ATP1A3_F GGCAGGCAGGGCAAAGAAAG SEQ ID NO: 50 ATP1A3_R GCTCTGTAACCCCCTGCCAGG SEQ ID NO: 51 IQGAP2_F GGACAGCACAGACAGCACCAG SEQ ID NO: 52 IQGAP2_R GCCAGCCAGCACCCATGTTTCAT SEQ ID NO: 53 GRM7_1F GAGCCCCTTAGCTGGGGTCTC SEQ ID NO: 54 GRM7_1R TACAAAGACCCTCACGCACCG SEQ ID NO: 55 GRM72F GTACCAGGCGGTGATCAGCTC SEQ ID NO: 56 GRM72R TGAAGAGCAAGTCCCCCAAGGA SEQ ID NO: 57 GRM7: rs779701_F CTTTGTGACCATTCCGGTTT SEQ ID NO: 58 GRM7: rs779701_R CGAAGCAGGGAGCTTTG SEQ ID NO: 59 GRM7_4F GGTGCATGCCTTCACAA SEQ ID NO: 60 GRM7_4R ATATCTGTCCTCTACCTCCAGAAGC SEQ ID NO: 61 SLC28A3_F GGTGGGAGAGGAGGAAGCTATT SEQ ID NO: 62 SLC28A3_R GGTAGTAGCTGCCAGGTTTCTGC SEQ ID NO: 63 KCNQ1_F GCAAAGAAAACTAACAAACTGGCAAA SEQ ID NO: 64 KCNQ1_R CTGATCCTGAGGGGCCCCAG SEQ ID NO: 65 A1713D_F GGGACAAGCTGAGGCTGTG SEQ ID NO: 66 A1713D_R GACACGAGGGGCACGTCC SEQ ID NO: 67 P402L_F AGCCCTGTTCACTCTGAGCTG SEQ ID NO: 68 P402L_R CACACCCAATCCCTTCTCCTCC

Primer sequences for discriminating genotypes related to senile hearing loss (continued) SEQ ID NO: Primer name Base sequence SEQ ID NO: 69 KCNQ4_1_F GTGTGCCACAAGAAGTAGCGAGC SEQ ID NO: 70 KCNQ4_1_R GTCTCCATGCAGCTCACCACC SEQ ID NO: 71 KCNQ4_2_F CACCAGTGCCCTCCAGTCTCA SEQ ID NO: 72 KCNQ4_2_R CCACTGACCGTGACATCGGGAT SEQ ID NO: 73 KCNQ4_3_F CCATCGCTCACCGACAGGGTTG SEQ ID NO: 74 KCNQ4_3_R CTTGGCCATCATCATCACAGAT SEQ ID NO: 75 KCNQ4_4_F CCTAACAGGAGCTCAGAAGGA SEQ ID NO: 76 KCNQ4_4_R CCCGAGTTCCTCAACCCCATCC SEQ ID NO: 77 MTHFR_1298F GTCCTCCTGGTTGGGCACCAG SEQ ID NO: 78 MTHFR_1298R GAACTTAAACAGAAGCTTGCATG SEQ ID NO: 79 MTHFR_677F TCATTATAATTTACACTTATTTTGTGTG SEQ ID NO: 80 MTHFR_677R GTACAGCTAGGGTAATTTTTGGCTT SEQ ID NO: 81 KCNQ4_F AGTACACTAAAAACCAAGACTTTGC SEQ ID NO: 82 KCNQ4_R CTGGTCTAGAGCCAGGGACTC SEQ ID NO: 83 GRHL2_F ACCTGGGGCATTGCTTCTAAAGC SEQ ID NO: 84 GRHL2_R GATTACAGGCATGAGCCACTGTG SEQ ID NO: 85 OTOF2_F ACTTGTCACTTACCAATCTGGC SEQ ID NO: 86 OTOF2_R AGAATTCCTCAAGAGGGCAGCCC SEQ ID NO: 87 OTOF1_F CCACCACTATGCCCCAAGAAGTC SEQ ID NO: 88 OTOF1_R CTCACCAATCTGCCCGTAGGC SEQ ID NO: 89 OTOF_F CTCCCCTCTCCGGCTCACCC SEQ ID NO: 90 OTOF_R AGACTTTACATTGATAAGATTCAGC

Example 2: Melting curve analysis for discriminating genotypes related to senile hearing loss

Genomic DNA was amplified and analyzed by melting curve to discriminate the base mutation region.

Single-stranded target nucleic acid generation for melting curve analysis was performed using a CFX96 ™ Real-Time system (BIO-RAD, USA). The primers and PNA probes prepared in Example 1 were used and the PCR conditions were as follows; 10 μl of 2 × qPCR PreMix (Seen Biomaterials, Korea), 0.5 μl of bidirectional primer, 0.5 μl of PNA and 3 μl of human genomic DNA (15 ng / μl) were added and distilled water was added to make a total volume of 20 μl Real-time PCR was performed.

 Real-time PCR was performed by denaturation at 95 ° C for 10 minutes, followed by 45 cycles of 95 ° C for 30 seconds, 58 ° C for 45 seconds, and 72 ° C for 45 seconds. Fluorescence was measured in real time. Melting curve analysis was carried out by denaturation at 95 ° C for 5 minutes, followed by gradually increasing the temperature to hybridize, then increasing the temperature from 30 ° C to 85 ° C by 0.5 ° C, maintaining the stationary state for 0.5 seconds, (Fig. 1).

As a result of melting curve analysis using human genomic DNA as a template, it was confirmed that a difference in melting temperature (Tm) according to base mutation occurred. To confirm this, Sanger sequencing was performed (FIG. 2).

Multiple analysis of the kit for predicting the risk associated with senile sensory organs disease was established and the Melting temperature of the PNA probe was matched with the base mutation (FIG. 3).

Table 3 shows the results of the test. Table 3 shows the results of the test. Table 3 shows the results of the test.

Elderly hearing loss related disease risk prediction kit Set Gene: Mutation Flour. Wild Mutant Heterozygote 1 set CDH23: R1916H FAM 74 ± 3 ° C 66 ± 3 ° C 74 ± 3 ° C, 66 ± 3 ° C CDH23: R1588W HEX 70 ± 3 ° C 51 ± 3 ° C 70 ± 3 ° C, 51 ± 3 ° C CDH23: P240L TxR 68 ± 3 ° C 51 ± 3 ° C 68 ± 3 ° C, 51 ± 3 ° C CDH23: D2202N Cy5 68 ± 3 ° C 61 ± 3 ° C 68 ± 3 ° C, 61 ± 3 ° C 2 sets PCDH15: rs7081730 FAM 64 ± 3 ° C 53 ± 3 ° C 64 ± 3 ° C, 53 ± 3 ° C PCDH20: rs78043697 HEX 57 ± 3 ° C 45 ± 3 ° C 57 ± 3 ° C, 45 ± 3 ° C ITGA8: rs1417664 TxR 62 ± 3 ° C 54 ± 3 ° C 62 ± 3 ° C, 54 ± 3 ° C ITGA8: rs2236579 Cy5 65 ± 3 ° C 56 ± 3 ° C 65 ± 3 ° C, 56 ± 3 ° C 3 sets COCH: G38D FAM 67 ± 3 ° C 51 ± 3 ° C 67 ± 3 ° C, 51 ± 3 ° C ATP1A3: E831K HEX 65 ± 3 ° C 49 ± 3 ° C 65 ± 3 ° C, 49 ± 3 ° C IQGAP2: rs457717 Cy5 65 ± 3 ° C 56 ± 3 ° C 65 ± 3 ° C, 56 ± 3 ° C 4 sets GRM7: rs161927 FAM 63 ± 3 ° C 57 ± 3 ° C 63 ± 3 ° C, 57 ± 3 ° C GRM7: rs11928865 HEX 61 ± 3 ° C 55 ± 3 ° C 61 ± 3 ° C, 55 ± 3 ° C GRM7: rs779701 TxR 65 ± 3 ° C 55 ± 3 ° C 65 ± 3 ° C, 55 ± 3 ° C GRM7: rs779706 Cy5 65 ± 3 ° C 56 ± 3 ° C 65 ± 3 ° C, 56 ± 3 ° C 5 sets SLC28A3: rs7032430 FAM 63 ± 3 ° C 52 ± 3 ° C 63 ± 3 ° C, 52 ± 3 ° C KCNQ1 HEX 64 ± 3 ° C 51 ± 3 ° C 64 ± 3 ° C, 51 ± 3 ° C CDH23: A1713D TxR 72 ± 3 ° C 57 ± 3 ° C 72 ± 3 ° C, 57 ± 3 ° C CDH23: P402L Cy5 69 ± 3 ° C 54 ± 3 ° C 69 ± 3 ° C, 54 ± 3 ° C 6 sets MTHFR: 1298AC FAM 62 ± 3 ° C 51 ± 3 ° C 62 ± 3 ° C, 51 ± 3 ° C MTHFR: 677CT HEX 64 ± 3 ° C 55 ± 3 ° C 64 ± 3 ° C, 55 ± 3 ° C KCNQ4: rs2149034 TxR 66 ± 3 ° C 59 ± 3 ° C 66 ± 3 ° C, 59 ± 3 ° C KCNQ4: rs4660468 Cy5 68 ± 3 ° C 55 ± 3 ° C 68 ± 3 ° C, 55 ± 3 ° C 7 sets KCNQ4: rs12143503 FAM 71 ± 3 ° C 51 ± 3 ° C 71 ± 3 ° C, 51 ± 3 ° C KCNQ4: rs13374844 HEX 65 ± 3 ° C 41 ± 3 ° C 65 ± 3 ° C, 41 ± 3 ° C KCNQ4: rs727146 TxR 68 ± 3 ° C 59 ± 3 ° C 68 ± 3 ° C, 59 ± 3 ° C GRHL2: rs10955255 Cy5 64 ± 3 ° C 55 ± 3 ° C 64 ± 3 ° C, 55 ± 3 ° C 8 sets OTOF: rs772729658 FAM 71 ± 3 ° C 52 ± 3 ° C 71 ± 3 ° C, 52 ± 3 ° C OTOF: rs368155547 HEX 67 ± 3 ° C 56 ± 3 ° C 67 ± 3 ° C, 56 ± 3 ° C OTOF: rs80356605 TxR 75 ± 3 ° C 66 ± 3 ° C 75 ± 3 ° C, 66 ± 3 ° C

Example 3: Analytical reproducibility test of genotype-type discrimination kit for senile hearing loss

In order to confirm the analytical reproducibility of the genetic type discrimination kit for the senile hearing loss, it was confirmed whether the reproducibility according to the interval of the experimenter, equipment, and analysis time was maintained.

In order to confirm the reproducibility between the experimenters, three experimenters independently performed experiments (FIG. 4), and it was confirmed that the reproducibility was high in all the markers.

In order to confirm the reproducibility between the instruments used, experiments were performed using the other three CFX96 real-time PCR instruments, and it was confirmed that all markers were included in the Tm value in the judgment table (FIG. 5).

In order to see the reproducibility according to the test time, three tests were performed at intervals of one week, and the Tm values of all the markers were appropriate to the judgment criteria and it was confirmed that the reproducibility with respect to the test time was high (FIG. 6).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

                         SEQUENCE LISTING <110> SeasunBio Materials   <120> Pepetide nucleic acid probe for genotyping age related hearing        loss (presbycusis) and Method using the same &Lt; 130 > P17-B321 <160> 90 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> DNA <213> Artificia Sequence <400> 1 cttgaaaatg aaaatg 16 <210> 2 <211> 17 <212> DNA <213> Artificia Sequence <400> 2 taaactgaca taacctg 17 <210> 3 <211> 16 <212> DNA <213> Artificia Sequence <400> 3 aatcaaggat tgatac 16 <210> 4 <211> 15 <212> DNA <213> Artificia Sequence <400> 4 gcaaatgaaa agtac 15 <210> 5 <211> 14 <212> DNA <213> Artificia Sequence <400> 5 tcactgccac agac 14 <210> 6 <211> 15 <212> DNA <213> Artificia Sequence <400> 6 atctccccga cctcc 15 <210> 7 <211> 17 <212> DNA <213> Artificia Sequence <400> 7 agacactttc ttcactg 17 <210> 8 <211> 14 <212> DNA <213> Artificia Sequence <400> 8 gaaatcggct ccct 14 <210> 9 <211> 16 <212> DNA <213> Artificia Sequence <400> 9 tcctttgttg gatgaa 16 <210> 10 <211> 15 <212> DNA <213> Artificia Sequence <400> 10 cttctcagcc aggta 15 <210> 11 <211> 13 <212> DNA <213> Artificia Sequence <400> 11 cgcacacatg gct 13 <210> 12 <211> 17 <212> DNA <213> Artificia Sequence <400> 12 ccctctccct catgttg 17 <210> 13 <211> 14 <212> DNA <213> Artificia Sequence <400> 13 ggtttctgtc ccac 14 <210> 14 <211> 17 <212> DNA <213> Artificia Sequence <400> 14 ttagcataaa gtgattc 17 <210> 15 <211> 13 <212> DNA <213> Artificia Sequence <400> 15 cgcacctcgt ccg 13 <210> 16 <211> 10 <212> DNA <213> Artificia Sequence <400> 16 gccccgcggg 10 <210> 17 <211> 11 <212> DNA <213> Artificia Sequence <400> 17 cagccggccc g 11 <210> 18 <211> 12 <212> DNA <213> Artificia Sequence <400> 18 ctggaccgcg ag 12 <210> 19 <211> 12 <212> DNA <213> Artificia Sequence <400> 19 ccacacggcc tg 12 <210> 20 <211> 14 <212> DNA <213> Artificia Sequence <400> 20 aacctgcctt acag 14 <210> 21 <211> 14 <212> DNA <213> Artificia Sequence <400> 21 tgaccacgac ctca 14 <210> 22 <211> 16 <212> DNA <213> Artificia Sequence <400> 22 gaagactact gaatac 16 <210> 23 <211> 17 <212> DNA <213> Artificia Sequence <400> 23 atttaaaaaa taagaca 17 <210> 24 <211> 17 <212> DNA <213> Artificia Sequence <400> 24 atacatataa aattagg 17 <210> 25 <211> 17 <212> DNA <213> Artificia Sequence <400> 25 tctattgcag tcccatc 17 <210> 26 <211> 14 <212> DNA <213> Artificia Sequence <400> 26 tccaagcctc tggt 14 <210> 27 <211> 13 <212> DNA <213> Artificia Sequence <400> 27 tcgctttcgg cag 13 <210> 28 <211> 17 <212> DNA <213> Artificia Sequence <400> 28 cgttagttca atgtttc 17 <210> 29 <211> 16 <212> DNA <213> Artificia Sequence <400> 29 aaattacagt aaaagc 16 <210> 30 <211> 17 <212> DNA <213> Artificia Sequence <400> 30 tgatttcaat tgcccta 17 <210> 31 <211> 22 <212> DNA <213> Artificia Sequence <400> 31 gccaaactga agagagaaat ta 22 <210> 32 <211> 23 <212> DNA <213> Artificia Sequence <400> 32 tagccaatcc taatcctgca tac 23 <210> 33 <211> 21 <212> DNA <213> Artificia Sequence <400> 33 caaggtcttt caggagacag g 21 <210> 34 <211> 24 <212> DNA <213> Artificia Sequence <400> 34 tgacacggga cttcaatgac agag 24 <210> 35 <211> 24 <212> DNA <213> Artificia Sequence <400> 35 cagctgtgtc atggcaagta aaga 24 <210> 36 <211> 23 <212> DNA <213> Artificia Sequence <400> 36 cacttccctg aagctcttca gag 23 <210> 37 <211> 24 <212> DNA <213> Artificia Sequence <400> 37 ctcttctctc tcaactgtag ttgc 24 <210> 38 <211> 24 <212> DNA <213> Artificia Sequence <400> 38 gtctcaatcc catagacatt aacc 24 <210> 39 <211> 23 <212> DNA <213> Artificia Sequence <400> 39 taccttaatg ggacatggtt acc 23 <210> 40 <211> 23 <212> DNA <213> Artificia Sequence <400> 40 caaggacacc tcaataatga tgc 23 <210> 41 <211> 25 <212> DNA <213> Artificia Sequence <400> 41 atcctatcaa ctgaaagtac tagag 25 <210> 42 <211> 23 <212> DNA <213> Artificia Sequence <400> 42 ggttcatgac ctttgcactt atg 23 <210> 43 <211> 22 <212> DNA <213> Artificia Sequence <400> 43 ctgcctctcc cacagagttg cc 22 <210> 44 <211> 21 <212> DNA <213> Artificia Sequence <400> 44 ggtgctgcag acctaccacc c 21 <210> 45 <211> 21 <212> DNA <213> Artificia Sequence <400> 45 ggctgtgtcc catgcaccca c 21 <210> 46 <211> 23 <212> DNA <213> Artificia Sequence <400> 46 cccttcctga ctggcttctt ctg 23 <210> 47 <211> 23 <212> DNA <213> Artificia Sequence <400> 47 gccagtgagt ggggccttac atc 23 <210> 48 <211> 24 <212> DNA <213> Artificia Sequence <400> 48 cctgcctgta acctgtttgt gtct 24 <210> 49 <211> 20 <212> DNA <213> Artificia Sequence <400> 49 ggcaggcagg gcaaagaaag 20 <210> 50 <211> 21 <212> DNA <213> Artificia Sequence <400> 50 gctctgtaac cccctgccag g 21 <210> 51 <211> 21 <212> DNA <213> Artificia Sequence <400> 51 ggacagcaca gacagcacca g 21 <210> 52 <211> 23 <212> DNA <213> Artificia Sequence <400> 52 gccagccagc acccatgttt cat 23 <210> 53 <211> 21 <212> DNA <213> Artificia Sequence <400> 53 gagcccctta gctggggtct c 21 <210> 54 <211> 21 <212> DNA <213> Artificia Sequence <400> 54 tacaaagacc ctcacgcacc g 21 <210> 55 <211> 21 <212> DNA <213> Artificia Sequence <400> 55 gtaccaggcg gtgatcagct c 21 <210> 56 <211> 22 <212> DNA <213> Artificia Sequence <400> 56 tgaagagcaa gtcccccaag ga 22 <210> 57 <211> 20 <212> DNA <213> Artificia Sequence <400> 57 ctttgtgacc attccggttt 20 <210> 58 <211> 17 <212> DNA <213> Artificia Sequence <400> 58 cgaagcaggg agctttg 17 <210> 59 <211> 17 <212> DNA <213> Artificia Sequence <400> 59 ggtgcatgcc ttcacaa 17 <210> 60 <211> 25 <212> DNA <213> Artificia Sequence <400> 60 atatctgtcc tctacctcca gaagc 25 <210> 61 <211> 22 <212> DNA <213> Artificia Sequence <400> 61 ggtgggagag gaggaagcta tt 22 <210> 62 <211> 23 <212> DNA <213> Artificia Sequence <400> 62 ggtagtagct gccaggtttc tgc 23 <210> 63 <211> 26 <212> DNA <213> Artificia Sequence <400> 63 gcaaagaaaa ctaacaaact ggcaaa 26 <210> 64 <211> 20 <212> DNA <213> Artificia Sequence <400> 64 ctgatcctga ggggccccag 20 <210> 65 <211> 19 <212> DNA <213> Artificia Sequence <400> 65 gggacaagct gaggctgtg 19 <210> 66 <211> 18 <212> DNA <213> Artificia Sequence <400> 66 gacacgaggg gcacgtcc 18 <210> 67 <211> 21 <212> DNA <213> Artificia Sequence <400> 67 agccctgttc actctgagct g 21 <210> 68 <211> 22 <212> DNA <213> Artificia Sequence <400> 68 cacacccaat cccttctcct cc 22 <210> 69 <211> 23 <212> DNA <213> Artificia Sequence <400> 69 gtgtgccaca agaagtagcg agc 23 <210> 70 <211> 21 <212> DNA <213> Artificia Sequence <400> 70 gtctccatgc agctcaccac c 21 <210> 71 <211> 21 <212> DNA <213> Artificia Sequence <400> 71 caccagtgcc ctccagtctc a 21 <210> 72 <211> 22 <212> DNA <213> Artificia Sequence <400> 72 ccactgaccg tgacatcggg at 22 <210> 73 <211> 22 <212> DNA <213> Artificia Sequence <400> 73 ccatcgctca ccgacagggt tg 22 <210> 74 <211> 22 <212> DNA <213> Artificia Sequence <400> 74 cttggccatc atcatcacag at 22 <210> 75 <211> 21 <212> DNA <213> Artificia Sequence <400> 75 cctaacagga gctcagaagg a 21 <210> 76 <211> 22 <212> DNA <213> Artificia Sequence <400> 76 cccgagttcc tcaaccccat cc 22 <210> 77 <211> 21 <212> DNA <213> Artificia Sequence <400> 77 gtcctcctgg ttgggcacca g 21 <210> 78 <211> 23 <212> DNA <213> Artificia Sequence <400> 78 gaacttaaac agaagcttgc atg 23 <210> 79 <211> 28 <212> DNA <213> Artificia Sequence <400> 79 tcattataat ttacacttat tttgtgtg 28 <210> 80 <211> 25 <212> DNA <213> Artificia Sequence <400> 80 gtacagctag ggtaattttt ggctt 25 <210> 81 <211> 25 <212> DNA <213> Artificia Sequence <400> 81 agtacactaa aaaccaagac tttgc 25 <210> 82 <211> 21 <212> DNA <213> Artificia Sequence <400> 82 ctggtctaga gccagggact c 21 <210> 83 <211> 23 <212> DNA <213> Artificia Sequence <400> 83 acctggggca ttgcttctaa agc 23 <210> 84 <211> 23 <212> DNA <213> Artificia Sequence <400> 84 gattacaggc atgagccact gtg 23 <210> 85 <211> 22 <212> DNA <213> Artificia Sequence <400> 85 acttgtcact taccaatctg gc 22 <210> 86 <211> 23 <212> DNA <213> Artificia Sequence <400> 86 agaattcctc aagagggcag ccc 23 <210> 87 <211> 23 <212> DNA <213> Artificia Sequence <400> 87 ccaccactat gccccaagaa gtc 23 <210> 88 <211> 21 <212> DNA <213> Artificia Sequence <400> 88 ctcaccaatc tgcccgtagg c 21 <210> 89 <211> 20 <212> DNA <213> Artificia Sequence <400> 89 ctcccctctc cggctcaccc 20 <210> 90 <211> 25 <212> DNA <213> Artificia Sequence <400> 90 agactttaca ttgataagat tcagc 25

Claims (14)

A PNA probe for discriminating an elderly hearing disorder-related genotype comprising any one selected from the group consisting of SEQ ID NOS: 1 to 30
The method according to claim 1,
Wherein the PNA probe hybridizes with any one gene selected from the group consisting of CDH23, OTOF, COCH, ATP1A3, GRHL2, ITGA8, IQGAP2, PCDH15, PCDH20, GRM7, MTHFR, KCNQ1, KCNQ4 and SLC28A3 PNA probes for discriminating genotypes associated with senile hearing loss.
The method according to claim 1,
The PNA probes include the CDH23 gene rs121908354, rs373168635, rs137937502, rs746971522, rs121908349, A1713D, rs80356605, rs80356605, rs772729658, rs368155547, COCH gene G38D, rs587777771 of ATP1A3 gene, rs10955255 of GRHL2 gene, rs2236579, rs1417664 of ITGA8 gene, Rs457717 of the IQGAP2 gene, rs7081730 of the PCDH15 gene, rs78043697 of the PCDH20 gene, rs11928865, rs779706, rs77901, rs161927, rs1801133, rs1801131 of the MTHFR gene, rs12277647 of the KCNQ1 gene, rs727147 of the KCNQ4 gene, rs4660468, rs2149034, rs13374844, rs12143503 And Rs7032430 of the SLC28A3 gene. The PNA probe for discriminating the genotype of the senile hearing disorder according to the present invention is characterized in that it hybridizes with any one selected from the group consisting of:
The method according to claim 1,
Wherein the PNA probe is coupled to a reporter and a quencher.
The method according to claim 1,
Wherein the PNA probe is 60-75 DEG C when the melting temperature is completely hybridized with the target gene, and 40-67 DEG C when the melting temperature is in the genotype of claim 3. 3. The PNA probe according to claim 1,
A nucleotide sequence of SEQ ID NO: 31 as a forward primer, a nucleotide sequence of SEQ ID NO: 32 as a reverse primer, SEQ ID NO: 33 as a forward primer, SEQ ID NO: 34 as a reverse primer, SEQ ID NO: 35 as a forward primer, SEQ ID NO: 36 as a reverse primer, A nucleotide sequence of SEQ ID NO: 37 as a forward primer, a nucleotide sequence of SEQ ID NO: 38 as a reverse primer, SEQ ID NO: 39 as a forward primer, SEQ ID NO: 40 as a reverse primer, A nucleotide sequence of SEQ ID NO: 41 as a forward primer, a nucleotide sequence of SEQ ID NO: 42 as a reverse primer, A nucleotide sequence of SEQ ID NO: 43 as a forward primer, a nucleotide sequence of SEQ ID NO: 44 as a reverse primer, A nucleotide sequence of SEQ ID NO: 45 as a forward primer, a nucleotide sequence of SEQ ID NO: 46 as a reverse primer, A nucleotide sequence of SEQ ID NO: 47 as a forward primer, a nucleotide sequence of SEQ ID NO: 48 as a reverse primer, SEQ ID NO: 49 as a forward primer, SEQ ID NO: 50 as a reverse primer, SEQ ID NO: 51 as a forward primer, SEQ ID NO: 52 as a reverse primer, A nucleotide sequence of SEQ ID NO: 53 as a forward primer, a nucleotide sequence of SEQ ID NO: 54 as a reverse primer, A nucleotide sequence of SEQ ID NO: 55 as a forward primer, a nucleotide sequence of SEQ ID NO: 56 as a reverse primer, A nucleotide sequence of SEQ ID NO: 57 as a forward primer, a nucleotide sequence of SEQ ID NO: 58 as a reverse primer, A nucleotide sequence of SEQ ID NO: 59 as a forward primer, a nucleotide sequence of SEQ ID NO: 60 as a reverse primer, A nucleotide sequence of SEQ ID NO: 61 as a forward primer, a nucleotide sequence of SEQ ID NO: 62 as a reverse primer, A nucleotide sequence of SEQ ID NO: 63 as a forward primer, a nucleotide sequence of SEQ ID NO: 64 as a reverse primer, A nucleotide sequence of SEQ ID NO: 65 as a forward primer, a nucleotide sequence of SEQ ID NO: 66 as a reverse primer, A nucleotide sequence of SEQ ID NO: 67 as a forward primer, a nucleotide sequence of SEQ ID NO: 68 as a reverse primer, A nucleotide sequence of SEQ ID NO: 69 as a forward primer, a nucleotide sequence of SEQ ID NO: 70 as a reverse primer, A nucleotide sequence of SEQ ID NO: 71 as a forward primer, a nucleotide sequence of SEQ ID NO: 72 as a reverse primer, The nucleotide sequence of SEQ ID NO: 73 as the forward primer, the nucleotide sequence of SEQ ID NO: 74 as the reverse primer; A nucleotide sequence of SEQ ID NO: 75 as a forward primer, a nucleotide sequence of SEQ ID NO: 76 as a reverse primer, The nucleotide sequence of SEQ ID NO: 77 as the forward primer, the nucleotide sequence of SEQ ID NO: 78 as the reverse primer; A nucleotide sequence of SEQ ID NO: 79 as a forward primer, a nucleotide sequence of SEQ ID NO: 80 as a reverse primer, A nucleotide sequence of SEQ ID NO: 81 as a forward primer, a nucleotide sequence of SEQ ID NO: 82 as a reverse primer, A nucleotide sequence of SEQ ID NO: 83 as a forward primer, a nucleotide sequence of SEQ ID NO: 84 as a reverse primer, A nucleotide sequence of SEQ ID NO: 85 as a forward primer, a nucleotide sequence of SEQ ID NO: 86 as a reverse primer, A nucleotide sequence of SEQ ID NO: 87 as a forward primer, a nucleotide sequence of SEQ ID NO: 88 as a reverse primer, And a nucleotide sequence of SEQ ID NO: 90 as a forward primer and a nucleotide sequence of SEQ ID NO: 89 as a forward primer, and a reverse primer and a nucleotide sequence of SEQ ID NO: 90, respectively.
6. A composition for discriminating the genotype of senile hereditary disease, comprising the PNA probe of any one of claims 1 to 5 and the primer pair of claim 6.
a) extracting gDNA from a specimen sample;
b) a pair of forward and reverse primers capable of amplifying a gene selected from the group consisting of CDH23, OTOF, COCH, ATP1A3, GRHL2, ITGA8, IQGAP2, PCDH15, PCDH20, GRM7, MTHFR, KCNQ1, KCNQ4 and SLC28A3 Amplifying the gene or a mutant thereof, and hybridizing the PNA probe with a PNA probe capable of hybridizing with the gene or a mutant thereof; And
c) analyzing the fusion curve of the hybridized reactant in the step b) to detect the gene or a mutant thereof;
The method comprising the steps of:
9. The method of claim 8,
The variants of the gene include the CDH23 gene rs121908354, rs373168635, rs137937502, rs746971522, rs121908349, A1713D, rs80356605, rs772729658, rs368155547, rs772729658, rs368155547 of COCH gene, rs587777771 of ATP1A3 gene, rs10955255 of GRHL2 gene, rs2236579 of ITGA8 gene, rs1417664 , Rs457717 of the IQGAP2 gene, rs7081730 of the PCDH15 gene, rs78043697 of the PCDH20 gene, rs11928865, rs779706, rs77901, rs161927, rs1801133, rs1801131 of the MTHFR gene, rs12277647 of the KCNQ1 gene, rs727146, rs4660468, rs2149034, rs13374844 of the KCNQ4 gene, and rs7032430 of the SLC28A3 gene.
9. The method of claim 8,
Wherein the PNA probe is any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 30.
9. The method of claim 8, wherein the primer pair
A nucleotide sequence of SEQ ID NO: 31 as a forward primer, a nucleotide sequence of SEQ ID NO: 32 as a reverse primer, SEQ ID NO: 33 as a forward primer, SEQ ID NO: 34 as a reverse primer, SEQ ID NO: 35 as a forward primer, SEQ ID NO: 36 as a reverse primer, A nucleotide sequence of SEQ ID NO: 37 as a forward primer, a nucleotide sequence of SEQ ID NO: 38 as a reverse primer, SEQ ID NO: 39 as a forward primer, SEQ ID NO: 40 as a reverse primer, A nucleotide sequence of SEQ ID NO: 41 as a forward primer, a nucleotide sequence of SEQ ID NO: 42 as a reverse primer, A nucleotide sequence of SEQ ID NO: 43 as a forward primer, a nucleotide sequence of SEQ ID NO: 44 as a reverse primer, A nucleotide sequence of SEQ ID NO: 45 as a forward primer, a nucleotide sequence of SEQ ID NO: 46 as a reverse primer, A nucleotide sequence of SEQ ID NO: 47 as a forward primer, a nucleotide sequence of SEQ ID NO: 48 as a reverse primer, SEQ ID NO: 49 as a forward primer, SEQ ID NO: 50 as a reverse primer, SEQ ID NO: 51 as a forward primer, SEQ ID NO: 52 as a reverse primer, A nucleotide sequence of SEQ ID NO: 53 as a forward primer, a nucleotide sequence of SEQ ID NO: 54 as a reverse primer, A nucleotide sequence of SEQ ID NO: 55 as a forward primer, a nucleotide sequence of SEQ ID NO: 56 as a reverse primer, A nucleotide sequence of SEQ ID NO: 57 as a forward primer, a nucleotide sequence of SEQ ID NO: 58 as a reverse primer, A nucleotide sequence of SEQ ID NO: 59 as a forward primer, a nucleotide sequence of SEQ ID NO: 60 as a reverse primer, A nucleotide sequence of SEQ ID NO: 61 as a forward primer, a nucleotide sequence of SEQ ID NO: 62 as a reverse primer, A nucleotide sequence of SEQ ID NO: 63 as a forward primer, a nucleotide sequence of SEQ ID NO: 64 as a reverse primer, A nucleotide sequence of SEQ ID NO: 65 as a forward primer, a nucleotide sequence of SEQ ID NO: 66 as a reverse primer, A nucleotide sequence of SEQ ID NO: 67 as a forward primer, a nucleotide sequence of SEQ ID NO: 68 as a reverse primer, A nucleotide sequence of SEQ ID NO: 69 as a forward primer, a nucleotide sequence of SEQ ID NO: 70 as a reverse primer, A nucleotide sequence of SEQ ID NO: 71 as a forward primer, a nucleotide sequence of SEQ ID NO: 72 as a reverse primer, The nucleotide sequence of SEQ ID NO: 73 as the forward primer, the nucleotide sequence of SEQ ID NO: 74 as the reverse primer; A nucleotide sequence of SEQ ID NO: 75 as a forward primer, a nucleotide sequence of SEQ ID NO: 76 as a reverse primer, The nucleotide sequence of SEQ ID NO: 77 as the forward primer, the nucleotide sequence of SEQ ID NO: 78 as the reverse primer; A nucleotide sequence of SEQ ID NO: 79 as a forward primer, a nucleotide sequence of SEQ ID NO: 80 as a reverse primer, A nucleotide sequence of SEQ ID NO: 81 as a forward primer, a nucleotide sequence of SEQ ID NO: 82 as a reverse primer, A nucleotide sequence of SEQ ID NO: 83 as a forward primer, a nucleotide sequence of SEQ ID NO: 84 as a reverse primer, A nucleotide sequence of SEQ ID NO: 85 as a forward primer, a nucleotide sequence of SEQ ID NO: 86 as a reverse primer, A nucleotide sequence of SEQ ID NO: 87 as a forward primer, a nucleotide sequence of SEQ ID NO: 88 as a reverse primer, And a nucleotide sequence of SEQ ID NO: 90 as a forward primer and a nucleotide sequence of SEQ ID NO: 90 as a reverse primer.
9. The method of claim 8,
Wherein the specimen is a DNA sample derived from human hair, sputum, blood, saliva, or urine.
9. The method of claim 8,
Characterized in that the melting curve analysis is carried out by the FMCA (Fluorescence Melting Curve Analysis) method.
9. The method of claim 8,
Wherein the amplification is performed by a real-time PCR method.

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