WO2017090915A1 - Sonde d'anp pour détecter une perte auditive héréditaire et procédé pour détecter de perte auditive héréditaire l'utilisant - Google Patents

Sonde d'anp pour détecter une perte auditive héréditaire et procédé pour détecter de perte auditive héréditaire l'utilisant Download PDF

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WO2017090915A1
WO2017090915A1 PCT/KR2016/012733 KR2016012733W WO2017090915A1 WO 2017090915 A1 WO2017090915 A1 WO 2017090915A1 KR 2016012733 W KR2016012733 W KR 2016012733W WO 2017090915 A1 WO2017090915 A1 WO 2017090915A1
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seq
primer
base sequence
forward primer
reverse primer
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PCT/KR2016/012733
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English (en)
Korean (ko)
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송민식
박희경
김경탁
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주식회사 시선바이오머티리얼스
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Priority to CN201680069509.9A priority Critical patent/CN108291260A/zh
Publication of WO2017090915A1 publication Critical patent/WO2017090915A1/fr

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

Definitions

  • the present invention relates to a PNA (Peptide Nucleic Acid) probe for detecting genetic hearing loss.
  • PNA Peptide Nucleic Acid
  • the neonatal screening test may be performed by an automatic auditory brainstem test (AABR) or
  • PCR polymerase chain reaction
  • PCR-RFLP restriction enzyme fragment length quantitative analysis
  • Line Probe line probe analysis
  • assay restriction enzyme fragment mass diversity analysis
  • PCR-RFMP restriction enzyme fragment mass diversity analysis
  • multiplex PCR multiplex PCR
  • the present invention provides a method for detecting genetic hearing loss.
  • PNA probes and primer pairs were designed and provided.
  • the present invention also provides a method for detecting mutation of genes related to hearing loss from amplified and simulated sample using the PNA probe and primer pair.
  • the present invention is directed to a PNA probe for the detection of genetically deaf hearing comprising any one sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.
  • the present invention also relates to a pair of primers for detecting genetic hearing loss selected from the group consisting of SEQ ID NOs 12 to 31.
  • the present invention also relates to a genetic deafness comprising the PNA probe and primer pair.
  • the present invention also provides a method for augmenting a gene comprising a) extracting a gDNA from a sample sample; b) a pair of forward and reverse primers capable of amplifying a gene or variant thereof selected from the group consisting of GJB2, SLC26A4, 12S rRNA, CDH23 and TMPRSS3. Amplifying the gene or variant thereof and hybridizing it with a PNA probe capable of hybridizing with the gene or variant thereof; and c) analyzing a melting curve of the semicoast that has been hybridized in step b). Detecting the variant; It relates to a method for detecting genetic hearing loss.
  • the PNA probe for detecting genetic hearing loss according to the present invention and a method for detecting genetic hearing loss using the same can quickly and easily identify genes or variants thereof related to hearing loss, and are the major causes of GJB2, SLC26A4 and 12S. Eleven mutations in the rRNA, CDH23, and TMPRSS3 genes are used to accelerate genetic hearing loss. It is expected to be used to diagnose and diagnose non-syndrome deafness and risk.
  • 1 is amplification for the detection of genetic hearing loss and PCR conditions for PNA FMCA.
  • Figure 2 shows the wild-type and mutant organisms of deafness related genes.
  • probe refers to a nucleic acid fragment of RNA or DNA that is short or baseline or hundreds of bases capable of specific binding with a gene or mRNA. Probe, short chain
  • It can be produced in the form of single stranded DNA probes, double stranded DNA probes, RNA probes, etc., and can be labeled for easier detection, but is not particularly limited thereto.
  • Timing of the present invention means that the complementary single-stranded nucleic acids form double-stranded nucleic acids.
  • the hybridization occurs or is part of a perfect match between the two nucleic acid strands. Mismatch bases can also occur.
  • the degree of complementarity required for oscillation can vary depending on the conditions of oscillation, and can be controlled in particular by temperature.
  • nucleic acid' of the present invention means a nucleic acid sequence to be detected and is annealed or hybridized with a primer or probe under conditions of shaking, annealing or amplification.
  • the present invention is directed to a PNA probe for detecting genetically deaf hearing, comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 11.
  • PNA Peptide Nucleic Acid
  • LNA Locked Nucleic Acid
  • MNA Mopholino Nucleic Acid
  • PNA-DNA binding ability is much better than DNA-DNA binding force, so there is a difference of 1 (15 ° C melting temperature (Tm) difference even in one nucleotide mismatch. Using this difference, single nucleotide polymorphism (SNP) is used. And InDel (insertion / deletion) nucleic acid changes can be detected.
  • Tm 15 ° C melting temperature
  • SNP single nucleotide polymorphism
  • Fluorescence Melting Curve Analysis is used as a method of analysis of the localization reaction, and fluorescence melting curve analysis is based on the melting temperature difference between the product produced after PCR reaction and the injected probe.
  • the probe design is very simple and is constructed using 11-18 mer base sequences containing SNPs.
  • Tm depends on the length of the PNA probe. The value can be adjusted, and PNA probes of the same length can change the probe to adjust the Tm value.
  • PNA Since PNA has a higher binding force than DNA and has a higher basic Tm value, it can be designed with a shorter length than DNA, so that even the nearest SNP can be detected.
  • the existing HRM method is very small because the difference in Tm value is about 0.5 o C. However, if a small temperature change is required and two or more SNPs appear, the PNA probe can be analyzed unaffected by SNPs other than the probe sequence.
  • the PNA probe is not limited, but GJB2, SLC26A4, 12S
  • the PNA probe is not limited, but 1555A> G of the V37I, 235delC, 299-300delAT, R143W, SLC26A4 gene of the GJB2 gene, T410M, L676Q, H723R, IVS7-2A> G, and 12S rRNA gene.
  • the PNA probe is not limited, but may be a reporter or
  • the quencher may be bound.
  • the reporter and PNA probe containing the quencher of the present invention generate a fluorescent signal that is localized with the target nucleic acid, and rapidly melt with the target nucleic acid at the proper melting temperature of the probe as the temperature rises.
  • the signal is quenched and the presence of the target nucleic acid can be detected through a high resolution melting curve analysis obtained from the fluorescence signal resulting from this temperature change.
  • PNA probe means that the target nucleic acid is present.
  • Tm melting temperature
  • Tm melting temperature
  • the probe of the present invention is capable of extinguishing reporters and reporter fluorescence at both ends.
  • the fluorescence of the quenchers can be combined and can include intercalating fluorophores.
  • the reporter is a six-carboxyfluorescein (FAM), HEX, Texas red, JOE, It may be one or more selected from the group consisting of TAMRA, CY5, CY3, Alexa680, and the quencher is preferably, but not limited to, using TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 or Dabysyl. Rating phosphors include Acridine homodimer and derivatives thereof, Acridine Orange and derivatives thereof,
  • Hexidium iodide and its derivatives bisbenzimide, Hoechst 33258 and its derivatives, Hoechst 33342 and its derivatives, Hoechst 34580 and its derivatives derivative,
  • LDS 751 LDS 751 and its derivatives, propidium iodide (PI) and its derivatives and cy-dyes derivatives.
  • the present invention also relates to a pair of primers for detecting genetic hearing loss selected from the group consisting of SEQ ID NOs 12 to 31.
  • the present invention relates to a composition for detecting genetic hearing loss comprising the PNA probe and the primer pair.
  • composition of the present invention is not limited to one or more of the primer pairs,
  • the PNA probe of SEQ ID NO: 1 and the primer pair of SEQ ID NO: 12/13 may contain the V37I of GJB2, a hearing loss related gene.
  • VNA variants of SEQ ID NOS: 2 to 3 and primer pairs of SEQ ID NOs: 14/15 are GJB2 or variants thereof (235delC, 299delAT)
  • PNA probes of SEQ ID NO: 4 and primer pairs of 16/17 are CDH23 or variants (P240L)
  • the PNA probe of SEQ ID NO: 5 and the primer pair of SEQ ID NO: 18/19 are SLC26A4 or a variant thereof (T410M)
  • the PNA probe of SEQ ID NO: 6 and the primer pair of SEQ ID NO: 20/21 are SLC26A4 or
  • the present invention also provides a method for augmenting a gene comprising a) extracting gDNA from a sample sample; b) a pair of forward and reverse primers capable of amplifying a gene or variant thereof selected from the group consisting of GJB2, SLC26A4, 12S rRNA, CDH23 and TMPRSS3. Amplifying the gene or variant thereof and using the PNA probe to be capable of hybridizing with the gene or variant thereof; and c) analyzing the melting curve of the reactant shaken in the step b). Or detecting a variant thereof.
  • a sample that can be used in the detection of genetically deafness of the present invention is DNA or RNA, and the molecule may be a double stranded or single stranded form. Or partial single-stranded formation. It is desirable to isolate strands by heat, alkali, formamide, urea and glycoxal treatments, enzymatic methods (eg helicase action) and binding proteins. Including but not limited to, for example, strand separation can be achieved by thermal treatment with a temperature of 80 to 105 ° C.
  • the detection method of the present invention provides a method for detecting target DNA or RNA in the target.
  • Tm melting temperature
  • the variants of the gene are not limited, but the V37I, 235delC, 299-300delAT, R143W, SLC26A4 gene of the GJB2 gene, T410M, L676Q, H723R, IVS7-2A> G, and 1555A> G of the 12S rRNA gene. , P240L of CDH23 gene and
  • It can be selected from the group consisting of A306T of TMPRSS3 gene.
  • Hearing loss due to genetic causes can be divided into syndrome-deaf and non-syndrome-deaf according to accompaniment with other symptoms. ⁇ 15-20% are autosomal dominant genes and less than 2% are present in genes present in the X chromosome and mitochondria Genetic deafness, which appears as non-syndrome deafness, is a complex genetic dysplasia, which has been reported to be related to group 120 days on human chromosomes, and 41 of these genes have been identified as causal genes. GJB2, GJB6, and SCL26A4 genes have been reported as major contributors to genetic deafness in many populations, and mutations in mitochondrial 12S rRNA genes represented 0.9% of all patients.
  • the PNA probe is not limited but may be any one sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.
  • the primer pair is not limited.
  • the sample sample is not limited, but may be a DNA sample derived from human sputum, blood, saliva, or urine.
  • bio-samples are included, and preferably, bio-samples are analyzed using the method of the present invention.
  • Biological samples of origin may be analyzed.
  • the samples may be derived from specific tissues or organs. Representative examples of tissues include connective, skin, muscle or nerve tissue. do.
  • organs include eyes, brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testicle, ovary, uterus, rectum, nervous system
  • the biological sample to be analyzed includes any cell, tissue, fluid, or any medium that can be well analyzed by the present invention, which is derived from a biological source. And samples from animals, humans, or foods prepared for consumption. Animal samples also include body fluid samples, including blood, serum, plasma, lymph, breast milk, urine, feces, and eye fluids. It includes, but is not limited to, saliva, semen, brain extracts (eg, brain crushes), spinal fluid, strata, spleen, and tonsil tissue extracts.
  • the melting curve is not limited, but the analysis is performed by FMCA (Fluorescence). Melting Curve Analysis.
  • the fusion curve analysis of the present invention is a method of analyzing the melting temperature of a double-stranded nucleic acid formed of target DNA or RNA and a probe. Such a method is performed by, for example, Tm analysis or analysis of the fusion curve of the double chain. For this reason, it is called fusion curve analysis.
  • Tm analysis or analysis of the fusion curve of the double chain. For this reason, it is called fusion curve analysis.
  • a probe complementary to the target sequence containing the variation of the detection target a target single-stranded DNA of the detection sample and a hybrid (double-stranded DNA) with the probe are formed. Then, the hybridization body is subjected to a heat treatment, and the dissociation (melting) of the hybrid according to the increase in the silver content is detected by a change in a signal such as absorbance. Then, based on the detection result, the Tm value is determined.
  • the Tm value (the reference value for the hybrid formation between the detection target sequence including the point mutation and the probe complementary thereto)
  • the Tm value the reference value for the hybrid formation between the detection target sequence including the point mutation and the probe complementary thereto
  • the Tm value the measurement value between the target single-stranded DNA of the detection sample and the probe. It can be determined that there is a mutation in the match, that is, the target DNA, if the measured value is equal to the evaluation standard value If the measured value is lower than the criterion value, it can be determined that there is no mutation in the mismatch, ie the target DNA.
  • 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 may be analyzed by using a probe containing a fluorescent substance. It can be a photon or an intercalating phosphor.
  • the above amplification is not limited, but in real time.
  • the real-time PCR method of the present invention allows the fluorescent material to be intercalated into a double stranded DNA chain during PCR, and increases the temperature with the amplification of the PCR product to release DNA double strands.
  • Analysis of the pattern of the fusion curve which reduces the amount of fluorescent material present between the double strands, in particular, the degree (Tm) of fusion (denaturation) of DNA, can be used to analyze the variation of nucleotide sequences between normal controls and mutations.
  • target probes for detecting the presence or absence of mutations at specific positions in the GJB2, SLC26A4, 12S rRNA, CDH23, and TMPRSS3 genes are shown in SEQ ID NOs: 1 to 11.
  • Dabcyl was synthesized by HPLC purification method in Panagene (Panagene, Korea), and the purity of all synthesized probes was confirmed by mass spectrometry, and the unnecessary secondary structure of the probe was avoided for more effective binding with the target nucleic acid.
  • GJB2_R143W-1 GGTGGACCTACACAAGCA 21 63.54 90 28 F GCA
  • PCR for melting curve analysis using the double stranded DNA and PNA probe synthesized above was performed using the CFX96 TM Real-Time System (BIO-RAD, USA).
  • IX qPCR PreMix (Eye Biomaterials, Korea), 0 ⁇ 5 ⁇ PNA probe to 7 ⁇ reverse primer, 7 ⁇ human gDNA (15ng / ⁇ ) or artificial synthesis to achieve a total volume of 20 ⁇ .
  • Target DNA (Bioneer, Korea) was added and real-time PCR was performed.
  • Four PNA probes were added with each PCR primer in one tube or well and proceeded in multiplex. Multiplex combinations were performed by adding primer sets and probes as shown in Table 4.
  • Setl is GJB2: V37I, GJB2: 299delAT, CDH23: P240L, GJB2: 235delC from the left, and Set2 is SLC26A4: T410M, SLC26A4.L676Q, SLC26A4.H723R, SLC26A from the left. : IVS7-2A> G, and Set3 is
  • the melting temperature obtained through PNA fluorescence melting curve analysis (FMCA) is shown in Table 5.
  • the combinations of phosphors and probes used in sets A, B and C for each multiplex analysis (multiplex) in Table 5 and the melting temperatures of the resulting mutant and nocturnal types are as follows. [Table 5]
  • PCR polymerase chain reaction

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Abstract

La présente invention concerne une sonde d'acide nucléique peptidique (ANP) pour détecter une perte auditive héréditaire et un procédé pour détecter une perte auditive héréditaire l'utilisant. Selon l'invention, la sonde d'ANP pour détecter une perte auditive héréditaire et le procédé pour détecter une perte auditive héréditaire l'utilisant permettraient la discrimination rapide et simple de gènes associés à la perte auditive ou de leurs mutants et peuvent être utilisés pour diagnostiquer une perte auditive héréditaire à un stade précoce et pour diagnostiquer une perte auditive non syndromique et un degré de risque au moyen de 11 mutations des gènes GJB2, SLC26A4, 12S ARNr, CDH23 and TMPRSS3, qui sont les principales causes d'une perte auditive héréditaire.
PCT/KR2016/012733 2015-11-26 2016-11-07 Sonde d'anp pour détecter une perte auditive héréditaire et procédé pour détecter de perte auditive héréditaire l'utilisant WO2017090915A1 (fr)

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Cited By (2)

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CN109055532A (zh) * 2018-09-12 2018-12-21 苏州贝康医疗器械有限公司 胚胎植入前遗传性耳聋基因检测用引物组合物、试剂盒及应用
CN112126677A (zh) * 2020-11-25 2020-12-25 北京迈基诺基因科技股份有限公司 耳聋单倍型基因突变无创检测方法

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CN111172250B (zh) * 2020-03-03 2022-01-14 肇庆医学高等专科学校 一种检测药物性耳聋基因的探针组合物及其应用
KR102286597B1 (ko) * 2020-03-25 2021-08-05 하이브리드테크놀로지 주식회사 타겟과 표지된 프로브의 결합 효율 개선 및 미결합 프로브와 결합 프로브의 효율적인 분리 방법

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055532A (zh) * 2018-09-12 2018-12-21 苏州贝康医疗器械有限公司 胚胎植入前遗传性耳聋基因检测用引物组合物、试剂盒及应用
CN109055532B (zh) * 2018-09-12 2022-03-18 苏州贝康医疗器械有限公司 胚胎植入前遗传性耳聋基因检测用引物组合物、试剂盒及应用
CN112126677A (zh) * 2020-11-25 2020-12-25 北京迈基诺基因科技股份有限公司 耳聋单倍型基因突变无创检测方法
CN112126677B (zh) * 2020-11-25 2021-02-19 北京迈基诺基因科技股份有限公司 耳聋单倍型基因突变无创检测方法

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