CN101497925B - Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof - Google Patents
Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof Download PDFInfo
- Publication number
- CN101497925B CN101497925B CN 200810204771 CN200810204771A CN101497925B CN 101497925 B CN101497925 B CN 101497925B CN 200810204771 CN200810204771 CN 200810204771 CN 200810204771 A CN200810204771 A CN 200810204771A CN 101497925 B CN101497925 B CN 101497925B
- Authority
- CN
- China
- Prior art keywords
- probe
- sequence
- dna
- optic neuropathy
- artificial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 58
- 108020005196 Mitochondrial DNA Proteins 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 208000032087 Hereditary Leber Optic Atrophy Diseases 0.000 title claims description 41
- 239000000523 sample Substances 0.000 claims abstract description 125
- 230000000869 mutational effect Effects 0.000 claims abstract description 40
- 238000012408 PCR amplification Methods 0.000 claims abstract description 18
- 206010061323 Optic neuropathy Diseases 0.000 claims abstract 5
- 208000020911 optic nerve disease Diseases 0.000 claims abstract 5
- 201000000639 Leber hereditary optic neuropathy Diseases 0.000 claims description 65
- 108020005187 Oligonucleotide Probes Proteins 0.000 claims description 21
- 239000002751 oligonucleotide probe Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 10
- 238000005070 sampling Methods 0.000 claims description 10
- 239000013641 positive control Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 8
- 239000013642 negative control Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 125000003368 amide group Chemical group 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 abstract description 10
- 230000035772 mutation Effects 0.000 abstract description 8
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 97
- 238000009396 hybridization Methods 0.000 description 27
- 101001109052 Homo sapiens NADH-ubiquinone oxidoreductase chain 4 Proteins 0.000 description 21
- 102100021506 NADH-ubiquinone oxidoreductase chain 4 Human genes 0.000 description 20
- 238000005406 washing Methods 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 239000006210 lotion Substances 0.000 description 10
- 230000000875 corresponding effect Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000001712 DNA sequencing Methods 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000007899 nucleic acid hybridization Methods 0.000 description 5
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 5
- 102220218475 rs878853857 Human genes 0.000 description 5
- 125000003172 aldehyde group Chemical group 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 238000005382 thermal cycling Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 238000012937 correction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 102200008142 rs878853856 Human genes 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000000123 temperature gradient gel electrophoresis Methods 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007334 copolymerization reaction Methods 0.000 description 2
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 208000001799 Hereditary Optic Atrophies Diseases 0.000 description 1
- 101000829171 Hypocrea virens (strain Gv29-8 / FGSC 10586) Effector TSP1 Proteins 0.000 description 1
- 206010052641 Mitochondrial DNA mutation Diseases 0.000 description 1
- 241000768494 Polymorphum Species 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000009223 counseling Methods 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000005357 flat glass Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 208000014188 hereditary optic neuropathy Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000004377 microelectronic Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002094 self assembled monolayer Substances 0.000 description 1
- 239000013545 self-assembled monolayer Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a gene chip, and discloses an integrated detection gene chip of an mtDNA mutation site related to Leber genetic optic neuropathy, as well as the preparation and the application thereof. A specific oligonucleotide detecting probe of the mtDNA mutational site related to the Leber genetic optic neuropathy and a specific oligonucleotide detecting probe of a mononucleotide polymorphyism site mtDNA 11719G>A are fixed on the carrier surface lattice of the gene chip. The gene chip has the advantages of time saving, low cost, simple operation and the like, has 32 mutation sites related to the Leber genetic optic neuropathy in a specific detecting clinical sample only through one-time PCR amplification and one-time crossing reaction, and is suitable for the gene diagnosis of the Leber genetic optic neuropathy.
Description
Technical field
The present invention relates to gene chip, be specifically related to detection gene chip relevant and preparation thereof and application with Leber hereditary optic neuropathy.
Background technology
Leber hereditary optic neuropathy (Leber ' s hereditary optic neuropathy, LHON) be a kind of modal hereditary optic atrophy disease, be characteristic with the acute or subacute central vision forfeiture in both sides, mainly involve adolescents in male.1871, Theodor Leber reported first a LHON family, and the detailed Clinical symptoms of describing LHON.Subsequently in one period; The genetics pattern that it is believed that LHON be associated with X chromosome (X-linked); The mode of inheritance of proposing LHON up to Erickson in 1972 is more prone to matrilinear inheritance, and he thinks that possibly to have certain potential Mitochondrial DNA (mtDNA) sudden change relevant with LHON.1988, Wallace and colleague reported first LHON thereof were relevant with MTND4 gene 11 778G＞A sudden change, have so far more than 30 kind of mtDNA point mutation be in the news relevant with LHON, they be distributed in not agnate widely all over the world and the crowd in.(http://www.mitomap.org) can retrieve the gene mutation site relevant with LHON from the MITOMAP DB, and wherein LHON patient more than 90% and three big primary mtDNA sudden changes (11778G＞A, 3460G＞A and 14484T＞C) relevant; Some rare mtDNA sudden changes also can cause LHON, like 14459G＞A, and 10663T＞C; 14568C＞T, 14482T＞A/G, 14495A＞G; 4171C＞A, 37336＞A etc.Though the pathogenesis of LHON is not illustrated so far fully, unquestionable, the primary sudden change is the pathogenesis basis of LHON.But only exist the primary sudden change also to be not enough to cause a disease; As carry 11778G＞A; The part family member of 3460G＞A or 14484T＞C can not show any clinical symptom, and other genetics factors (like Secondary cases mtDNA sudden change, nuclear regulatory gene) and environmental factors also possibly brought into play keying action in the physiopathology process of LHON.The different familys of in addition, carrying identical primary mutational site can show different penetrances, age of onset, severity and visual loss process.Thereby, be necessary that (like rare sudden change and Secondary cases sudden change) carried out examination to the paathogenic factor beyond the primary sudden change, further illustrating the relation of mtDNA sudden change and LHON, for diagnosing and treatment provides guidance.
The appearance of gene diagnosis technology has changed that only to rely on typical family history and Clinical symptoms clinically in the past be basis for estimation, has improved distributing the diagnosis rate of LHON disease.So far the LHON disease does not still have highly effective therapy, and it is prevention and the effective measure that reduce this sick incidence that pre-marital genetic counseling and molecular genetics detect.Many mtDNA sudden change detection methods have been set up; Like restriction fragment length polymorphism (Restriction fragment length poly morphism; RFLP), single strand conformation polymorphism (Single-strand conformation polymorphism; SSCP), the allele specific oligonucleotide spot marking (Allele-specific oligonucleotide dot blot), temperature gradient electrophoresis (Temperaturegradient gel electrophoresis; TGGE), the transient temperature gradient electrophoresis (Temporal temperaturegradient gel electrophoresis, TTGE), denaturing gradient gel electrophoresis (Denaturant gradient gelelectrophoresis, DGGE) with sex change performance liquid chromatography (Denaturing high performance liquidchromatography; DHPLC) etc.; But still there is certain limitation in these technology in practice, and are high like RFLP cost, workload is big, and some important mutational site is not because of being detected at the identification range of used restriction endonuclease; Additive method wants complicated with respect to RFLP; Main is, these methods once can only detect to one or several mutational site mostly, and detected result often needs the dna sequencing checking.Total length mtDNA sequencing technologies is a present LHON disease gene diagnosis method the most reliably, but this method workload is big, expensive, is difficult to the universal utilization in routine clinical diagnostic test chamber.
At present; The Chinese invention patent relevant with the present invention has three; Denomination of invention is respectively: and Leber hereditary optic neuropathy detection method and test kit thereof (application number: 96116296.1), 200410027623.4) and the gene diagnosis kit of Leber hereditary optic neuropathy and detection method (application number: 200510006097.8) thereof Leber ' s hereditary optic neuropathy patient's Mitochondrial DNA Mutation quantitative detecting method (application number:.All only (partly or entirely detecting among the 11778G＞A, 3460G＞A and 14484T＞C), and not detecting to rare sudden change or Secondary cases sudden change still has certain limitation to these patents in utilization to three kinds of primary sudden changes of LHON.
Gene chip is the new and high technology that integrates physics, chemistry, Materials science and life science that develops rapidly in recent years; Utilize the intensification and parallel handling principle of microelectronic chip technology; The biological sample of the special sequence that has biological significance in a large number is solidificated in stromal surface such as sheet glass and silicon chip in an orderly manner; And utilize the micro-reaction volume; Once hybridization just can be carried out special, quick, accurate detection to many gene expression doses, sudden change and polymorphum, has succeedd in fields such as genetic expression, transgenation, Study on gene polymorphism and gene diagnosises and has used.Because gene chip has these unique advantages and makes it obtain certain utilization aspect plastosome gene test; As utilize the express spectra microarray that the expression situation of chondriogen is detected; Inquire into the pathogenesis of mitochondriopathy, or utilize primer extension reaction or direct nucleic acid hybridization on the oligonucleotide microarray to detect mitochondrial single nucleotide polypeptide property (SNP) etc.
Because the mtDNA mutational site quantity relevant with LHON is more; Conventional gene diagnosis method has been difficult to meet the demands, thereby, research and develop a kind of novel gene chip; Be used for integrated detection, have potential social benefit and clinical value undoubtedly LHON related mtDNA mutational site.
Summary of the invention
One of the object of the invention is to provide a kind of Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip and preparation method thereof.
Another object of the present invention is to provide the method for use of a kind of above-mentioned Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip.
Design of the present invention is following:
Set up the gene chip in relevant 32 mutational sites of a kind of Leber of detection hereditary optic neuropathy.This gene chip comprises detection system and supervisory system.Detection system is according to 32 Mitochondrial DNA (mtDNA) mutational sites relevant with Leber hereditary optic neuropathy of MITOMAP DB (http://www.mitomap.org) report; Design corresponding locus specificity oligonucleotide probe; Utilize amino covalent attachment that oligonucleotide probe is fixed on chip surface with aldehyde radical; Probe and fluorescently-labeled sample pcr amplification product are hybridized, and can judge according to hybridization signal having or not sudden change to take place and mutation type.Monitoring systems and detection system are positioned at same an array; Comprise positive control, negative control and parallel control; Positive control is the equal amount of mixture of each probe of detection system; Negative probe is a sampling liquid, and parallel control is the common mtDNA 11719G of gook＞A mononucleotide polymorphism site probe, can judge the quality of hybridization according to supervisory system.
Specifically; According to correction Cambridge mtDNA reference sequences (revised Cambridge Reference Sequence; RCRS) with the 32 kinds of mtDNA point mutation relevant (being respectively: T3394C, G3460A, G3635A, G3733A, A4136G, T4160C, C4171A, T4216C, A4435G, C4640A, A4917G, G5244A, G7444A, G9738T, G9804A, T10663C, G11696A, G11778A, G13708A, G13730A, G14459A, C14484A, A14495G, T14498C, A14495G, T14498C, C14568T, A14596T, A14693G, G15257A, G15812A, A15951G) and the common mtDNA11719G＞A pleomorphism site of gook of MITOMAP DB (http://www.mitomap.org) report with Leber hereditary optic neuropathy; 33 pairs of probes (wherein 32 pairs of probes are to 32 Leber hereditary optic neuropathy relevant mutational sites, and 1 pair of probe is to 11719G＞A pleomorphism site) and 11 pairs of PCR primers have been designed altogether.11 pairs of primers are divided into three groups and under same thermal cycling, carry out the multiplex PCR amplification, can amplify the target DNA fragment that comprises above-mentioned 32 Leber hereditary optic neuropathy related mtDNA mutational sites and 11719G＞A pleomorphism site specifically.Fixing above-mentioned 33 pairs of probes on a chip; Only with a PCR and a hybridization; Just can confirm timely and accurately at 6-8 hour whether the examinee exists the said mutation site; Thereby seeking advice from for treatment and genetics provides unambiguous evidence, and can further illustrate these mutational sites in Chinese population distribution and to the influence of the cause of disease, pathogeny, clinical treatment and prognosis.
One aspect of the present invention discloses a kind of Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip, and dot matrix is fixed with the specific oligonucleotide probe in the mtDNA mutational site relevant with Leber hereditary optic neuropathy and the specific oligonucleotide probe of mtDNA 11719G＞A mononucleotide polymorphism site on the gene chip carrier surface.
The principle of design of above-mentioned specific oligonucleotide probe is: according to the 32 kinds of LHON related mtDNA mutational sites and the 11719G＞A pleomorphism site of MITOMAP DB report; With reference to correction Cambridge mtDNA reference sequences (revisedCambridge Reference Sequence; RCRS); Make the Tm value of different probe be positioned at (45 ± 5) ℃ as far as possible; Make probe length be positioned at 14-20bp, the gene locus that every pair of probe is detected is positioned near probe sequence middle part or the middle part, the combining site that makes probe and corresponding strand target gene as far as possible near 5 ' of strand target gene hold between the central region with the guarantee hybridization efficiency.But mtDNA mutational site and 11719G＞A pleomorphism site that specific oligonucleotide probe specific recognition Leber hereditary optic neuropathy is relevant.
Above-mentioned specific oligonucleotide probe is selected from one or more in the oligonucleotide probe that sequence is SEQ ID NO.1～65.
Preferable, gene chip carrier surface dot matrix is fixed with the oligonucleotide probe that sequence is SEQ ID NO.1～65, and this chip can be used for special detection LHON relevant mtDNA sudden change and 11719G＞A pleomorphism site.
Preferably, 5 ' end of above-mentioned oligonucleotide probe has amido modified group, so that combine with aldehyde group modified chip, probe 5 ' end adds the Poly T that the preceding paragraph 15 gathers, sterically hindered when reducing hybridization.
The said gene chip also can be fixed with negative control and positive control.Negative control is a sampling liquid, and positive control is the equivalent mixed liquor of selected each oligonucleotide probe.
The said gene chip carrier can be slide glass, preferred aldehyde group modified slide glass.
Second aspect present invention discloses the preparation method of above-mentioned Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip, comprises the following steps:
With being selected from sequence is that the oligonucleotide probe 5 ' end of SEQ ID NO.1～65 adds the Poly T that the preceding paragraph 15 gathers; And carry out amido modified to 5 ' end; With deionized water each probe is diluted respectively; And mix with the sampling liquid equal-volume respectively, obtaining final concentration is the oligonucleotide probe solution of 12.5～50uM, adopts conventional method that oligonucleotide probe solution is also fixing in slide surface with negative control and positive control dot matrix.
The effect of above-mentioned sampling liquid is to optimize the quality of chip manufacturing, can select various sampling liquids commercially available or that dispose voluntarily for use.
The third aspect of the invention discloses the method for use of above-mentioned Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip, comprises the following steps:
1) collects patient's peripheral blood, sample drawn DNA;
2) pcr amplification of sample DNA and mark: with the sample DNA is template, selects suitable primer PCR amplification and mark sudden change correlated series to be measured;
3) adopt aforementioned gene chip in hybridizing detection after the sex change of sample PCR product: after sample pcr amplification product equal-volume is mixed; Mix (as the sample pcr amplification product is mixed with 1: 2～1: 1 volume ratio with hybridization solution) with an amount of volume hybridization solution; Will with the PCR product sex change behind the hybridization solution mixing; Get the dot matrix area that an amount of volume drops in chip and hybridize, the hybridization back is with washing lotion washing, the intensity of certification mark signal again.Strength ratio through the certification mark signal is right, can judge whether to take place corresponding sudden change.
Above-mentioned hybridization solution is the conventional hybridization solution that is used for nucleic acid hybridization reaction, can select various hybridization solutions commercially available or that dispose voluntarily for use.
Pcr amplification product sex change after the above-mentioned hybridization solution dissolving can be adopted conventional denaturation method, like 95 ℃ of sex change.
Washing lotion is used for the pcr amplification product that does not mate is fully removed from chip, can select the conventional washing lotion that is used for nucleic acid hybridization in this area for use.
Preferable, in the above-mentioned steps 3, hybridization temperature is 35-40 ℃; Hybridization time is 30-60 minute.
During the washing lotion washing, washed 5-15 minute with primary wash liquor earlier, washed 5-15 minute with secondary cleaning water again.Preferably, primary wash liquor is 2xSSC+0.1w%SDS (being the basis in the washing lotion gross weight); Secondary cleaning water is 0.2xSSC+0.1w%SDS; An elution requirement is 30 ℃ of washings 10 minutes; The secondary elution requirement is 30 ℃ of washings 5 minutes.
Improved, the primer in the above-mentioned steps 2 is selected from the nucleotide sequence that sequence is SEQ ID NO.66～87.Whenever align in the anti-primer, have at least one to be labeled, mark can adopt conventional mark, like fluorescent mark etc., has used Cy5 fluorescence molecule mark in the embodiment of the invention, and has used laser copolymerization collection scanner scanning fluorescence signal intensity.
Better; Pcr amplification in the above-mentioned steps 2 is multiplex PCR amplification: with sequence be the nucleotide sequence of ID NO.66～71, ID NO.72～79, ID NO.80～87 respectively as the primer of three groups of multiplex PCRs, under same thermal cycling, be that template is carried out pcr amplification with the sample DNA.
Fourth aspect of the present invention discloses the application of above-mentioned detection Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip in the detection kit in preparation Leber hereditary optic neuropathy related mtDNA mutational site.
Fifth aspect present invention discloses the detection kit in a kind of Leber hereditary optic neuropathy related mtDNA mutational site, comprises the said gene chip, and the pcr amplification primer in Leber hereditary optic neuropathy related mtDNA mutational site.
Preferable, the pcr amplification primer in above-mentioned Leber hereditary optic neuropathy related mtDNA mutational site is to be selected from one or more in the nucleotide sequence that sequence is SEQ ID NO.66～87.
Preferably, the pcr amplification primer in above-mentioned Leber hereditary optic neuropathy related mtDNA mutational site is the nucleotide sequence of SEQ IDNO.66～87.Each primer can be distinguished independent packaging.
Also can comprise the conventional reagent that other need be used in the test kit in the test kit use, like hybridization solution, nucleic acid hybridization washing lotion etc.Preferable, above-mentioned washing lotion comprises primary wash liquor and secondary cleaning water, wherein primary wash liquor is 2xSSC+0.1w%SDS; Secondary cleaning water is 0.2xSSC+0.1w%xSDS.
Gene chip of the present invention can be used for detecting the relevant mtDNA mutational site of Leber hereditary optic neuropathy, can once detect 32 kinds of mtDNA mutational sites simultaneously.Preferred version only needs 1 pcr amplification and 1 hybridization, can the specific detection clinical sample in 32 kinds with the relevant mutational site of Leber hereditary optic neuropathy.Gene chip of the present invention have save time, cost and advantage such as easy and simple to handle, be prepared into pcr amplification from sample, obtain detected result; Can in 6-8 hour, accomplish; Shortened Diagnostic Time greatly, can carry out preliminary examination to doubtful Leber hereditary optic neuropathy patient's mtDNA clinically, to define no known two mutants to the patient; Thereby be that the consulting of treatment of diseases or genetics provides certain clue, have better clinical popularization value.Gene chip of the present invention also has very high sensitivity and specificity, compares with traditional molecular biology method, and shorten detection time greatly, reaches the purpose of LHON clinical diagnosis and differential diagnosis.
Description of drawings
Fig. 1 chip point sample cloth system of battle formations, the positive contrast of P, composition is the equal amount of mixture of SEQ ID NO.1～65 probes, the negative contrast of N, composition is a sampling liquid.
Fig. 2 P1-11778G＞A uniformity two mutants chip detection and dna sequencing result
Heterogeneous two mutants chip detection of Fig. 3 P2-11778G＞A and dna sequencing result
Fig. 4 P3-3460G＞A and 3394T＞C uniformity two mutants chip detection and dna sequencing result
Fig. 5 P4-14484T＞C uniformity two mutants chip detection and dna sequencing result
Embodiment
Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment only are used to explain the present invention, and unrestricted scope of the present invention.The reagent of the experimental technique of unreceipted actual conditions and undeclared prescription is according to people such as normal condition such as Sambrook in the following example; Molecular cloning: the condition of the condition described in the test handbook (New York:Cold Spring Harbor LaboratoryPress, 1989) or manufacturers's suggestion is carried out or is disposed.
The preparation of embodiment 1 probe design and chip
(1) design of probe on the chip:
32 kinds of LHON related mtDNA mutational sites and 11719G＞A pleomorphism site according to MITOMAP DB report; With reference to correction Cambridge mtDNA reference sequences (revised Cambridge Reference Sequence; RCRS); Design also screening obtains 33 pairs of oligonucleotide probes, makes the Tm value of different probe be positioned at (45 ± 5) ℃ as far as possible, makes probe length be positioned at 14-20bp; The gene locus that every pair of probe is detected is positioned near probe sequence middle part or the middle part, the combining site that makes probe and corresponding strand target gene as far as possible near 5 ' of strand target gene hold between the central region with the guarantee hybridization efficiency.5 ' end of probe has amido modified group, so that combine with aldehyde group modified chip; The comprehensive parameters of different probe (probe length, GC% and Tm value) is approaching as far as possible, in order to reduce the sterically hindered of when hybridization, when synthetic, adds the Poly T (connecting arm) that the preceding paragraph 15 gathers at the oligonucleotide 5 ' end of 33 pairs of probes.The positive control probe is chosen the equal amount of mixture of each probe, and negative control probe is chosen sampling liquid.The 33 pairs of probes such as the table 1 of detection system and parallel control are listed:
The amido modified probe of table 1 detection system and parallel control
(2) preparation of chip:
Sampling liquid: be Micro-Spotting Solution (2 *), available from TeleChem Internation company, room temperature preservation.
Probe is responsible for synthetic by Takara company;, and mix with the sampling liquid equal-volume probe dilution with deionized water, making the probe final concentration is 12.5uM; Through Cartesian Tech.; PROSYS 5510A chip manufacturing system dot matrix is in aldehyde group modified slide surface, and 3 points of the parallel point of each probe of detection system and parallel control place under 70% relative humidity, the room temperature condition and to fix in 48-72 hour.Take out the back in boiling water bath 30 seconds, thorough drying in the air, the gene chips that detect 32 kinds of relevant sudden changes of Leber hereditary optic neuropathy have promptly been processed in 4 ℃ of preservations, and the chip point sample cloth system of battle formations is seen Fig. 1.
The sudden change of embodiment 2 samples detects
(1) preparation of sample, mark and processing:
Extract Leber hereditary optic neuropathy patient or suspected patient sample DNA with the DNA of Qiagen company extraction agent box, subsequent use.
The pcr amplification of sample and mark are handled: under same thermal cycling; Through three groups of multiplex PCRs (is that the nucleotide sequence of ID NO.66～71, ID NO.72～79, ID NO.80～87 is respectively as the primer of three groups of multiplex PCRs with sequence), 11 target genes that increase, these 11 target genes are contained relevant with Leber hereditary optic neuropathy 32 mtDNA mutational sites and 11719G＞A pleomorphism site.(amplification system comprises 2.5ul 10*PCR buffer, the dATP of each 200uM, dGTP, dCTP, dTTP, 1.5mM MgCl in 25ul PCR reaction system
2, 1.5U DNA TagE (Takara), and the primer that between 0.05-0.5uM, does not wait; Add the 100ng sample DNA in each reaction, prepare 11 fluorescently-labeled sample DNA purpose fragments through following thermal cycling process: at first at 94 ℃ of sex change 5min, with 94 ℃; 35s, 61 ℃, 55s; 72 ℃, 90s circulation 35 times is extended 10min at 72 ℃ at last.Above-mentioned PCR primer is synthetic by Takara company, wherein, has at least 5 ' of a primer to hold in the every pair of forward primer and the reverse primer and adds cy5 fluorescence molecule mark, and primer sequence such as table 2 are listed.
Table 2PCR primer
Annotate: F representes forward primer, and R representes reverse primer.
From three groups of multiplex PCR amplified productions, get 10 μ l respectively,, use target gene as chip hybridization with 30ul hybridization solution mixing.
(2) hybridization and signal detection
Get the chip hybridization of embodiment 2 steps (1) and use target gene 15ul, 95 ℃ of sex change 10min, ice bath 3-5min rapidly; Getting chip hybridization target gene after the 10ul sex change drips in the dot matrix zone on the gene chips surface of 32 kinds of relevant sudden changes of detection Leber hereditary optic neuropathy of embodiment 1 preparation; Covered places 37 ℃ of wet boxes to hybridize 45min, and 30 ℃ in washing lotion 1 (2 * SSC then; Swing 0.1%SDS) and wash 10min; (0.2 * SSC swings in 0.1%SDS) and washes 5min, and nitrogen dries up in washing lotion 2 again.(Inc.) aforementioned wash-out of scanning and dried hybridization hybrid chip at the 635nm place scan intensity and were made as for 700 (being 920 to the maximum) for GenePix 4000B, Axon Instruments with laser copolymerization collection scanner; Through the filtration background signal of punctuating, extract each point mean fluorecence signal strength values.Cooperate its software kit analysis, finally obtain the chip detection result.To diagnose ratio (ratio of wild-type probe strength of signal MV and corresponding positions point mutation type probe signals average strength) as analyzing the standard whether sudden change exists; If diagnosis ratio is being wild-type more than 3; Being the uniformity sudden change below 0.3, between 0.3 and 3 heterogeneous sudden change.(the ratio criterion is with reference to Du W; Marsac C; Kruschina M; Ortigao F.Florentz C.Functionalized self-assembled monolayer on gold fordetection of human mitochondrial tRNA gene mutations.Anal Biochem, 2003,322 (1): 14-25 obtains)
32 kinds of relevant gene chips that suddenly change of detection Leber hereditary optic neuropathy with embodiment 1 preparation detect with 12 routine Leber hereditary optic neuropathy patients 20 routine normal healthy controls respectively as stated above: each normal healthy controls does not all detect 32 kinds of relevant sudden changes; 8 examples detect 11778G＞A uniformity sudden change among the 12 routine patients, and 1 example detects the heterogeneous sudden change of 11778G＞A, and 1 example detects 3460G＞A and 3394T＞C uniformity sudden change simultaneously, and 1 example detects 14484T＞C uniformity sudden change; All normal healthy controls and patient all detect 11719G＞A SNP.Chip detection result and dna sequencing result fit like a glove.
4 examples contain different mutants patient sample chip scintigram example and corresponding mutational site dna sequencing result shown in Fig. 2-5, and the chip in corresponding site diagnosis ratio such as table 3 are listed:
Table 3 sudden change diagnosis ratio
W/m refers to wild-type probe fluorescence signal intensity MV and corresponding positions point mutation.
The assembling of embodiment 3 test kits
Press embodiment 1 preparation gene chip, the PCR primer packing with SEQ ID NO.66～87 of embodiment 2 records makes test kit with packing after the gene chip that makes, hybridization solution, the assembly of nucleic acid hybridization washing lotion.
Sequence table
< 110>Shanghai Inst. of Microsystem and Information Technology, Chinese Academy of Sci of Medical University Of Anhui
< 120>Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip and preparation thereof and application
<130>PCNWX081297
<160>87
<170>PatentIn?version?3.1
<210>1
<211>20
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>1
attctaggct?atatacaact 20
<210>2
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>2
attctaggcc?atatacaac 19
<210>3
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>3
gttttatggc?gtcagcg 17
<210>4
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>4
agttttatgg?tgtcagcga 18
<210>5
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>5
acctctagcc?tagcc 15
<210>6
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>6
acctctaacc?tagcc 15
<210>7
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>7
tctcatatga?agtcacc 17
<210>8
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>8
tctcatataa?agtcacc 17
<210>9
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>9
tcgggggtat?gctgt 15
<210>10
<211>14
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>10
tcgggggcat?gctg 14
<210>11
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>11
ggtgtatgag?ttggtc 16
<210>12
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>12
ggtgtatggg?ttggt 15
<210>13
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>13
tttttcatag?gaggtg 16
<210>14
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>14
tttttcatat?gaggtg 16
<210>15
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>15
tggagacata?tcatataag 19
<210>16
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>16
tggagacatg?tcatataag 19
<210>17
<211>14
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>17
ttcggggtat?gggc 14
<210>18
<211>14
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>18
ttcggggcat?gggc 14
<210>19
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>19
gctgccatca?agtatt 16
<210>20
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>20
gctgccataa?agtatt 16
<210>21
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>21
cctcactaaa?cgtaag 16
<210>22
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>22
cctcactaga?cgtaag 16
<210>23
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>23
gctaaccggc?ttttt 15
<210>24
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>24
gctaaccagc?ttttt 15
<210>25
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>25
cttttttgtc?tagattt 17
<210>26
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>26
cttttttgtt?tagattt 17
<210>27
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>27
cctacaagcc?tcaga 15
<210>28
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>28
cctacaatcc?tcaga 15
<210>29
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>29
ttttgtagcc?acagg 15
<210>30
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>30
ttttgtaacc?acagg 15
<210>31
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>31
cggcaaagac?tagtat 16
<210>32
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>32
cggcaaaggc?tagta 15
<210>33
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>33
tgagaatgac?tgcgc 15
<210>34
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>34
tgagaatgat?tgcgc 15
<210>35
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>35
gatgtaagcc?cgtgg 15
<210>36
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>36
gatgtaagtc?cgtgg 15
<210>37
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>37
tatgatgcga?ctgtg 15
<210>38
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>38
ttatgatgtg?actgtg 16
<210>39
<211>14
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>39
aacgcctggc?agcc 14
<210>40
<211>14
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>40
aacgcctgac?agcc 14
<210>41
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>41
tcgcaggatt?tctca 15
<210>42
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>42
tcgcagaatt?tctca 15
<210>43
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>43
tactacagcg?atggc 15
<210>44
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>44
tactacagtg?atggc 15
<210>45
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>45
ggaatgatgg?ttgtct 16
<210>46
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>46
ggaatgattg?ttgtct 16
<210>47
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>47
ggaatgatcg?ttgtct 16
<210>48
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>48
gggaatgatg?gttgt 15
<210>49
<211>14
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>49
gggaatggtg?gttg 14
<210>50
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>50
aatttattta?ggggga 16
<210>51
<211>16
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>51
aatttattca?ggggga 16
<210>52
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>52
tttaatttat?ttagggg 17
<210>53
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>53
tttaatttgt?ttagggg 17
<210>54
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>54
tgttagcggt?gtggt 15
<210>55
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>55
tgttagcagt?gtggt 15
<210>56
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>56
ccttctccta?tttatgggg 19
<210>57
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>57
ccttctccta?tatatgggg 19
<210>58
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>58
gtcgtggttg?tagtc 15
<210>59
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>59
gtcgtggctg?tagtc 15
<210>60
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>60
actcagtaga?cagtccc 17
<210>61
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>61
actcagtaaa?cagtccc 17
<210>62
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>62
tagcatccgt?actatac 17
<210>63
<211>17
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>63
tagcatccat?actatac 17
<210>64
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>64
ttccaaggac?aaatc 15
<210>65
<211>15
<212>DNA
<213>artificial?sequence
<220>
< 223>probe
<400>65
ttccaagggc?aaatc 15
<210>66
<211>21
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>66
cagccgctat?taaaggttcg?t 21
<210>67
<211>21
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>67
tggcaggagt?aatcagaggt?g 21
<210>68
<211>21
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>68
tcttcaatca?gccacatagc?c 21
<210>69
<211>21
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>69
tctcggtaaa?taaggggtcg?t 21
<210>70
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>70
accaatccta?cctccatcg 19
<210>71
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>71
ccaaggagtg?agccgaagt 19
<210>72
<211>23
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>72
tagaagaacc?ctccataaac?ctg 23
<210>73
<211>23
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>73
tagcgtcttg?tagacctact?tgc 23
<210>74
<211>20
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>74
acacccactc?cctcttagcc 20
<210>75
<211>22
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>75
gttgggacga?ttagttttag?ca 22
<210>76
<211>22
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>76
cgtccttgcc?ctattactat?cc 22
<210>77
<211>20
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>77
ttgggtgcta?atggtggagt 20
<210>78
<211>22
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>78
gaccctactt?ctaacctccc?tg 22
<210>79
<211>20
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>79
cacctcaact?gcctgctatg 20
<210>80
<211>22
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>80
gcaccacgac?cctactacta?tc 22
<210>81
<211>21
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>81
gggatgatga?ggctattgtt?t 21
<210>82
<211>22
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>82
cgaaaccaaa?taattcaagc?ac 22
<210>83
<211>22
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>83
gaaagttgag?ccaataatga?cg 22
<210>84
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>84
caaacgcctg?agccctatc 19
<210>85
<211>23
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>85
gaatccgagt?atgttggaga?aat 23
<210>86
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>86
catcggcatt?atcctcctg 19
<210>87
<211>19
<212>DNA
<213>artificial?sequence
<220>
< 223>primer
<400>87
gttgtttgat?cccgtttcg 19
Claims (3)
1. the detection kit in a Leber hereditary optic neuropathy related mtDNA mutational site; Comprise Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip; Pcr amplification primer with Leber hereditary optic neuropathy related mtDNA mutational site; The gene chip carrier surface dot matrix of said Leber hereditary optic neuropathy related mtDNA mutational site integrated detection gene chip is fixed with the oligonucleotide probe that sequence is SEQ ID NO.1～65; Said and pcr amplification primer Leber hereditary optic neuropathy related mtDNA mutational site are selected from one or more in the nucleotide sequence that sequence is SEQ ID NO.66～87; And whenever align and have at least one to be labeled in the anti-primer; 5 ' end of said oligonucleotide probe has amido modified group, and probe 5 ' is held the Poly T that has a section 15 to gather.
2. the detection kit in Leber hereditary optic neuropathy related mtDNA mutational site according to claim 1; It is characterized in that; Also be fixed with negative control and positive control on the said gene chip, wherein, positive control is the equivalent mixed liquor of selected each oligonucleotide probe.
3. the preparation method of integrated detection gene chip described in the detection kit in the said Leber hereditary of claim 2 optic neuropathy related mtDNA mutational site comprises the following steps:
With being selected from sequence is that the oligonucleotide probe 5 ' end of SEQ ID NO.1～65 adds the Poly T that the preceding paragraph 15 gathers; And carry out amido modified to 5 ' end; With deionized water each probe is diluted respectively; And mix with the sampling liquid equal-volume respectively, obtaining final concentration is the oligonucleotide probe solution of 12.5～50 μ M, and oligonucleotide probe solution is also fixing in slide surface with negative control and positive control dot matrix.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810204771 CN101497925B (en) | 2008-12-17 | 2008-12-17 | Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810204771 CN101497925B (en) | 2008-12-17 | 2008-12-17 | Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101497925A CN101497925A (en) | 2009-08-05 |
| CN101497925B true CN101497925B (en) | 2012-05-02 |
Family
ID=40945196
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810204771 Expired - Fee Related CN101497925B (en) | 2008-12-17 | 2008-12-17 | Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101497925B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146443B (en) * | 2011-01-06 | 2013-01-16 | 中国科学院昆明动物研究所 | Specific primer for detecting Leber hereditary optic neuropathy mitochondrial DNA mutation G10680A |
| CN102703577B (en) * | 2011-01-28 | 2014-05-28 | 珠海出入境检验检疫局检验检疫技术中心 | Gene chip and detection method thereof |
| CN102286629B (en) * | 2011-09-08 | 2013-01-02 | 中南大学湘雅医院 | Hereditary spastic paraplegia gene diagnosis chip |
| RU2566271C9 (en) * | 2014-06-25 | 2016-01-20 | Федеральное государственное бюджетное учреждение "Научно-исследовательский институт глазных болезней" Российской академии медицинских наук (ФГБУ "НИИГБ" РАМН) | Diagnostic technique for hereditary optic neuropathy |
| CN104805210B (en) * | 2015-04-30 | 2017-02-22 | 浙江大学 | Gene detection method of Leber's hereditary optic neuropathy, (LHON) gene chip and kit |
| CN114250296B (en) * | 2022-02-09 | 2024-02-20 | 深圳市妇幼保健院 | Primer group for detecting Leber hereditary optic neuropathy based on nucleic acid mass spectrum |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1345939A (en) * | 2000-09-29 | 2002-04-24 | 上海博德基因开发有限公司 | Novel polypeptide-human mitochondrial DNA polymerase 29.7 and polynucleotide for encoding said polypeptide |
| CN1606621A (en) * | 2000-10-31 | 2005-04-13 | 日立化学研究中心 | Method for collecting and using nuclear mRNA |
| CN101016564A (en) * | 2006-12-08 | 2007-08-15 | 武汉大学 | Micro-array chip with forty-five gene locus for detecting mitochondria diabetes |
| CN101177702A (en) * | 2007-10-23 | 2008-05-14 | 安徽医科大学 | Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method |
-
2008
- 2008-12-17 CN CN 200810204771 patent/CN101497925B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1345939A (en) * | 2000-09-29 | 2002-04-24 | 上海博德基因开发有限公司 | Novel polypeptide-human mitochondrial DNA polymerase 29.7 and polynucleotide for encoding said polypeptide |
| CN1606621A (en) * | 2000-10-31 | 2005-04-13 | 日立化学研究中心 | Method for collecting and using nuclear mRNA |
| CN101016564A (en) * | 2006-12-08 | 2007-08-15 | 武汉大学 | Micro-array chip with forty-five gene locus for detecting mitochondria diabetes |
| CN101177702A (en) * | 2007-10-23 | 2008-05-14 | 安徽医科大学 | Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method |
Non-Patent Citations (2)
| Title |
|---|
| DiMauro S.Mitochondrial encephalomyopathies.《Arch Neurol》.1993,第50卷(第11期),1197−1208. * |
| 杜卫东.Leber遗传性视神经病变线粒体DNA原发性突变位点的分析.《疾病控制杂志》.2006,第10卷(第6期),574-579. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101497925A (en) | 2009-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20240141443A1 (en) | Biomarkers and uses thereof | |
| CN101497925B (en) | Leber's hereditary optic neuroretinopathy related mtDNA mutant site integrated detection gene chip, as well as preparation and use thereof | |
| CN101619352B (en) | Double-probe gene mutation detecting method based on allele special amplification as well as special chip and kit thereof | |
| WO2009043178A1 (en) | Biomarkers for autism spectrum disorders | |
| WO2008070074A2 (en) | Genetic markers of schizophrenia | |
| CN111676283B (en) | Application of mitochondrial DNA single nucleotide polymorphism related to occurrence of high altitude pulmonary edema | |
| CN106834434B (en) | Nucleic acid, kit and method for detecting COX-1, COX-2 and GPIIIa gene polymorphism | |
| KR101068939B1 (en) | DNA chip and PNA chip for the dystrophin gene deletion, duplication, point mutation and DMD/BMD screening test therethrough | |
| CN101177702B (en) | Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method | |
| AU2014203213A1 (en) | Diagnostic markers for ankylosing spondylitis and uses thereof | |
| CN111944912B (en) | Skin gene detection method | |
| WO2021135559A1 (en) | Primer group for detecting human red blood cell abo blood type genotyping, kit and application thereof | |
| US20120148574A1 (en) | Diagnostic Markers for Ankylosing Spondylitis | |
| CN100348732C (en) | Quantitative detection of Leber's genetic optic nerve disease | |
| CN109402249A (en) | A kind of method and kit detecting CYP2C19 gene pleiomorphism | |
| Prior et al. | A model for molecular screening of newborns: simultaneous detection of Duchenne/Becker muscular dystrophies and cystic fibrosis | |
| CN113322317A (en) | Primer pair, probe set and kit for mitochondrial obesity gene mutation detection | |
| CN108410966B (en) | Method for detecting insertion and deletion of ACE gene | |
| CN101525620B (en) | COPPOCK cataract disease-causing gene and detecting method thereof | |
| WO2023283838A1 (en) | Composition for detecting sickle-cell disease, kit, and application | |
| CN107236801A (en) | The detection kit that a kind of step reverse transcription RT qPCR people MTHFR is expressed with MTRR gene pleiomorphisms | |
| CN107828879B (en) | PKP2 gene mutation detection kit and detection method thereof | |
| CN114774552A (en) | Diagnosis marker and diagnosis reagent for nevoid basal cell carcinoma syndrome and application | |
| CN116479111A (en) | Congenital pupil-free small eyeball-cataract syndrome screening method and screening kit | |
| KR20220137382A (en) | Genetic polymorphic markers for determining wrinkle type of skin and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120502 Termination date: 20151217 |
|
| EXPY | Termination of patent right or utility model |