WO2013042993A2 - Trousse de puce à adn-sprex pour la détection d'allèles mica à résolution élevée - Google Patents

Trousse de puce à adn-sprex pour la détection d'allèles mica à résolution élevée Download PDF

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WO2013042993A2
WO2013042993A2 PCT/KR2012/007607 KR2012007607W WO2013042993A2 WO 2013042993 A2 WO2013042993 A2 WO 2013042993A2 KR 2012007607 W KR2012007607 W KR 2012007607W WO 2013042993 A2 WO2013042993 A2 WO 2013042993A2
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mica
seq
dna
probe
allele
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PCT/KR2012/007607
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Korean (ko)
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WO2013042993A3 (fr
WO2013042993A9 (fr
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김태규
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가톨릭대학교 산학협력단
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Publication of WO2013042993A9 publication Critical patent/WO2013042993A9/fr
Publication of WO2013042993A3 publication Critical patent/WO2013042993A3/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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a sequence A (MHC class I polypept i de-related sequence A) associated with a class I polypeptide of a human histocompatibility antigen complex using a DNA chip prepared according to a sequence specific primer refractory extension (SPREX) method.
  • SPREX sequence specific primer refractory extension
  • the present invention relates to a high-resolution MICA allele detection SPREX-DNA chip kit capable of selecting high resolution MICA alleles.
  • MHC Major Histocompatibility Complex
  • HLA Human Leukocyte Antigen
  • MHC class I chain-related (MIC) gene is located in the C class I region of the sixth chromosome, and a locus having a total of seven similar sequences at the MIC locus was found and was named from MICA to MICG. Of these, only MICA and MICB are functional genes.
  • MIC proteins are similar in structure to human leukocyte antigen (HLA) class I proteins and are known to be very polymorphic as HLA. Alleles show differences in allele distributions.
  • the MICA gene is a surface glycoprotein that is responsible for innate immunity and is expressed in vascular endothelial cells, branched cells, and fibroblasts, but not in peripheral blood lymphocytes. It has been reported to be associated with diseases such as ankylosing spondylitis, Behcet's disease, psoriasis, insulin-dependent diabetes mellitus, Adison disease, primary sclerotic cholangitis, and ulcerative colitis.
  • MICA has been reported to analyze high resolution allele types using sequencing (PCR-SSP, PCR-SBT, PCR-SS0P) based on polymerase chain reaction.
  • the type analysis method is performed for the polymorphism of the MICA gene, mainly to analyze the MICA allele type by analyzing the microsatellite in axon 2, 3, 4,.
  • these methods require large amounts of genes used for amplification (old samples, etc. have difficulty acquiring large amounts of genes), may be error in reading the sequence, and may take a long time to amplify. There are disadvantages.
  • the present invention provides a DNA chip for detecting the MICA allele comprising a probe of SEQ ID NO: 1 to 78 that can specifically bind to the MICA (MHC class i polypeptide-related sequence A) allele to provide.
  • the present invention also provides a step for constructing a ligonucleotide probe of SEQ ID NOs: 1 to 7 8 of 19 to 30 mer in length such that the position representing the polymorphism of the MICA allele is located at 1 to 3 base sites at the 3 'terminal site. ;
  • the present invention provides a method for producing a DNA chip for MICA allele detection comprising the step of binding the constructed oligonucleotide probe to a solid surface.
  • the present invention also provides a MICA allele selection kit comprising a DNA chip for detecting the MICA allele of the present invention.
  • the invention also provides a MICA allele selection kit comprising a DNA chip for detecting the MICA allele of the present invention.
  • a method for screening an MICA allele comprising a fourth step of analyzing a fluorescent signal of a polymerized probe.
  • the present invention enables high resolution screening of MICA alleles, which is one of the major histocompatibility antigen complexes in humans, using probes designed to contain sites that exhibit polymorphisms of MICA alleles. '
  • the present invention can distinguish the major histocompatibility antigen complex at the gene level, it can be widely used for more accurate histocompatibility determination.
  • FIG 1 shows the arrangement of the MICA allele probe on the MICA allele detection DNA chip of the present invention.
  • Figure 2 is a photograph showing the results of testing the MICA allele type using the DNA chip for MICA allele detection of the present invention.
  • the present invention relates to a MICA allele detection DNA chip comprising a probe of SEQ ID NOS: 1 to 78 that can specifically bind to MICACMHC class I polypept i de-related sequence A) alleles.
  • DNA chip for detecting the MICA allele of the present invention includes a probe capable of non-specifically binding to the MICA allele, the probe non-specifically binds to the MICA allele, in particular a position that indicates polymorphism of the MICA allele (base ) Is an oligonucleotide having a length of 19 to 30 mer including a 3 'terminal portion, and has a feature capable of selecting MICA alleles at high resolution.
  • the "high resolution of MICA alleles” refers to subtypes of subclasses of the MICA alleles, for example MICA * 002: 01, MICA * 008: 01, MICA * 007: 01, and the like.
  • “Low resolution screening” means that MICAs can be screened by subclasses of subtypes of genes such as MICA * 002, MICA * 008, MICAO07, etc. More specific selection will be called.
  • the term "SPREX (sequence specific primer refractory; extension)" as used herein includes a site showing polymorphism of a specific gene after processing the DNA of a specific gene amplified from the DNA of a test sample into a single chain.
  • SPREX sequence specific primer refractory; extension
  • the SPREX is distinguished in that it uses a probe containing a site (base) showing polymorphism of a specific gene, and the APEX (arrayed primer extension) uses a probe that does not include a site showing polymorphism of a specific gene.
  • the probe for the MICA allele which can be detected using the DNA chip for detecting the MICA allele of the present invention is characterized by consisting of the MICA selection probe specifically described in SEQ ID NOs: 1 to 78.
  • the DNA chip for detecting the MICA allele of the present invention may further include a positive or negative control probe for comparing the binding of the probe.
  • a positive or negative control probe for comparing the binding of the probe.
  • One or more sequences of SEQ ID NOS: 79-84 can be used as the positive or negative control probe. More specifically, one or more MICA positive control probes selected from the group consisting of the sequences set forth in SEQ ID NOs: 79 to 81 and 84, and negative control probes consisting of the sequences set forth in SEQ ID NOs: 82 or 83 can be used.
  • the present invention also provides a method for constructing an oligonucleotide probe of SEQ ID NOs: 1 to 78, 19 to 30 mer in length, such that the position representing the polymorphism of the MICA allele is located at 1 to 3 base sites at the 3 'terminal site; And it relates to a method for producing a DNA chip for MICA allele detection comprising the step of binding the oligonucleotide probe constructed on the solid surface.
  • the MICA allele detection probe has a position indicating polymorphism of the MICA allele at 1 to 3 base sites at the 3 'end, 18 thymines (cTTTP), 6 C3 ⁇ 4 chains and amines at the 5' end. It may be prepared by the step of constructing to include an (amine) group. Specifically, the probe may be those described in SEQ ID NOs: 1 to 78.
  • the oligonucleotides can be chemically synthesized by methods known per se in the art.
  • the DNA chip for detecting the MICA allele of the present invention can be fixed to a solid surface by a known method, for example, by binding the constructed DNA probe to the surface of an aldehyde-bonded solid through a siphonbase reaction. It can be prepared by reducing the aldehyde remaining without binding to the amine on the solid surface to which the DNA probe is bound, but is not particularly limited thereto.
  • the reduction of the aldehyde may be carried out using a reducing agent such as NaBH 4 , but is not particularly limited thereto.
  • the solid surface may be a substrate, a resin, a plate (eg, a multiwell plate), a filter, a cartridge, a column, or a porous material.
  • the substrate may be a nickel-PTFE (polytetrafluoroethylene) substrate, a glass substrate, an apatite substrate, a silicon substrate, a gold, silver or alumina substrate, or the like.
  • nickel-PTFE polytetrafluoroethylene
  • the resin includes agarose particles, silica particles, copolymers of acrylamide and N, N'-methylenebisacrylamide, polystyrene crosslinked divinylbenzene particles, particles crosslinked with epichlorohydrin, and cells. Loose fiber, allyldextran and ⁇ , ⁇ '-methylenebisacrylamide cross-linked polymer, monodisperse synthetic polymer, monodisperse hydrophilic polymer, Sepharose or Toyopearl, etc. Moreover, resin which combined various functional groups with these resin can be used.
  • the present invention also relates to a MICA allele selection kit comprising a DNA chip for detecting the MICA allele of the present invention.
  • the MICA allele selection kit may further include a primer set consisting of an antisense primer of SEQ ID NO: 85 and a sense primer of SEQ ID NO: 86 for specifically amplifying the MICA allele.
  • the sense primer may further include a phosphate group at the 5 'end.
  • the phosphate group is bound to the 5 'end of the amplification product through a polymerization chain reaction, so that the exonuclease recognizes the 5' end of the phosphate group to separate the double strand into a single strand.
  • the primer set is a specific sequence designed to amplify the axon region of the MICA, and more specifically, may amplify the axon 2, 3, 4, and 5 regions of the MICA.
  • the MICA allele selection kit is a specific sequence designed to amplify the axon region of the MICA, and more specifically, may amplify the axon 2, 3, 4, and 5 regions of the MICA.
  • Lambda axonucleases (1 ambda exonuc 1 ease) for separating single strands of amplified products by polymerization chain reaction;
  • the DNA chip may further include a fluorescently labeled dideoxy nucleotide capable of detecting an amplification product bound to the DNA chip.
  • the fluorescently labeled dideoxy nucleotide may be cyanine 5 clCTP. However, it is not particularly limited thereto.
  • the invention also provides cyanine 5 clCTP. However, it is not particularly limited thereto.
  • the invention also provides cyanine 5 clCTP. However, it is not particularly limited thereto.
  • the invention also provides cyanine 5 clCTP. However, it is not particularly limited thereto.
  • a second fragment 1 separating the amplified DNA into single strands and cutting to a suitable length;
  • a method for screening a MICA allele comprising a fourth step of analyzing the fluorescent signal of a polymerized probe.
  • the first step is amplification of the DNA sample.
  • the DNA collected from the sample to be tested is amplified using the primers of the MICA gene selection kit of the present invention.
  • the primer may be a primer capable of amplifying a MICA gene region such as axon 2, 3, 4, and 5 sites.
  • the second step is to separate the amplified DNA into single strands, and to process the separated single strands into appropriate lengths.
  • the method of separating into single strands is not particularly limited, it is preferable to use a method using an enzyme. More specifically, lambda exonuclease (lamda exonuclease) is treated with a single strand may be separated.
  • the method of cleaving a single strand is not particularly limited and may be cleaved using an enzyme. More specifically, deoxidase (shrimp in isolated single chain) Alkaline phosphatase) can be used to dephosphorylate the remaining dNTPs and can be cleaved into single chains of 50 to 100 bp in length by treatment with uracil DNA glycosylase (UDG).
  • UDG uracil DNA glycosylase
  • Crab is a hybridization and polymerization step using the processed DNA single chain DNA chip. The processed DNA, fluorescently labeled dideoxy nucleotides and polymerases are added to the DNA chip containing the MICA allele specific probe for hybridization and polymerization.
  • the synthesis and polymerization may be carried out at 50 to 65 ° C for 20 to 60 minutes, but is not particularly limited thereto.
  • washing and drying steps may be further performed to remove unreacted DNA from the DNA chip in order to increase the analysis of the fluorescent signal of the probe polymerized with fluorescently labeled dideoxy nucleotides.
  • the unreacted DNA may be removed by sequentially adding NaOH and alconox mixed aqueous solution and tertiary distilled water to the DNA chip, but are not particularly limited thereto.
  • the fourth step is to analyze the fluorescence signal of the polymerized probe.
  • the DNA chip was scanned at a wavelength range of 667 nm by using a scanner to examine the probe site where the polymerization reaction of the DNA chip was performed.
  • the MICA allele type of the DNA sample can be analyzed by selecting a subclass of the MICA allele for the DNA sample.
  • the present invention will be described in more detail with reference to examples according to the present invention, but the scope of the present invention is not limited to the examples shown.
  • a single salt chain having a sequence of 19 to 30 mer in length was constructed by positioning the polymorphism of the MICA allele at 1 to 3 bases at the 3 'end and constructing a single base chain.
  • the probe was constructed to include 18 thymine (( ⁇ ), 6 CH 2 chains and amine groups at the 5 'end of the.
  • Tables 1 to 2 below show the probes constructed above, Table 1 is a list of MICA selection probes, and Table 2 is a list of MICA negative / positive control probes.
  • Probes 1 to 78 I Probe Number Base Sequence (5'-3 ') Sequence Number I
  • Probe 57 5 '-CACTGACCTGGCGTCAGGATGGGCT (SEQ ID NO: 57)
  • Probe 58 5' -CTTTGAGCCACGACACCCAGCAGTC (SEQ ID NO: 58) ⁇ JJ 5 '-AACCTACCAGACCTGGGTGGCCACT (SEQ ID NO: 59)
  • Probe 60 5 '-AGACCTGGGTGGCCACCAGGATTTGCG (SEQ ID NO: 60)
  • Probe 61 5 '-GAGACCTGGGTGGCCACYAGGA1TTGCCG (SEQ ID NO: 61)
  • Probe 62 5 '-GTGGCCACCAGGA1TTGCCAAGGAA (SEQ ID NO: 62)
  • Probe 64 5 '-CAGGATTTGCCAAGGAGAGGAGCAA (SEQ ID NO: 64)
  • Probe 65 5 '-GA1TTGCCAAGGAGAGGAGCAGAGT (SEQ ID NO: 65)
  • Probe 66 5 '-CACCTGCTACATGGAACACAGCGGG (SEQ ID NO: 66)
  • Probe 69 5 1 -CACAGCGGGMTCACRGCACTCACC (SEQ ID NO: 69)
  • Probe 74 5 '-CTGCTATTTTTGTTATTA TATTTTCTA (SEQ ID NO: 74)
  • Probe 75 5 '— TrGTTATTATTATTTTCTATGTC; (SEQ ID NO: 75)
  • Probe 76 5 '-TATTGTTATTATTAT TTCTAYGTCT (SEQ ID NO: 76)
  • Probe 77 5 '-AGAAGAAAACATCAGCTGCAGAGGG (SEQ ID NO: 77)
  • the probes prepared above were dissolved in a buffer solution (350mM sodium bicarbonate, H 9 ⁇ 0) at 50 ⁇ , respectively, and the arrayer was coated on the surface of a slide coated with aldehydes (CS-100, CEL, Houston, TX, USA). (MicroGrid II, BioRobotics, USA) was used to drip the 150-degree interval 340 m in size, and then the siphon base reaction was performed.
  • CS-100 CS-100, CEL, Houston, TX, USA
  • the mixture was washed sequentially with a 0.2% ( ⁇ v / v) sodium dodecyl sulfate (SDS) solution and tertiary distilled water, followed by NaBH 4 solution (O.lg NaBH 4 , 30mC, P hosphate buffered).
  • NaBH 4 solution O.lg NaBH 4 , 30mC, P hosphate buffered.
  • saline (PBS), 10 ml, ethanol) was immersed for 30 minutes to reduce the amine unbound aldehyde residue, washed with tertiary distilled water and dried to prepare a DNA chip.
  • DNA isolated from blood was subjected to specific primers capable of amplifying 2m3, dGTP, dCTP, 0.8mM dTTP, 0.2mM dUTP, and axons 2, 3 ⁇ 4 and 5 of the MICA gene, polymerization buffer and polymerization.
  • 1 to 5 minutes exons 2, 3, 4 and 5 of the MICA gene at 95 ° C once using a reaction enzyme 10 sec at 95 ° C, 30 seconds at 65 ° C, one minute to 8 times at 72 ° C , was amplified by 10 seconds at 95 ° C, 30 seconds at 63 ° C, 1 minute 32 times at 72 ° C, at 72 ° C for 10 minutes, repeat once.
  • the base sequence of the primer used is as follows:
  • MICA-R 5 1 -GATGCTGCCCCCAnCCCnCCCAA-3 '' (SEQ ID NO: 85)
  • MICA-F 5'—P-CGTTCTTGTCCCmGCCCGTGTGC ⁇ 3 '(SEQ ID NO: 86)
  • Amplified DNA was treated with lambda exonuc lease (LOunitsA) and isolated into single chains. The separated single chains were treated with a deshrimp alkaline phosphotase to dephosphorylate the remaining dNTPs, and treated with uracil DNA glycosylase (UDG) to obtain single stranded DNA of 50 to 100 bp in length. .
  • the processed sample gene was added to the DNA chip prepared in Example 1, and labeled with dCTP (NEN Life Science) and polymerase (Thermo Sequenase, AP Biotech., USA) labeled with cyanine 5 (Cyanine 5) fluorescent material. ) was added and reacted at 60 ° C. for 30 minutes. Subsequently, the DNA chip was washed sequentially with 50mM NaOH and 0.1% alconox mixed solution for 10 minutes, followed by 5 minutes with tertiary distilled water, and then dried. The dried DNA chip was subjected to 100 ⁇ using a scanner Scanning was performed in the wavelength range of 667 kHz with a pixel size of.
  • Figure 1 shows the probe position for the MICA allele on the chip of Example 1 above.
  • Figure 2 is a photograph showing the results of testing the MICA allele type, each of the 18 samples have 16 MICA alleles, the increase, 4 are selected as a subclass of MICA alleles.
  • Figure 2 is MICA * 004 / * 010, * 007: 01 / * 008: 01, * 016 / * 018, * 001 / * 008: 01 of the DNA sample determined by the DNA chip of Example 1 * 010 / * 027, * 002: 01 / * 011, * 002 : 01 / * 012 : 01, * 008: 01 / * 011, * 004 / * 018, * 008 : 01 / * 041, * 002 : 01 / * 015, * 008 : 01 / * 045, * 001 / * 016, * 017 / * 019, * 004 / * 011, * 010 / * 027, * 007 : 01 / * 008 :
  • FIGS. 3 to 5 show a table for reading the genotype of the MICA using the DNA chip prepared in Example 1, wherein 1 to 78 of the first row of FIGS. 3 to 5 are the probe numbers, The first column represents the MICA allele type, '0' means 'signal' due to MICA high resolution SPREX chip, fluorescent material (Cyanine 5-dCTP) and fragmented amplification product, and 'X' means 'no signal''Means that.
  • MICA allele types can be typed using the probes 1 to 78 of the present invention.
  • the present invention enables high resolution screening of MICA alleles, which is one of the major histocompatibility antigen complexes in humans, using probes designed to contain sites that exhibit polymorphisms of MICA alleles.
  • the present invention can distinguish the major histocompatibility antigen complex at the gene level can be widely used for accurate histocompatibility determination.

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Abstract

La présente invention concerne une trousse de puce à ADN-SPREX pour la détection d'allèles MICA à résolution élevée. Plus particulièrement, la trousse de puce à ADN-SPREX de la présente invention utilise une puce à ADN-extension réfractaire d'amorce spécifique à une séquence (SPREX) pour choisir des allèles MICA à résolution élevée qui est une séquence A associée à un polypeptide du complexe majeur d'histocompatibilité (CMH) de classe I humain.
PCT/KR2012/007607 2011-09-23 2012-09-21 Trousse de puce à adn-sprex pour la détection d'allèles mica à résolution élevée WO2013042993A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005185177A (ja) * 2003-12-25 2005-07-14 Canon Inc Ｈｌａ−ｍｉｃａアレルを同定するためのプローブセット及び特定方法
KR20120007193A (ko) * 2010-07-14 2012-01-20 가톨릭대학교 산학협력단 고해상도 ＨＬＡ 대립유전자 ＳＰＲＥＸ-ＤＮＡ ｃｈｉｐ 키트

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Publication number Priority date Publication date Assignee Title
AU2004219820B2 (en) 2003-03-10 2008-06-12 Japan Science And Technology Agency Marker for detecting mesenchymal stem cell and method of distinguishing mesenchymal stem cell using the marker

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005185177A (ja) * 2003-12-25 2005-07-14 Canon Inc Ｈｌａ−ｍｉｃａアレルを同定するためのプローブセット及び特定方法
KR20120007193A (ko) * 2010-07-14 2012-01-20 가톨릭대학교 산학협력단 고해상도 ＨＬＡ 대립유전자 ＳＰＲＥＸ-ＤＮＡ ｃｈｉｐ 키트

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MENDOZA-RINCON J. ET AL.: 'Characterization of the MICA polymorphism by sequence- specific oligonucleotide probing' IMMUNOGENETICS vol. 49, no. 6, 1999, pages 471 - 478 *
O'MEARA D. ET AL.: 'SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays' NUCLEIC ACIDS RES. vol. 30, no. 15, 2002, page E75 *

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WO2013042993A3 (fr) 2013-06-27
KR20130032837A (ko) 2013-04-02
WO2013042993A9 (fr) 2013-05-16

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