WO2013042993A9

WO2013042993A9 - Sprex-dna chip kit for detecting high-resolution mica alleles

Info

Publication number: WO2013042993A9
Application number: PCT/KR2012/007607
Authority: WO
Inventors: 김태규
Original assignee: 가톨릭대학교 산학협력단
Priority date: 2011-09-23
Filing date: 2012-09-21
Publication date: 2013-05-16
Also published as: WO2013042993A3; KR101374247B1; KR20130032837A; WO2013042993A2

Abstract

The present invention relates to a SPREX-DNA chip kit for detecting high-resolution MICA alleles. More particularly, the SPREX-DNA chip kit of the present invention uses a sequence specific primer refractory extension (SPREX)-DNA chip to select high-resolution MICA alleles which is a human major histocompatibility complex (MHC) class I polypeptide-related sequence A.

Description

[Person ^ years old

[Name of invention]

S P R E X ᅳ D N A chip kit for high resolution M CA CA allele detection

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. The present invention relates to a high-resolution MICA allele detection SPREX-DNA chip kit capable of selecting high resolution MICA alleles.

Background Art

After allograft, the genetic location that is thought to be the cause of the rejection of the transplant was identified as the Major Histocompatibility Complex (MHC), and this MHC locus is not only an important role in tissue histocompatibility, It contains a large number of polymorphic genes that serve as another early function of the immune system. In the MHC gene group of humans, genes such as HLA-A, B, and C exist in the class I region, and HLA-DR, DQ, and DP genes exist in the MHC class II region. Class I gene's protein structure is made up of α chain and β ₂ microglobulin while showing the binding to achieve a form of crystal (crystal) crystal, a flux without being combined with β ₂ microglobulin (flexible) type.

Human Leukocyte Antigen (HLA) is a cell surface antigen that is primarily involved in the rejection of grafts, and its importance and the need for accurate tests as transplantation develops as a treatment for intractable diseases with the development of modern medicine Is steadily increasing. In recent years, methods for diagnosing by DNA test have been developed regarding the HLA gene. Meanwhile, the 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. Currently, 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,. However, 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.

Therefore, there is a need for a method for easily selecting high resolution MICA alleles.

Prior Art Documents

[Patent literature]

1. Republic of Korea Patent Publication No. 2009-00316, 2009.03.27

[Non-patent literature]

1. Thesis of Cha, Chung-Hwan, Department of Medicine, University of Ulsan, 2008, "Relationship of MIC Alleles and Haplotypes in Koreans with Severe Acute Exacerbation of MICA

2. Sohn et al., 2009, “MICA polymorphism and ha lotype with HLA-B and HLA ᅳ 腿 1 in Koreans "

3. Welsh Blood Service, Welsh Trans lant at ion and Immunogenet ics Laboratory, Ree et al., 2005, "Tying System for the Major Histocompatibility Complex Class I Chain Related Genes A and B Using Polymerase Chain React ion with Sequence-Specific Primers "

4. Yao et al., Labor fur Immunogenet ik, Germany, 1999, "Typing System for the Major Histocompat ibi 1ity Complex Class I Chain Related Genes A and B Using Polymerase Chain React ion with Sequence-Spec i f i c Primers"

5. Dissertation, Zhang, Texas, Dallas, Southwestern Medical University Zhang et al., 2000, "Typing for All Known MICA Alleles by Group-Specific PCR and SSOP"

[Detailed Description of the Invention]

[Technical problem]

It is an object of the present invention to provide a DNA chip for detecting MICA alleles and a kit comprising the same for selecting high resolution MICA alleles found in Koreans. Another object of the present invention is to provide a method for selecting an MICA allele using the selection kit.

Technical Solution

In order to achieve the above object, 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. ; And

It 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

Amplifying the MICA allele DNA in a DNA sample using an antisense primer of SEQ ID NO: 85 and a sense primer of SEQ ID NO: 86;

Separating the amplified DNA into single strands and cutting it into appropriate lengths; A third step of adding the cleaved DNA, fluorescently labeled dideoxy nucleotide, and polymerase to the DNA chip of claim 1 to perform hybridization and polymerization; And

Provided is a method for screening an MICA allele comprising a fourth step of analyzing a fluorescent signal of a polymerized probe.

Advantageous Effects

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. ^'

Since the present invention can distinguish the major histocompatibility antigen complex at the gene level, it can be widely used for more accurate histocompatibility determination.

[Brief Description of Drawings]

Figure 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.

3 to 5 show a table for reading the MICA genotype using the DNA chip for detecting the MICA allele of the present invention. [Form for implementation of invention]

Hereinafter, the configuration of the present invention will be described in detail.

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.

As used herein, 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. ᅳ-Also, 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. By means of hybridization and polymerization using a probe, a method of typing a specific gene by analyzing a fluorescent signal of a probe polymerized with fluorescently labeled dideoxy nucleotides. 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.

In addition, 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. 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 C¾ 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.

In addition, 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. The thing which coated polymer etc. to the board | substrate is mentioned.

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 Ν, Ν'-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. In addition, the MICA allele selection kit

Lambda axonucleases (1 ambda exonuc 1 ease) for separating single strands of amplified products by polymerization chain reaction;

Dephosphoryase and uracil DNA glycosylase (UDG) for cleaving the isolated single strand to an appropriate length; and

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

A first step of amplifying the MICA allele DNA in a DNA sample using an antisense primer of SEQ ID NO: 85 and a sense primer of SEQ ID NO: 86;

A second fragment 1 separating the amplified DNA into single strands and cutting to a suitable length; A third step of adding the cleaved polysaccharide, fluorescently labeled dideoxy nucleotide and polymerase to the DNA chip of the U term to perform hybridization and polymerization; And

A method for screening a MICA allele comprising a fourth step of analyzing the fluorescent signal of a polymerized probe.

The method of screening the MICA allele of the present invention will be described in detail as follows.

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 that can amplify the 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.

At this time, 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.

In addition, when the separated single strand is too long, the probability of generating an error in the subsequent hybridization reaction with the DNA chip increases, so in order to prevent this, it is recommended to cut the separated single strand to an appropriate length.

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). 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.

In addition, 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.

DNA chips from which unbanned DNA has been removed can be dried and used for analysis. The fourth step is to analyze the fluorescence signal of the polymerized probe.

After the reaction, 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.

Through the above test, the MICA allele type of the DNA sample can be analyzed by selecting a subclass of the MICA allele for the DNA sample. Hereinafter, 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.

Example l Preparation of DNA Chip for MICA Allele Detection

(Creation of probe)

Various alleles referenced to the MICA allele sequence reported in the HLA database The diversity sequence of the submerged ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾R ^ P gene was designed to find the diversity sequence for each position.

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 ((ΠΤΡ), 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.

Table 1

MICA Screening Probes: Probes 1 to 78 I Probe Number Base Sequence (5'-3 ') Sequence Number I

5'-ctt ccccagAGCCCCACAGTCTTCG (SEQ ID NO: 1)

2 5'-agAGCCCCACAGTCTTCCT (SEQ ID NO: 2)

5 '-tt ccccagAGCCCCACAGTCTTCG (SEQ ID NO: 3)

5 ¹ -TATAACCTCACGGTGCTGTCCT (SEQ ID NO: 4)

5 ¹ -TATAACCTCACGGTGCTGTCCG (SEQ ID NO: 5)

5-GGATCTGTGCAGTCAGGGTTTCTCA (SEQ ID NO: 6)

5 -GATGGATCTGTGCAGTCAGGGnTCTCG (SEQ ID NO: 7)

5'-GGATCTGTGCAGTCAGGGTTTCTT (SEQ ID NO: 8)

5 '-TGCAGTCAGGGTTTCTYRCTGAGGT (SEQ ID NO: 9)

5 '-TGCAGTCAGGGITTCTCGCTGAGGG (SEQ ID NO: 10)

5 '-TGGATGGTCAGCCOTCCTGCGCTA (SEQ ID NO: 11)

5 '-GTGGGCAGAAGATGTCCTGGGAAAT (SEQ ID NO: 12)

5 '-GTGGGCAGAAGATGTCCTGGGAAAC (SEQ ID NO: 13)

5 '-AGACATGGGACAGAGAGACCAGA (SEQ ID NO: 14)

5 '-tgggtgggggcagGCTTGCATTCCC (SEQ ID NO: 15)

5 '-t gggt gggggcagGCTTGCATTCCT (SEQ ID NO: 16)

5 '' -gggggcagGCTTGCATTCCCTCCA (SEQ ID NO: 17)

5 '-cagGCTTGCATTCCCTCC (SEQ ID NO: 18)

5 '-GATCCATGAAGACAACAGCACCAGG (SEQ ID NO: 19)

5 '-GATCCATGAAGACAACAGCACCAAG (SEQ ID NO: 20)

2 5 '-AGGAGCTCCCAGCATTTCTACTACG (SEQ ID NO: 21)

22 5 '-AGGAGCTCCCAGCATTTCTACTATG (SEQ ID NO: 22) Squeeze l ± t OQ

口 5 '-TCCCAGCATTTCTACTACGATGGGG (SEQ ID NO: 23) Probe 24 5' -TCCCAGCATTTCTACTACGATAGGG (SEQ ID NO: 24) Probe 25 5 '-GGGGAGCTCnCCTCTCCCAAAACG (SEQ ID NO: 25) Probe 26 5' —CTCTTCCTCCCCCAMACSTGGAGA (SEQ ID NO: 26 AACC) SEQ ID NO: 27) Probe 28 5 '-TTCCTCTCCCAAAACCTGGAGACTA (SEQ ID NO: 28) Probe 29 5' -nCCTCTCCCAAAACSTGGAGSCTG (SEQ ID NO: 29) Probe 30 5 '-AACCTGGAGACTGAGGAATGGACAA (SEQ ID NO: 30) Probe 31 5' -AGAGCTCAGACCn 5 '-AGAGCTCAGACOTGGCCATGAACA (SEQ ID NO: 32) Probe 33 5' -AGGAATTTCnGAAGGAAGATGCCA (SEQ ID NO: 33) Number 36) Probe 37 5 '-TACGGCGATATCTARAATCCAG (SEQ ID NO: 37) Probe 38 5' -CTACGGCGATATCTARAATCCRGCG (SEQ ID NO: 38) Probe 39 5 '-CTACGGCGATATCTAGAATCCAGCA (SEQ ID NO: 39) Probe 40 5' -AATCCRGCRTAGTCCTGAG GAGAAC (SEQ ID NO: 40) Probe 41 5 '-AATCCRGCGTAGTCCTGAGGAGAAG (SEQ ID NO: 41) Probe 42 5' -CCCATGGTGAATGTCACCCGCAGC (SEQ ID NO: 42) Probe 43 5 '-CCCATGGTGAATGTCACCCGCAGT (SEQ ID NO: 43) Probe 44 5' -GGTGAATGCTCCCCCCC Ramp hand 45 5 '-GGTGAATGTCACCCGCAGCGAGGCA (SEQ ID NO: 45) probe 46 5' -CAGCGAGGCCTCAGAGGGCAACATT (SEQ ID NO: 46) ACCGTGACATGCAGGGCHCCA (SEQ ID NO: 49) Probe 50 5 '-TGACATGCAGGGOTCYRGCnCTA (SEQ ID NO: 50) ) Probe 54 5'-TCTRTCCCYGGAATATCAYACTGAG (SEQ ID NO: 54) Probe 55 5 '-TCTATCCCCGGAATATCAYACTGAC (SEQ ID NO: 55) Probe 56 5' -YACTGASCTGGCGTCAGGATGGGGT (SEQ ID NO: 56). Probe 57 5 '-CACTGACCTGGCGTCAGGATGGGCT (SEQ ID NO: 57) Probe 58 5' -CTTTGAGCCACGACACCCAGCAGTC (SEQ ID NO: 58) 口 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 63 5 '-YAGGATTTGCYRAGGAGAGGAGCAG (SEQ ID NO: 63)

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 67 5 '-CACCTGCTACATGGAACACAGCAGG (SEQ ID NO: 67)

Probe 68 5 ¹ -ACATGGAACACAGCRGGAATCACAG (SEQ ID NO: 68)

Probe 69 5 ¹ -CACAGCGGGMTCACRGCACTCACC (SEQ ID NO: 69)

70 70 ¹ -CACAGCGGGAATCACAGCACTCACG (SEQ ID NO: 70)

E w 71 5 ¹ -GACAnCCATGITTCTGCTGTTGCTGCTGC (SEQ ID NO: 71;

el 一 level

ㅁ ㅁ 72 5 '' -CATGTTTCTGCTGnGCTGCTGG (SEQ ID NO: 72)

ㅁ ㅁ 73 5 '' -ATTTTTGmTTAnATTITCTACG (SEQ ID NO: 73)

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)

78 5 ¹ -AGAAGAAAACATCAGCTGCAGATGG (SEQ ID NO: 78)

1) Lowercase: Intron sequence. Underlined MICA polymorphism site. R = A + G, Y = C + T, M = A + C, K = G + T, S = G + C

2) 'G' delet ion (between T and G) at the second position from the 3 'end

Table 2

MICA Bisexual / Body

(Manufacture of DNA Chip)

The probes prepared above were dissolved in a buffer solution (350mM sodium bicarbonate, H 9 ᅳ 0) at 50 μΜ, 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. Subsequently, 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). 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.

Example 2 MICA Allele Selection Using 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 ᅳ 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. At this time, the base sequence of the primer used is as follows:

1) Antisense Primer: MICA-R

MICA-R: 5 ¹ -GATGCTGCCCCCAnCCCnCCCAA-3 '' (SEQ ID NO: 85)

2) Sense primer: MICA-F

MICA-F: 5'—P-CGTTCTTGTCCCmGCCCGTGTGC ᅳ 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 隱 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. Thus, 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 ： 01, * 002 ： 01 / * 015 genotype test results.

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. Thus, it can be seen that hundreds of MICA allele types can be typed using the probes 1 to 78 of the present invention. _,

Industrial Applicability

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.

Claims

[Range of request]

[Claim 1]

DNA chip for MICA allele detection comprising a probe of SEQ ID NOs: 1 to 78 capable of specifically binding to MHC (MHC class I polypept i de-related sequence A) allele

[Claim 2]

According to claim 11,

DNA for MICA allele detection further comprising one or more MICA allele positive control probes selected from the group consisting of the sequences set forth in SEQ ID NOs: 79-81 and 84 or a MICA allele negative control probe of SEQ ID NOs: 82 or 83 Chip ᅳ

[Claim 3]

Constructing a raised nucleotide probe of SEQ ID NOs: 1 to 78 of 19 to 30 mer in length so that the position representing the polymorphism of the MICA allele is located at 1 to 3 base sites at the 3 'terminal site; A method for producing a DNA chip for detecting MICA alleles, comprising binding an oligonucleotide probe to a solid surface.

[Claim 4]

The method of claim 3,

The probe is a method for producing a DNA chip for detecting MICA alleles further comprising 18 thymine (dTTP), 6 CH ₂ chain and an amine group at the 5 'end.

[Claim 5]

MICA allele selection key comprising the DNA chip for detecting the MICA allele of item 1

[Claim 6]

The method of claim 5,

MICA allele selection kit further comprising 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.

[Claim 7]

The method of claim 6,

Sense primer MICA allele selection kit further comprises a phosphate (phosphate) group at the 5 'end.

[Claim 8]

The method of claim 5,

Lambda axonucleases (1 ambda exonuc 1 ease) for separating single strands of amplification products by polymerase chain reaction;

MICA allele selection kit further comprises a fluorescently labeled dideoxy nucleotide that can detect the amplification product bound to the DNA chip.

[Claim 9]

The method of claim 8,

Fluorescently labeled dideoxy nucleotides are cyanine 5 (Cyanine 5) dCTP MICA allele selection kit.

[Claim 10]

Amplifying the MICA allele DNA in a DNA sample using an antisense primer of SEQ ID NO: 85 and a sense primer of SEQ ID NO: 86; Separating the amplified DNA into single strands and cutting it into appropriate lengths in two steps;

A third step of adding the cleaved DNA, a fluorescently labeled dideoxy nucleotide, and a polymerase to the DNA chip of claim 1 to perform hybridization and polymerization; And

A method for screening a MICA allele comprising four steps to analyze the fluorescent signal of the polymerized probe. ^'

[Claim 11]

The method of claim 10, wherein the second step is

Amplified DNA is treated with lambda exonuclease to separate into single strands; And

The isolated single strand is treated with desphosphinase (shrimp alkaline phosphatase) and uracil DNA glycosylase (UDG) to cut the length of 50 to lOObp.

[Claim 12]

The method of claim 10,

Hybridization and polymerization reactions are screened for MICA alleles at 50 to 65 ^° C. for 20 to 60 minutes.