KR101374247B1

KR101374247B1 - SPREX(Sequence Specific Primer Refractory Extension)-DNA chip kit for high resolution detection of MICA allele

Info

Publication number: KR101374247B1
Application number: KR1020120104973A
Authority: KR
Inventors: 김태규
Original assignee: 가톨릭대학교기술지주 주식회사
Priority date: 2011-09-23
Filing date: 2012-09-21
Publication date: 2014-03-13
Also published as: WO2013042993A3; WO2013042993A9; WO2013042993A2; KR20130032837A

Abstract

The present invention relates to a SPREX-DNA chip kit for detecting a high resolution MICA allele, and more specifically, to a class I polypeptide of a human major histocompatibility antigen complex using a DNA chip by a sequence specific primer refractory extension (SPREX) method. The MICA allele, a related MHC class I polypeptide-related sequence A, can be selected at high resolution.

Description

Sequence Specific Primer Refractory Extension (SPREX-DNA chip kit for high resolution detection of MICA allele)

The present invention provides a MICA allele which is a sequence A (MHC class I polypeptide-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. It relates to a high resolution MICA allele detection SPREX-DNA chip kit that can be screened at high resolution.

After allografts, the genetic location believed to be the cause of the rejection of the transplant was identified as the Major Histocompatibility Complex (MHC), and this MHC locus plays an important role in tissue histocompatibility as well as in the immune system. It also contains a large number of polymorphic genes that perform another initial function. HLA-A, B, and C genes are present in the class I region of the human MHC gene group, and HLA-DR, DQ, and DP genes are present in the MHC class II region. The protein structure of the class I gene is composed of α chain and β ₂ microglobulin, which forms a crystal and shows crystal crystals, while β ₂ microglobulin is not bound and is in a flexible form.

Human Leukocyte Antigen (HLA) is a cell surface antigen that is primarily involved in the rejection of graft rejection, and the importance of precise and accurate testing is increasing with the development of modern medicine as a treatment for intractable diseases. The situation is increasing continuously. 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 MHC class I region of the chromosome 6, and a locus having a total of seven similar sequences at the MIC locus was found and named from MICA to MICG. Of these, only MICA and MICB are functional genes. MIC protein is similar in structure to human leukocyte antigen (HLA) class I protein and is known to be very polymorphic as HLA. Allele distributions differ between species. The MICA gene is a surface glycoprotein 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-SSOP) 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 exon 2, 3, 4, or 5. However, these methods require a large amount of genes used for amplification (old samples, etc. have difficulty acquiring a large amount of genes), there may be errors in reading the gene sequence, and it may take a long time to amplify. There is this.

Therefore, there is a need for the development of a method that can easily select high resolution MICA alleles.

1. Republic of Korea Patent Publication No. 2009-0031671, 2009.03.27

1. Thesis of Cha, Chung-Hwan, Department of Medicine, University of Ulsan, 2008, "Relationship of MIC Alleles and Haplotypes in Koreans with Severe Acute Exacerbations of MICA and Chronic Hepatitis B." 2. Sohn et al., 2009, "MICA polymorphism and haplotype with HLA-B and HLA-DRB1 in Koreans" 3. Welsh Blood Service, Ree et al., Welsh Transplantation and Immunogenetics Laboratory, 2005, "Typing System for the Major Histocompatibility Complex Class I Chain Related Genes A and B Using Polymerase Chain Reaction with Sequence-Specific Primers" 4. Yao et al., Labor fur Immunogenetik, Munich, Germany, 1999, "Typing System for the Major Histocompatibility Complex Class I Chain Related Genes A and B Using Polymerase Chain Reaction with Sequence-Specific Primers" 5. Dissertation, Zhang et al., 2000, "Typing for All Known MICA Alleles by Group-Specific PCR and SSOP"

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.

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 NOS: 1 to 78 that can specifically bind to the MICA (MHC class I polypeptide-related sequence A) allele do.

The present invention also provides a method of 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 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 present 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;

A second step of separating the amplified DNA into single strands and cutting to an appropriate length;

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

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

The present invention can select high resolution MICA alleles, which is one of the major histocompatibility antigen complexes in humans, using probes constructed to include sites showing 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.

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.

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 an MICA (MHC class I polypeptide-related sequence A) allele.

DNA chip for detecting the MICA allele of the present invention includes a probe capable of non-specifically binding to the MICA allele, wherein the probe nonspecifically binds to the MICA allele, in particular the position (base) showing the polymorphism of the MICA allele Is an oligonucleotide having a length of 19 to 30 mer including a 3 'terminal portion, and has a feature of selecting MICA alleles at high resolution.

As used herein, "able to screen high resolution MICA alleles" means subtypes of subclasses of the MICA alleles, for example MICA * 002: 01, MICA * 008: 01, MICA * 007: 01, and the like. "Low resolution" means that the subclass of MICA alleles, for example, MICA * 002, MICA * 008, MICA * 007, etc. It will be called an embodied selection.

In addition, the "sequence specific primer refractory extension" (SPREX) referred to in the present specification refers to a probe containing a site showing polymorphism of a specific gene after processing a DNA of a specific gene amplified from the DNA of a test sample into a single chain. Means a method of typing a specific gene by analyzing the fluorescent signal of a probe polymerized with fluorescently labeled dideoxy nucleotides by synthesizing and polymerizing. The SPREX 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 of 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 is such that the position indicating the polymorphism of the MICA allele is located at 1 to 3 base sites at the 3 'end, 18 thymine (dTTP) at the 5' end, 6 CH ₂ chains, and It may be prepared by the step of constructing to include an amine (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 shif base reaction, the DNA The probe may be prepared by reducing aldehyde remaining on the solid surface to which the probe is bound without binding to the amine, 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, in which these substrates are coated with a polymer or the like. .

The resin is agarose particles, silica particles, copolymers of acrylamide and N, N'-methylenebisacrylamide, polystyrene crosslinked divinylbenzene particles, particles crosslinked with dextran with epichlorohydrin, cellulose fiber And cross-linked polymers of allyldextran and N, N'-methylenebisacrylamide, monodisperse synthetic polymers, monodisperse hydrophilic polymers, Sepharose or Toyopearl, and the like, and these resins Resin which combined various functional groups to 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 polymerase 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 exon region of the MICA, and more specifically, may amplify the exon 2, 3, 4 and 5 regions of the MICA.

In addition, the MICA allele selection kit

Lambda exonuclease for separating amplified products by polymerase chain reaction into single strands;

Dephosphatase 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 dCTP, but is not particularly limited thereto.

The present invention also

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

Referring to the MICA allele selection method of the present invention in detail step by step.

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 MICA gene regions, such as exon 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, it is preferable to treat lambda exonuclease to separate it into single strands.

In addition, when the separated single strand is too long, the probability of generating an error in the subsequent 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 for cleaving a single strand is not particularly limited, and it is preferable to cut with an enzyme. More specifically, the separated single chain is treated with desulfase (shrimp alkaline phosphatase) to dephosphorylate the remaining dNTPs, and treated with uracil DNA glycosylase (UDG) to a single chain having a length of 50 to 100 bp. Can be cut

The third step is the step of hybridization and polymerization using a DNA chip of the processed DNA single chain.

The processed DNA, fluorescently labeled dideoxy nucleotides, and polymerase 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 ℃ 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 degree of 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 is not particularly limited thereto.

DNA chips free of unreacted DNA can be dried and used for analysis.

The fourth step is to analyze the fluorescent 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 of the DNA chip was performed.

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

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited to the examples shown.

Example 1 Fabrication of DNA Chip for MICA Allele Detection

(Creation of probe)

Based on the MICA allele sequences reported in the HLA database, the diversity sequences of various alleles with known sequences were found to design the diversity sequences for each position.

A single base chain having a nucleotide sequence of 19 to 30 mer was constructed by placing a position representing the polymorphism of the MICA allele at 1 to 3 base sites at the 3 'end site, and at the 5' end of the constructed single base chain. The probe was constructed to contain 18 thymine (dTTP), 6 CH ₂ chains and amine groups.

Tables 1-2 below show the probes constructed above, Table 1 is a list of probes for MICA screening, and Table 2 is a list of probes for MICA negative / positive control.

(Manufacture of DNA Chip)

The probes prepared above were dissolved in a buffer solution (350 mM sodium bicarbonate, pH 9.0) at 50 μM, respectively, and an arrayer (MicroGrid) was applied to the surface of an aldehyde-coated slide (CS-100, CEL, Houston, TX, USA). II, BioRobotics, USA) was used to drip the size 150 μm, interval 340 μm, and then the seed base reaction was performed. Subsequently, the mixture was washed sequentially with a 0.2% (w / v) sodium dodecyl sulfate (SDS) solution and tertiary distilled water, followed by NaBH ₄ solution (0.1 g NaBH ₄ , 30 ml phosphate buffered saline (PBS). Immersed in 10 ml ethanol 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, polymerization buffer and polymerization to amplify the exon 2, 3, 4 and 5 sites of 2 mM dATP, dGTP, dCTP, 0.8 mM dTTP, 0.2 mM dUTP and MICA gene. Using the enzyme, exons 2, 3, 4 and 5 of the MICA gene were once for 5 minutes at 95 ° C, 10 seconds at 95 ° C, 30 seconds at 65 ° C, 8 minutes at 72 ° C and 10 at 95 ° C. Second, amplification was repeated for 30 seconds at 63 ° C., 1 minute at 72 ° C., and 10 minutes at 72 ° C. once. In this case, the base sequence of the primer used is as follows:

1) Antisense Primer: MICA-R

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

2) Sense Primer: MICA-F

MICA-F: 5'-P-CGTTCTTGTCCCTTTGCCCGTGTGC-3 '(SEQ ID NO: 86)

The amplified DNA was treated with lambda exonuclease (lambda exonuclease, 10 units / μl) and separated into a single chain. The separated single chains were treated with 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 dCTP (NEN Life Science) and polymerase (Thermo Sequenase, AP Biotech., USA) labeled with a cyanine 5 fluorescent material. After addition, it was made to react at 60 degreeC for 30 minutes. Subsequently, the DNA chip was sequentially washed with 50 mM NaOH and 0.1% alconox aqueous solution for 10 minutes, and then sequentially washed with distilled water for 5 minutes, and dried. The dried DNA chip was 100 mm in size using a scanner (Biochip Scanner, Nanostorage, Korea). The pixel size was scanned in the 667 nm wavelength range.

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, four of which are selected as a subclass of MICA alleles. Therefore, FIG. 2 shows MICA * 004 / * 010, * 007: 01 / * 008: 01, * 016 / * 018, * 001 / * 008: 01, * 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 tables for reading genotypes of MICA, respectively, using the DNA chip prepared in Example 1, wherein 1 to 78 of the first row of FIGS. 3 to 5 are probe numbers, and the first The 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' it means. As such, it can be seen that hundreds of MICA allele types can be typed using the probes 1-78 of the present invention.

Attach an electronic file to a sequence list

Claims

A DNA chip for detecting an MICA allele comprising a probe of SEQ ID NOs: 1 to 78 capable of specifically binding to an MHC (MHC class I polypeptide-related sequence A) allele.

The method of claim 1,
A DNA chip for detecting MICA alleles, further comprising at least one MICA allele positive control probe 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 NO: 82 or 83.

Constructing an oligonucleotide 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, and the constructed oligonucleotide A method of manufacturing a DNA chip for detecting MICA alleles comprising the step of binding a probe to a solid surface.

The method of claim 3,
The probe is a method for producing a DNA chip for detecting MICA alleles further comprises 18 thymine (dTTP), 6 CH ₂ chain and amine (amine) groups at the 5 'end.

MICA allele selection kit comprising the DNA chip for detecting the MICA allele of claim 1.

6. The method of claim 5,
An 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.

The method according to claim 6,
Sense primer MICA allele selection kit further comprises a phosphate (phosphate) group at the 5 'end.

6. The method of claim 5,
Lambda exonuclease for separating amplified products by polymerase chain reaction into single strands;
Dephosphatase and uracil DNA glycosylase (UDG) for cleaving the isolated single strand to an appropriate length; And
MICA allele selection kit further comprises a fluorescently labeled dideoxy nucleotide capable of detecting the amplification product bound to the DNA chip.

9. The method of claim 8,
Fluorescent labeled dideoxy nucleotides are Cyanine 5 (Cyanine 5) dCTP MICA allele selection kit.

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 step of separating the amplified DNA into single strands and cutting to an appropriate length;
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 an MICA allele comprising a fourth step of analyzing a fluorescent signal of a polymerized probe.

The method of claim 10, wherein the second step is
Amplified DNA is separated into a single strand by processing lambda exonuclease (lambda exonuclease); And
The isolated single strand is treated with desphosphinase (shrimp alkaline phosphatase) and uracil DNA glycosylase (uracil DNA glycosylase, UDG) to cut the length of 50 to 100bp method for screening the MICA allele.

11. The method of claim 10,
The method of screening the MICA alleles is carried out for 20 to 60 minutes at 50-65 ° C.