JP2009261358A - Method for typing allele polymorphism of human gene including hla-drb1 gene, and kit used in the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a method for typing a human gene allele polymorphism and a kit for typing the gene allele polymorphism capable of improving throughput efficiency and reducing costs.
The present invention provides a method for typing an allelic polymorphism of a human gene. The typing method of the present invention includes a first set of oligonucleotide primers for detecting an HLA-DRB1 gene allele polymorphism and a set of oligonucleotide primers for detecting an allele polymorphism of another gene different from HLA-DRB1. A container, a second set of gene amplification primers for detecting an allele polymorphism of the HLA-DRB1 gene, and a set of oligonucleotide primers for detecting an allele polymorphism of another gene different from HLA-DRB1 and the other gene Providing a second container containing.
[Selection figure] None

Description

  The present invention relates to a method for typing an allele polymorphism of a human gene containing an HLA-DRB1 gene and a kit used therefor, and more specifically, to typing of an allele polymorphism of an HLA-A, HLA-B and HLA-DRB1 gene The present invention relates to a method and a kit used therefor.

  Human leukocyte type antigen (HLA), the major human histocompatibility complex (MHC), is expressed on the surface of many types of cells, including leukocytes. There are class I molecules and class II molecules in HLA, class I molecules present peptide fragments derived from endogenous proteins to T cell receptors, and class II molecules present peptide fragments derived from foreign proteins to T cell receptors. . There are numerous gene mutations in the HLA gene. When organ transplantation is performed between human individuals with different allele polymorphisms, an immune reaction occurs between the transplanted tissue and the host, and there is a high risk of deteriorating therapeutic results such as rejection of the transplanted tissue. Therefore, in the treatment involving blood transfusion or organ transplantation, an HLA allele polymorphism is typed between an organ donor and a transplant applicant, and a transplantation request having an allele polymorphism that matches the HLA allele polymorphism of the organ donor. It is necessary to transplant it to a person. In addition, allele polymorphisms of HLA are associated with the onset and prognosis of various diseases including infectious diseases. Accordingly, as so-called “individual medical care”, which provides appropriate treatment according to the genetic background of individual patients, is expected to increase the demand for typing allele polymorphisms of HLA.

  In the WHO (World Health Organization) notation method for allele polymorphism typing, the 1st and 2nd digits indicate the serological level, the 3rd and 4th digits have genetic mutations, In addition, it means determination of a genotype having an amino acid mutation, and the fifth and sixth digits mean determination of a genotype having a genotype mutation but having no amino acid mutation. The currently popular method for typing allele polymorphisms of HLA at the 4-digit level is the Luminex® method of fluorescent microbead array systems. The gene detection method using the complementary hybridization method with the bead surface as a solid phase has already existed, but in the Luminex method, the polystyrene beads used are colored with a fluorescent substance, and the color tone and concentration of the fluorescent substance ( It is a technique that can be distinguished into 100 types by measuring (fluorescence amount). The normal bead method can detect only one type of bead per tube, that is, one type of probe, but the Luminex method can detect a maximum of 100 types of probes with a single tube. It is possible to obtain twice the processing capacity and information amount.

Patent Literature 1 discloses a method for typing gene allelic polymorphisms for three molecules of two class I molecules HLA-A and HLA-B and one class II molecule HLA-DRB1 using the Luminex method. 1 is disclosed. In Patent Document 1, after mixing a probe solid phase bead and an amplification product, the denaturation of the amplification product into a single strand is referred to as thermal denaturation by heating, and after denaturation, the temperature is reduced to an optimal complementary hybridization temperature. And a genotyping method improved by the Luminex method, characterized in that complementary hybridization is continuously performed.
JP 2005-160355 A

  According to the method disclosed in Patent Document 1, it was possible to perform a gene amplification reaction by adding all primers to one test tube for detecting allele polymorphisms of HLA-A and HLA-B molecules. However, for the detection of the HLA-DRB1 allelic polymorphism, the stability of gene amplification distinguishing between the HLA-DRB1 gene and similar gene groups (DRB3, DRB4, DRB5, etc.) and the complementary of the amplification product and the probe In order to improve the S / N ratio of hybridization, it is necessary to divide the primer into two sets and perform the gene amplification reaction in two test tubes, and also perform the reaction with the specific probe in separate containers. there were. That is, a gene amplification reaction is performed using four test tubes per subject. This is a major obstacle to improving the throughput efficiency and cost of HLA allele polytyping.

  In order to improve throughput efficiency and reduce costs, it is necessary to develop an HLA gene allele polymorphism typing method for performing gene amplification reaction in a smaller number of test tubes and a kit for typing the gene allele polymorphism.

  The present invention provides a method for typing an allelic polymorphism of a human gene. The allele polymorphism typing method of the present invention comprises (1) a first set of oligonucleotide primers for detecting an HLA-DRB1 gene allele polymorphism and an oligonucleotide primer for detecting an allele polymorphism of another gene different from HLA-DRB1. And a second set of gene amplification primers for detecting an allele polymorphism of the HLA-DRB1 gene, and for detecting an allele polymorphism of another gene different from HLA-DRB1 and the other gene. Providing a second container containing a set of oligonucleotide primers; (2) mixing sample DNA from the same individual with each of the first and second sets of primers; and (3) first And a step of incubating the second container to produce a gene amplification product, and (4) said other gene A set of carriers on which the allele detection probe is immobilized and a first set of carriers on which the HLA-DRB1 gene allele detection probe is immobilized are mixed with the gene amplification product in the first container. Mixing a set of carriers immobilizing the allele detection probe and a second set of carriers immobilizing the HLA-DRB1 gene allele detection probe with the gene amplification product in the second container; (5) first And incubating the second container to form a complementary hybrid of the gene amplification product of each container with one of the allele detection probes, and (6) for each of the first and second containers on the carrier. The primer incorporated in the gene amplification product formed in a complementary hybridization with the immobilized allele detection probe A step of detecting whether the allele detection probe is complementary hybridized with the gene amplification product, and the allele detection probe has one carrier or one or more nucleotide sequence probes immobilized on one carrier. In step (6), the primer incorporated in the gene amplification product complementary to the allele detection probe immobilized on the carrier or the allele complementary to the gene amplification product. When detecting the detection probe, the nucleotide sequence of the gene amplification product is specified.

  In the allelic polymorphism typing method of the present invention, the other gene and the further gene are all selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP and HLA-DQ. There may be at least one gene selected.

  In the allele polymorphism typing method of the present invention, the other gene may be an HLA-A gene, and the further gene may be an HLA-B gene.

  In the typing method of the present invention, the first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers of nucleotide sequences listed in SEQ ID NOs: 24 to 32, and the HLA-DRB1 gene allele polymorphism detection The second set of gene amplification primers for use consists of primers having the nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the first set of carriers on which the probes for detecting HLA-DRB1 gene alleles are immobilized are listed in SEQ ID NOs: 153 to 179 The second set of carriers on which the probe for detecting the HLA-DRB1 gene allele is immobilized is immobilized on a probe comprising the nucleotide sequence listed in SEQ ID NOs: 180 to 203. May consist of a supported carrier That.

  In the typing method of the present invention, the first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers of nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the HLA-DRB1 gene allele polymorphism detection The second set of gene amplification primers comprises nucleotide sequence primers listed in SEQ ID NOs: 24-32, and the first set of carriers on which the probes for detecting HLA-DRB1 gene alleles are immobilized are listed in SEQ ID NOs: 180-203 The second set of carriers on which probes for detecting the HLA-DRB1 gene allele are immobilized are probes of nucleotide sequences listed in SEQ ID NOs: 153 to 179. May consist of different carriers.

  In the typing method of the present invention, the HLA-A gene allele polymorphism detection gene amplification primer set comprises the nucleotide sequence primers listed in SEQ ID NOs: 1 to 13, and the HLA-B gene allele polymorphism detection The set of gene amplification primers for use consists of primers of nucleotide sequences listed in SEQ ID NOs: 14 to 23, and the set of carriers on which the allele detection probe of the HLA-A gene is immobilized is listed in SEQ ID NOs: 38 to 85 The nucleotide set probes listed in SEQ ID NOs: 86 to 152 are immobilized on the set of carriers on which the HLA-B gene allele detection probe is immobilized. May consist of different carriers.

  The present invention provides a kit for typing allele polymorphisms of HLA-A, HLA-B and HLA-DRB1 genes using two containers. The typing kit of the present invention includes (1) a first set of gene amplification primers for detecting an HLA-A gene allele polymorphism and a first set of gene amplification primers for detecting an HLA-DRB1 gene allele polymorphism. A second PCR reaction comprising a PCR reaction vessel, (2) a set of gene amplification primers for detecting an allele polymorphism of the HLA-B gene, and a second set of gene amplification primers for detecting an allele polymorphism of the HLA-DRB1 gene A first PCR reaction comprising a container, (3) a set of carriers on which an HLA-A gene allele detection probe is immobilized, and a first set of carriers on which an HLA-DRB1 gene allele detection probe is immobilized A combination with a carrier set on which an allele detection probe to be added to a container is immobilized; and (4) an allele detection probe for the HLA-B gene. An allele detection probe to be added to the second PCR reaction vessel, which is composed of a set of carriers on which the HB-DRB1 gene allele detection probe is immobilized, is immobilized. Combination with a carrier set.

  In the typing kit of the present invention, one of the first and second sets of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism includes primers having nucleotide sequences listed in SEQ ID NOs: 31 and 32, And the other of the second set includes primers of the nucleotide sequence listed in SEQ ID NO: 37, and is listed in SEQ ID NO: 204 depending on the presence or absence of gene amplification by the primer of the nucleotide sequence listed in SEQ ID NO: 31, 32 and / or 37 In some cases, different alleles of the second and third nucleotide sequences of the codon encoding the 86th amino acid of the amino acid sequence of the HLA-DRB1 gene product to be differentiated may be distinguished.

  In the typing kit of the present invention, the first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers of nucleotide sequences listed in SEQ ID NOs: 24 to 32, and the HLA-DRB1 gene allele polymorphism The second set of detection gene amplification primers is composed of primers having nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the first set of carriers on which probes for detecting HLA-DRB1 gene alleles are immobilized are SEQ ID NOs: 153 to 179. The probe of the nucleotide sequence listed is composed of a carrier on which the probe is immobilized, and the second set of carriers on which the probe for detecting the HLA-DRB1 gene allele is immobilized is a probe composed of the nucleotide sequence listed in SEQ ID NOs: 180 to 203. A place consisting of a structured carrier There is.

  In the typing kit of the present invention, the first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers of nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the HLA-DRB1 gene allele polymorphism The second set of detection gene amplification primers is composed of primers having the nucleotide sequences listed in SEQ ID NOs: 24 to 32, and the first set of carriers on which the probes for detecting HLA-DRB1 gene alleles are immobilized are SEQ ID NOs: 180 to 203. The second set of carriers on which probes for detecting the nucleotide sequences listed are immobilized and the probes for detecting HLA-DRB1 gene alleles are immobilized are probes for the nucleotide sequences listed in SEQ ID NOs: 153 to 179. May consist of a supported carrier. .

  In the typing kit of the present invention, the HLA-A gene allele polymorphism detection gene amplification primer set comprises nucleotide sequence primers listed in SEQ ID NOs: 1 to 13, and the HLA-B gene allele polymorphism A set of gene amplification primers for detection consists of primers having nucleotide sequences listed in SEQ ID NOs: 14 to 23. A set of carriers on which the allele detection probe of the HLA-A gene is immobilized is listed in SEQ ID NOs: 38 to 85. A set of carriers on which the probe for detecting the allele of the HLA-B gene is immobilized is immobilized on the nucleotide sequence probes listed in SEQ ID NOs: 86 to 152. In some cases.

  In addition to the HLA-DRB1 gene, the human gene that is the target of allele polymorphism typing in the method and typing kit for allelic polymorphism of the human gene of the present invention includes HLA-A, HLA-B, Genes involved in histocompatibility such as HLA-C, HLA-DP, HLA-DQ, genes involved in the metabolism of cytochrome P450 and other drugs, chemokine receptors CCR3 and CCR5 and others involved in resistance to infections such as HIV Including any gene that has an allele polymorphism associated with human genetic traits such as genes, single nucleotide polymorphisms (SNPs) and other diseases, but is not limited to these, It refers to any gene that may perform allelic polymorphism typing.

  For allelic polymorphisms of the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DRB1 genes, see EMBL (http://www.ebi.ac.uk/imgt/hla/ docs / relnotes.html), ASEATTA (The Australian and South East Asian, Tissue Typing Assoc. Inc., http://www.asettata.org.au/html. square.umin.ac.jp/JSHI/hla_data/data.html). The set of gene amplification primers for detecting the allele polymorphisms of the HLA-A, HLA-B and HLA-DRB1 genes of the present invention and the probes for detecting alleles of the HLA-A, HLA-B and HLA-DRB1 genes, Regarding genes, when the frequency of allelic polymorphism in Japanese is 0.005% or more, any nucleotide sequence may be used as long as it can be typed without discrimination between homo / heterologues. Absent.

  In the present specification, gene amplification refers to the template DNA having a nucleotide sequence complementary to each of the primers, using a pair of or more oligonucleotide primers and a template DNA. This refers to any genetic engineering technology that can specifically replicate the region sandwiched between the PCR, SMAP (SMart Amplification Process), LAMP (Loop-Mediated Isothermal Amplification) Including, but not limited to.

  The set of gene amplification primers for detecting an allele polymorphism of the HLA-A gene of the present invention includes, but is not limited to, a set of oligonucleotides having nucleotide sequences listed in SEQ ID NOs: 1 to 13. The set of gene amplification primers for detecting an allele polymorphism of the HLA-B gene of the present invention includes, but is not limited to, a set of oligonucleotides having nucleotide sequences listed in SEQ ID NOs: 14 to 23. The set of gene amplification primers for detecting an allele polymorphism of the HLA-DRB1 gene of the present invention includes, but is not limited to, a set of oligonucleotides having nucleotide sequences listed in SEQ ID NOs: 24 to 37. The gene amplification primer for detecting an allele polymorphism of the present invention may be labeled with biotin, digoxin or other low molecular weight compounds. The product amplified by the labeled primer is complementary-hybridized with a probe complementary to the nucleotide sequence of the amplified product, immobilized on a carrier, and specifically bound to the low molecular weight compound. Detected by combining with.

  The probe for detecting an allele of HLA-A of the present invention includes, but is not limited to, oligonucleotides having nucleotide sequences listed in SEQ ID NOs: 38 to 85. The HLA-B allele detection probe of the present invention includes, but is not limited to, oligonucleotides having the nucleotide sequences listed in SEQ ID NOs: 86 to 152. The probe for detecting an allele of the HLA-DRB1 gene of the present invention includes, but is not limited to, oligonucleotides having the nucleotide sequences listed in SEQ ID NOs: 153 to 203.

  The carrier of the present invention can withstand heat treatment for denaturing the gene amplification product upon complementary hybridization with the probe, and is a PCR product bound to the carrier by complementary hybridization with the probe. It may be made of any material provided that it is possible to identify the probe immobilized on the carrier when detecting the presence of. In the present invention, there are cases where two types, three types, or four or more types of probes bind to one carrier, and only one type of probe binds to one carrier. A preferred carrier is a polystyrene carrier called “MicroPlex Microspheres” available from Luminex. In “MicroPlex Microspheres”, two types of fluorescent materials are colored in combinations of 10 different concentrations. Therefore, a total of 100 types of carriers can be identified and identified. The surface of “MicroPlex Microspheres” is coated with avidin or a carboxyl group, and can bind and solid phase an oligonucleotide modified with biotin or an amino group. Immobilizing the probes for detecting alleles of the HLA-A, HLA-B, and HLA-DRB1 genes of the present invention on a carrier involves modifying the terminal portion of each oligonucleotide with an amino group and coating it with a carboxyl group. This is accomplished by carrying out a covalent bond reaction with “MicroPlex Microspheres”.

  In the typing method and typing kit of the present invention, a part of the allele is determined by which probe the gene amplification product obtained from the set of primers for detecting the allele of a human gene and the sample DNA binds. Another part of the allele is determined by the presence or absence of a gene amplification product derived from the primer. For example, the allele polymorphism of the HLA-DRB1 gene is GGT, wherein the codon encoding the 86th amino acid of the amino acid sequence of the gene product of HLA-DRB1 genotype DRB1 * 010101 listed in SEQ ID NO: 204 is GGT It can be roughly classified into two groups depending on whether or not. The amino acid sequence of the gene product of genotype DRB1 * 010101 and the amino acid sequence of the gene product of genotype DRB1 * 010201 indicate that the former codon encoding the 86th amino acid encodes glycine with GGT. Other than that, the codon encoding the 86th amino acid of GTG encodes valine with GTG. Therefore, if a gene amplification product derived from the primer is obtained by making the 3 ′ end of some primers include the nucleotide sequence corresponding to the 86th amino acid, it is complementary to the nucleotide sequence of the primer. It can be seen that the sample DNA has a specific sequence, and if such a gene amplification product is not obtained, it can be seen that the sample DNA does not have a sequence complementary to the nucleotide sequence of the primer. Of the primers for detecting alleles of the HLA-DRB1 gene of the present invention listed in Table 1, each of D2R1, D2R2 and D2R3 having the nucleotide sequences listed in SEQ ID NOs: 31, 32 and 37 depends on the presence or absence of a gene amplification product. A polymorphism of the nucleotide sequence at the site corresponding to the 86th amino acid of the HLA-DRB1 gene product can be detected.

  The HLA-DRB1 gene allele detection primer set of the present invention is divided into two sets and added to each of the two containers. The separation into two sets is performed on the condition that the gene amplification reaction proceeds stably and the S / N ratio of complementary hybridization between the amplification product and the probe does not interfere with typing. The array can be distributed to any set. Preferably, the oligonucleotides of the nucleotide sequences listed in SEQ ID NOs: 24 to 32 are set as one set, and the oligonucleotides of the nucleotide sequences listed in SEQ ID NOs: 33 to 37 are set as another set. Either one of these two sets of preferred HLA-DRB1 gene allele detection primers is added to the same container as the HLA-A gene allele detection primer, and the other is an HLA-B gene allele detection primer. Add to the same container.

  Hereinafter, the present invention will be described in detail. The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims.

Samples and Chromosomal DNA Extraction Chromosomal DNA was extracted and purified from a total of 95 healthy Japanese subjects using DNA extraction kits from Medical Biological Laboratory and Fuji Film Co., Ltd. DNA was quantified and purity was measured by measuring absorbance at 260 nm and 280 nm, and the concentration was adjusted to 10-20 ng / μL. The HLA gene allele polymorphisms of these specimens are known by determination by another method such as an existing determination method (Genosearch HLA ver. 2) sold by the Institute of Medical Biology.

Allele polymorphism detection primer Table 1 shows a list of primers used for the primer (A / PS1 amplification system) added to the first container and the primer (B / PS2 amplification system) added to the second container. It is. The primer added to the first container is a set of gene amplification primers for detecting an allele polymorphism of the HLA-A gene (SEQ ID NOs: 1 to 13) and a first gene amplification primer for detecting the allele polymorphism of the HLA-DRB1 gene. It is a set (SEQ ID NO: 24 to 32). The primers to be added to the second container are a set of gene amplification primers for detecting the allele polymorphism of the HLA-B gene (SEQ ID NOs: 14 to 23) and the gene amplification primers for detecting the allele polymorphism of the HLA-DRB1 gene. Two sets (SEQ ID NOs: 33 to 37). As the gene amplification primer for detecting an allele polymorphism of this example, an oligonucleotide whose 5 ′ end was labeled with biotin was used.

PCR Amplification Reaction A / PS1 amplification system and B / PS2 amplification system primers were mixed in the first and second containers at appropriate concentrations, respectively. The amplification buffer was a final concentration of 50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 2% DMSO, 0.2 mM dNTPs. To this, 0.5-1.0 μM of the above primer set, 25 U / mL Taq DNA polymerase and 50-100 ng of sample genomic DNA were added to make a total volume of 25 μL. The first and second containers are attached to a thermal cycler GeneAmp9700 (Appliedbiosystems), held at 94 ° C. for 2 minutes, then kept at 94 ° C. for 30 seconds, and kept at 65 ° C. for 30 seconds The PCR amplification reaction was carried out under the condition that the thermal cycle consisting of a step of keeping at 72 ° C. for 30 seconds was repeated 40 times.

Probes for detection of allele polymorphisms Tables 2-1 and 2-2 are complementary to the amplification products obtained by performing PCR amplification reaction in the first container using the allele polymorphism detection primers of the A / PS1 amplification system. Is a list of probes that hybridize to Tables 3-1 and 3-2 show the probes that hybridize complementarily with the amplification products obtained by performing the PCR amplification reaction in the second container using the B / PS2 amplification system allele polymorphism detection primer. It is a list.

Immobilization of allelic polymorphic probes on fluorescent microsphere particles Luminex's xMAP ™ microsphere particles are polystyrene microbead particles containing two types of fluorescent dyes with different fluorescence wavelengths at 10 different concentrations. One group includes 100 kinds of particles. Therefore, it is possible to detect whether or not complementary hybridization with up to 100 types of probes occurs in the products amplified in each of the first and second containers. There are 48 types of probes for detecting allele polymorphisms of the HLA-A gene, and 27 types of probes for detecting allele polymorphisms of the HLA-DRB1 gene. Each of these probes is assigned to be immobilized on any one of 100 types of microsphere particles of the first group, and the amino group labeled at the end of each probe and the carboxyl coated on the surface of the microsphere particles Both are immobilized by a coupling reaction with the group. This work was performed on 48 types of probes for detecting allele polymorphisms of HLA-A genes and a first set of 27 types of probes for detecting allele polymorphisms of HLA-DRB1 genes, and particles in which these probes were immobilized Was prepared. Similarly, with respect to the 67 types of HLA-B gene allele polymorphism detection probes and the second set of 24 types of HLA-DRB1 gene allele polymorphism detection probes, 100 types of microarrays of the second group are used. Assigned to be immobilized on any one of the sphere particles, particles with these probes immobilized thereon were prepared. The prepared first and second groups of microsphere particles contain about 350 particles per measurement in a hybridization buffer containing TMAC (tetramethylammonium chloride), EDTA, sarcosine and Tris buffer. As described above, the particle concentration was adjusted and redispersed to prepare first and second particle mixed solutions, respectively.

Complementary hybridization between PCR amplification product and array polymorphism detection probe Hybridization reaction buffer (3.75 M TMAC, 62.5 mM TB pH 8.0, 0.5 mM EDTA, 0.125% N-lauroyl sarcosine) To 50 μL, and 5 μL of the above bead mix and 5 μL of the PCR amplification product were added to make 50 μL. These were mounted on GeneAmp9700, and the hybrid formation reaction was carried out under the condition that the PCR amplification product was heat denatured by incubating at 95 ° C. for 2 minutes and then kept at 52 ° C. for 30 minutes.

Detection of PCR amplification product immobilized on particles After complementary hybridization, streptavidin-labeled phycoerythrin (SA-PE, MOSS) was added to PBS-Tween (1 × PBS (pH 7.5), 0.01% Tween). A solution diluted 100 times in 20) (hereinafter referred to as “SA-PE reaction solution”) was added in an amount of 50 μL. Centrifugation was performed at 1800 × g for 2 minutes using a swing-type plate centrifuge, and the supernatant was removed. The SA-PE reaction solution was added again by 50 μL and centrifuged under the above conditions to remove the supernatant. After adding 50 μL of SA-PE reaction solution each time, pipetting the microsphere particles and the reaction solution, setting them in GeneAmp 9700, keeping them warm at 52 ° C. for 10 minutes, then Luminex 100 system (for 96 samples) ) And measured. Allele polymorphism typing was performed using the allele polymorphism typing software to which the obtained reaction pattern was input.

Results PCR amplification was performed on 95 samples of genomic DNA derived from Japanese, and HLA-A, HLA-B and HLA-DRB1 genotyping was performed by the Luminex method in two containers using the above experimental conditions. Tables 4-1 to 6-4 show probe determination patterns of HLA-A, HLA-B, and HLA-DRB1 gene allele polymorphism typing, respectively. This method is capable of four-digit typing for alleles (see Tables 4-1 to 6-4) that have a gene frequency of 0.005% or more in the Japanese population, for homo and hetero combinations. is there. In addition, the determination of A29 and A32 that is frequently observed in a group other than Japanese is also a method that allows type determination.

  Tables 7-1 and 7-2 show representative examples of the results of allelic polymorphism typing of HLA-A, HLA-B and HLA-DRB1 genes according to the method of the present invention.

  In the method of the present invention, in order to perform PCR amplification reaction in two containers, the set of HLA-DRB1 gene allele polymorphism detection primers was divided into two sets, a first set and a second set. As a result, when the result of complementary hybridization of the first set of PCR amplification products with the probe does not match the result of complementary hybridization of the second set of PCR amplification products with the probe, for example, Table 8- As shown in 1 and 8-2, errors frequently occur in which the allele polymorphism of the HLA-DRB1 gene cannot be determined. Therefore, the present invention can prevent mistakes in allelic polymorphic typing due to sample mistakes.

Claims (11)

(1) a first container including a first set of oligonucleotide primers for detecting an HLA-DRB1 gene allele polymorphism and a set of oligonucleotide primers for detecting an allele polymorphism of another gene different from HLA-DRB1, and an HLA A second container comprising a second set of gene amplification primers for detecting an allele polymorphism of the DRB1 gene and a set of oligonucleotide primers for detecting an allele polymorphism of another gene different from HLA-DRB1 and the other gene And a step of preparing
(2) mixing the sample DNA derived from the same individual with each of the first and second sets of primers;
(3) performing incubation of the first and second containers to generate a gene amplification product;
(4) The carrier set on which the allele detection probe of another gene is immobilized and the first set of carriers on which the HLA-DRB1 gene allele detection probe is immobilized are mixed with the gene amplification product in the first container. Mixing a set of carriers on which the allele detection probe of another gene is immobilized and a second set of carriers on which the HLA-DRB1 gene allele detection probe is immobilized with the gene amplification product in the second container When,
(5) performing incubation of the first and second containers, and causing the gene amplification product of each container to form a complementary hybrid with any one of the allele detection probes;
(6) For each of the first and second containers, either the primer incorporated in the gene amplification product complementary to the allele detection probe immobilized on the carrier or a complementary hybrid to the gene amplification product Detecting whether the allele detection probe is formed,
In the allele detection probe, a probe having one or more nucleotide sequences is immobilized on one carrier,
In step (6), either the primer incorporated in the gene amplification product complementary to the allele detection probe immobilized on the carrier or the allele detection probe complementary to the gene amplification product. A method for typing an allelic polymorphism of a human gene, wherein the nucleotide sequence of the gene amplification product is specified when detecting this.   The other gene and the further gene are at least one gene selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP and HLA-DQ. The typing method of the allele polymorphism of Claim 1 characterized by these.   The allele polymorphism typing method according to claim 2, wherein the other gene is an HLA-A gene, and the further gene is an HLA-B gene.   The first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers having nucleotide sequences listed in SEQ ID NOs: 24-32, and the second set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism. The set consists of primers of nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the first set of carriers on which probes for detecting HLA-DRB1 gene alleles are immobilized are probes of nucleotide sequences listed in SEQ ID NOs: 153 to 179. The second set of carriers consisting of an immobilized carrier and immobilizing probes for detecting the HLA-DRB1 gene allele is composed of a carrier on which probes comprising nucleotide sequences listed in SEQ ID NOs: 180 to 203 are immobilized. A characteristic of claims 1 to 3 Allele polymorphism typing method according to any Re.   The first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers having nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the second set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism. The set consists of primers of nucleotide sequences listed in SEQ ID NOs: 24-32, and the first set of carriers on which the probes for detecting HLA-DRB1 gene alleles are immobilized are probes of nucleotide sequences listed in SEQ ID NOs: 180-203. A second set of carriers comprising an immobilized carrier and having immobilized probes for detecting the HLA-DRB1 gene allele is characterized by comprising a carrier on which probes having nucleotide sequences listed in SEQ ID NOs: 153 to 179 are immobilized. Any one of claims 1 to 3 Allele polymorphism typing method as set forth.   The set of gene amplification primers for detecting the allele polymorphism of the HLA-A gene comprises primers of nucleotide sequences listed in SEQ ID NOs: 1 to 13, and the set of gene amplification primers for detecting the allele polymorphism of the HLA-B gene is The carrier set comprising the primers of the nucleotide sequences listed in SEQ ID NOs: 14 to 23 and having the allele detection probe of the HLA-A gene immobilized thereon is fixed with the probes of the nucleotide sequences listed in SEQ ID NOs: 38 to 85 The carrier set to which the probe for detecting the allele of the HLA-B gene is immobilized is composed of a carrier on which probes having nucleotide sequences listed in SEQ ID NOs: 86 to 152 are immobilized. The allele polymorphic tie pin according to any one of claims 1 to 5. Method. (1) a first PCR reaction vessel comprising a set of gene amplification primers for detecting an allele polymorphism of the HLA-A gene and a first set of gene amplification primers for detecting an HLA-DRB1 gene allele polymorphism;
(2) a second PCR reaction vessel comprising a set of gene amplification primers for detecting an allele polymorphism of the HLA-B gene and a second set of gene amplification primers for detecting an allele polymorphism of the HLA-DRB1 gene;
(3) Addition to the first PCR reaction vessel comprising a set of carriers immobilizing the probe for detecting the allele of the HLA-A gene and a first set of carriers immobilizing the probe for detecting the HLA-DRB1 gene allele A combination with a carrier set on which an allele detection probe is immobilized,
(4) Addition to the second PCR reaction vessel comprising a set of carriers on which the probes for detecting the alleles of the HLA-B gene are immobilized and a second set of carriers on which the probes for detecting the alleles of HLA-DRB1 gene are immobilized And a combination of a carrier set on which an allele detection probe for immobilization is immobilized, and for typing allele polymorphisms of HLA-A, HLA-B and HLA-DRB1 genes using two containers kit.   One of the first and second sets of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism includes primers of nucleotide sequences listed in SEQ ID NOs: 31 and 32, and the other of the first and second sets is Of the HLA-DRB1 gene product listed in SEQ ID NO: 204, depending on the presence or absence of gene amplification with primers of the nucleotide sequence listed in SEQ ID NO: 31, 32 and / or 37. The kit for typing allele polymorphism according to claim 7, wherein alleles having different sequences of the second and third nucleotides of the codon encoding the 86th amino acid of the amino acid sequence are distinguished.   The first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers having nucleotide sequences listed in SEQ ID NOs: 24-32, and the second set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism. The set consists of primers of nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the first set of carriers on which probes for detecting HLA-DRB1 gene alleles are immobilized are probes of nucleotide sequences listed in SEQ ID NOs: 153 to 179. The second set of carriers consisting of an immobilized carrier and immobilizing probes for detecting the HLA-DRB1 gene allele is composed of a carrier on which probes comprising nucleotide sequences listed in SEQ ID NOs: 180 to 203 are immobilized. 9. Array according to claim 8, characterized in that Polymorphism typing kit for.   The first set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism comprises primers having nucleotide sequences listed in SEQ ID NOs: 33 to 37, and the second set of gene amplification primers for detecting the HLA-DRB1 gene allele polymorphism. The set consists of primers of nucleotide sequences listed in SEQ ID NOs: 24-32, and the first set of carriers on which the probes for detecting HLA-DRB1 gene alleles are immobilized are probes of nucleotide sequences listed in SEQ ID NOs: 180-203. A second set of carriers comprising an immobilized carrier and having immobilized probes for detecting the HLA-DRB1 gene allele is characterized by comprising a carrier on which probes having nucleotide sequences listed in SEQ ID NOs: 153 to 179 are immobilized. The allele polymorphism according to claim 8 Kit for typing.   The set of gene amplification primers for detecting the allele polymorphism of the HLA-A gene comprises primers of nucleotide sequences listed in SEQ ID NOs: 1 to 13, and the set of gene amplification primers for detecting the allele polymorphism of the HLA-B gene is The carrier set comprising the primers of the nucleotide sequences listed in SEQ ID NOs: 14 to 23 and having the allele detection probe of the HLA-A gene immobilized thereon is fixed with the probes of the nucleotide sequences listed in SEQ ID NOs: 38 to 85 The carrier set to which the probe for detecting the allele of the HLA-B gene is immobilized is composed of a carrier on which probes having nucleotide sequences listed in SEQ ID NOs: 86 to 152 are immobilized. The allele polymorphic typi according to any one of claims 8 to 10. Grayed kit.