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    • 专利摘要:
    DNA typing method, and primer sets for DNA typing The purpose of the present invention is to provide a method and kit for highly accurate DNA typing in which the ambiguity derived from the phase ambiguity is eliminated. the present invention provides a method for hla DNA typing, which is characterized by comprising: (1) a step of preparing a set of primers that can respectively anneal with an upstream region and a downstream region of each of the hla-a, hla-b, hla-c, hla-dqa1, hla-dqb 1, hla-dpa1 and hla-dpb1 genes in the nucleotide sequence of the human genome, and also prepare a set of primers that can loop respectively with exon 2 and an untranslated 3' lateral region in hla-drb1; (2) a step of performing the pcr amplification of a sample to be tested (dna) using the primer sets; (3) a step of determining the nucleotide sequence for an amplified pcr product; and (4) an optional step of performing a homology search in a database.
    公开号:BR112014001258B1
    申请号:R112014001258-0
    申请日:2012-05-18
    公开日:2021-07-13
    发明作者:Takashi Shiina；Shingo Suzuki；Yuki Ozaki；Shigeki Mitsunaga；Hidetoshi Inoko
    申请人:Genodive Pharma Inc.；
    IPC主号:
    专利说明:

    Technical Field
    [0001] The present invention relates to a method and a kit for DNA typing of an HLA gene using a massive parallel sequencer. Previous Technique
    [0002] The human leukocyte antigen (HLA), which represents the major human histocompatibility complex (MHC), presents peptides derived from foreign proteins, such as pathogens and self-protein-derived peptides for T cells. In this way, HLA is deeply involved in inducing immune responses. As the main HLAs, six types of antigens are known, namely, class I molecules (HLA-A, HLA-B, HLA-C), which are expressed in almost all cells, and class II molecules (HLA -DR, HLA-DQ, HLA-DP), which are mainly expressed in immune system cells.
    [0003] The HLA class I antigen consists of a highly polymorphic α chain and a substantially non-polymorphic β2 microglobulin; while the HLA class II antigen consists of a highly polymorphic b chain and a less polymorphic α chain. The α chains of class I molecules are encoded by the HLA-A, HLA-B and HLA-C genes. The β chains of class II antigens are encoded by the HLA-DRB1, HLA-DQB1 and HLA-DPB1 genes, while the α chains are encoded by the HLA-DRA1, HLA-DQA1 and HLA-DPA1 genes. At the genetic level, in HLA class I antigens, exon 2 and exon 3 of a gene encoding an α chain are highly polymorphic; whereas, in HLA class II antigens, exon 2 of a gene encoding a β chain is highly polymorphic.
    [0004] A region of the gene encoding an HLA is located on the short arm of human chromosome 6 at 6p21.3. A class I region (HLA-A, HLA-C and HLA-B, etc.) a class III region and a class II region (HLA-DRA, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA -DPA1, HLA-DPB1, etc.) are arranged in this order from the telomere side towards the centromere side. Many genes are encoded at an extremely high density and the association of these genes with transfusion, transplantation and various diseases has been reported. In the class III region, no HLA gene is present and complement component genes and tumor necrosis factors (TNF) etc. are present.
    [0005] In a region of the HLA-DRB gene that encodes a β chain of an HLA-DR antigen, it was confirmed that 5 types of structural polymorphisms are present. In the DR1 type and in the DR10 type, pseudogenes such as HLA-DRB6 and HLA-DRB9, in addition to HLA-DRB1, are located on the same chromosome. In the DR2 type, an HLA-DRB5 gene (DR51) and pseudogenes such as HLA-DRB6 and HLA-DRB9, in addition to HLA-DRB1, are located on the same chromosome. In types DR3, DR5 and DR6, an HLA-DRB3 gene (DR52) and pseudogenes such as HLA-DRB2 and HLA-DRB9, in addition to HLA-DRB1, are located on the same chromosome. In types DR4, DR7 and DR9, an HLA-DRB4 (DR53) gene and pseudogenes such as HLA-DRB7, HLA-DRB8 and HLA-DRB9, in addition to HLA-DRB1, are located on the same chromosome. Unlike these, in the DR8 type, no HLA-DRB gene, except HLA-DRB1, is located on the same chromosome.
    [0006] In the exon of each allele, several regions presenting polymorphism are present. In many cases, a nucleotide sequence (sequence of amino acids) present in a particular polymorphic region is commonly present in a plurality of alleles. In short, each HLA allele is specified by a plurality of polymorphic regions in combination. In an HLA class I antigen, not only a polymorphic region in the exon, but also exon 2 or exon 3 having the same nucleotide sequence is sometimes commonly present in a plurality of alleles.
    [0007] Since a highly polymorphic region is present in an HLA, the number of allele types is known to be extremely large and their notation has been defined: that is, a first field (two-digit level) is for discrimination of the serologic HLA types, a second field (4-digit level) is for discrimination of alleles with an amino acid substitution in the same serologic HLA type, a third field (6-digit level) is for discrimination of alleles with a substitution of base not following an amino acid mutation and a fourth field (8-digit level) is for discrimination of alleles with a base substitution in an intron, which is outside the genetic region that encodes an HLA molecule.
    [0008] In bone marrow transplantation, it is said that if the HLA type of a patient intending to receive a transplant completely matches the HLA type of a donor at the 4-digit level, the transplant success rate improves and the frequency of severe GVHD decreases. On the other hand, if the HLA types do not match at the 4-digit level, the risk of causing a failure, such as a reject response, increases. Therefore, accurate and highly accurate HLA typing is extremely important from a clinical point of view as well.
    [0009] As a method of DNA typing in an HLA gene, an SBT (sequence based typing) method and an SSO (sequence specific oligonucleotide) - Luminex method based on a polymerase chain reaction (PCR) are predominant trend .
    [00010] These conventional DNA typing methods have an advantage, which is that they quickly perform typing of many samples; however, they sometimes fail to accurately determine a polymorphic region and a cis/trans positional relationship of exons on a chromosome in the case of a class I gene. Because of this, phase ambiguity occurs, and highly accurate HLA typing at times was not easily accomplished.
    [00011] Since conventional methods are DNA typing methods using PCR based primarily on the exon regions of each gene, base substitutions in an intron region and a promoter region are neglected, with the result that there was a risk of failure to detect a null allele, which has the same genetic structure as other genes that express HLA, but whose expression is suppressed. Related Technique Patent Document
    [00012] Patent Document 1: JP H11-216000 A Non-Patent Document
    [00013] Non-Patent Document 1: Lind C., et al., Human Immunology, Vol. 71, pages 1033-1042 (2010) Invention Summary Technical problem
    [00014] An object of the present invention is to provide a method and a kit for highly accurate DNA typing in which the ambiguity derived from the phase ambiguity is eliminated. Solution to Problem
    [00015] The present inventors have lately conceived an idea to recently design a PCR primer capable of specifically amplifying HLA genes, such as HLA class I molecules, including HLA-A, HLA-B and HLA-C and, and HLA molecules class II, including HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1, defining appropriate PCR conditions and applying a massive parallel sequencing technique. Based on these new ideas, they studied repeatedly with the aim of achieving the above object. As a result, the present invention has been realized.
    [00016] More specifically, the present invention provides a method for HLA DNA typing, including the following steps: (1) a step of preparing a set of primers that respectively specifically loop with an upstream region and an upstream region. downstream of each of the HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 genes in the human genome sequence, and a set of primers that respectively specifically anneal with the exon 2 region and an exon 3' untranslated region of HLA-DRB1; (2) a step of amplifying a test sample (DNA) by a PCR using the primer sets; (3) a step of determining the nucleotide sequences of the products amplified by PCR; and (4) a step of performing a homology search in a database. Advantageous Effects of the Invention
    [00017] Since the method of the present invention provides all the nucleotide sequences necessary for DNA typing of a single molecule HLA gene, it is a modern DNA typing method in which the phase ambiguity due to the cis positional relationship /trans not evident is dropped. Because of this, highly accurate matching of HLAs between a patient seeking to receive a transplant and a post-transplant donor candidate is performed.
    [00018] Once all the nucleotide sequences of an HLA gene, including the peripheral regions such as a promoter region, exon regions and intron regions are determined, a null allele, which is not expressed or has the expression suppressed, and a new allele can be detected. Brief Description of the Drawings [Figure 1] (a) A diagram showing the relationship between the structure of an HLA class I gene and the structure of the HLA class I molecule; and (b) A diagram showing the structure of a promoter region of an HLA class I gene cited from "Transplantation/transfusion Examination", supervised by Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juuji, Kodan-sha Scientific, 2004, page 35. [Figure 2] (a) A diagram showing the relationship between the structure of an HLA class II gene and the structure of the HLA class II molecule; and (b) A diagram showing the structure of a promoter region of an HLA class II gene cited from "Transplantation/transfusion Examination", supervised by Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juuji, Kodan-sha Scientific, 2004, pages 46 and 47. [Figure 3] A diagram showing a region of the HLA-DR gene, cited from "Transplantation/transfusion Examination", supervised by Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juuji, Kodan-sha Scientific, 2004, page 48. [Figure 4 ] An electrophoretic agarose gel pattern showing amplification states of the PCR products amplified in Example 1. [Figure 5] A diagram schematically showing the structure of an HLA gene and the position to which a PCR primer is designed to bind ( SEQ ID No. of the designated primer in the indicated region is indicated in parentheses). [Figure 6] An agarose gel electrophoretic pattern showing the amplification states of the amplified PCR products of an HLA gene in Example 2. [Figure 7] An agarose gel electrophoretic pattern of the amplified PCR products obtained by three types of DNA extraction methods in Example 3. Ways to Carry Out the Invention
    [00019] Now, the DNA typing method of the present invention will be described more specifically in steps. (1) Step of preparing a starter set
    [00020] In the DNA typing method of the present invention, first, a set of primers that respectively specifically loop with an upstream region and a downstream region of each of the genes HLA-A, HLA-B, HLA- C, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 in the human genome sequence, and a set of primers that respectively specifically anneal with the exon 2 region and an exon 3' untranslated region of HLA- DRB1 are prepared.
    [00021] The genome sequence of human chromosome 6 (6p21.3), in which an HLA gene is present, has already been elucidated and the association of the gene structure and the structure of an expression product (HLA molecule) has been revealed (see Figure 1 and Figure 2).
    [00022] More specifically, each of the HLA-A, HLA-B and HLA-C genes, which are called classical HLA class I molecules, contains 7 or 8 exons (Figure 1(a)). Outside of exon 1, two types of enhancers and a promoter region are present to control expression (Figure 1(b)).
    [00023] It is also known that many polymorphic regions are present in exon 2, 3 and 4. Thus, PCR was performed by using primers prepared especially based on exons 2 and 3 in conventional DNA typing methods. Therefore, a phase ambiguity problem occurred, as mentioned above.
    [00024] However, the HLA-DR, HLA-DQ and HLA-DP genes, which are called classic HLA class II molecules, consist of α-chains and β-chains, where each of the genes contains 5 to 6 exons (Figure 2(a)). Outside of exon 1, a promoter region is present to control expression (Figure 2(b)).
    [00025] It is also known that many polymorphic regions are present in exon 2 and exon 3. Thus, PCR was performed by using primers specially prepared based on exon 2 in conventional DNA typing methods. In this sense, there was a phase ambiguity problem, as mentioned above.
    [00026] In the present invention, a set of primers that can amplify (by PCR) all regions of a gene (including not only exons, but also introns, 5' and 3' untranslated regions and a promoter region) in each of the classical class I molecules (HLA-A, HLA-B, HLA-C) and classical class II molecules (HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1); and a set of primers that can amplify (by PCR) the regions of the HLA-DRB1 gene including exon 2 to a 3' untranslated region are prepared, and the PCR products obtained by PCR amplification using the primer sets are submitted to next-generation sequencing (described later). Therefore, uncertainties such as phase ambiguity can be eliminated and the presence or absence of a null allele can be accurately detected.
    [00027] Specifically, the sets of PCR primers listed in Table 1 through Table 4 below are prepared.
    [00028] In Table 1, SEQ ID Nos. 1 to 3 represent a set of PCR primers specifically amplifying an HLA-A gene, which is an MHC class I α chain. These set of primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-A gene (including the promoter, exons and introns) and which span all regions in the sequence of the human genome (reference sequence: hg19).
    [00029] SEQ ID No. 1 has a nucleotide sequence corresponding from position 29.909,487 to position 29.909,514 in a human genome sequence (reference sequence: hg19).
    [00030] SEQ ID No. 2 has a nucleotide sequence corresponding from position 29.909,487 to position 29.909,514 in a human genome sequence (reference sequence: hg19).
    [00031] SEQ ID No. 3 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 29,914,925 to position 29,914,952 in a human genome sequence (reference sequence: hg19).
    [00032] The length of a PCR product obtained using these primer sets is estimated to be about 5500 bases (bp).
    [00033] In Table 1, SEQ ID Nos. 4 and 5 represent a set of PCR primers specifically amplifying an HLA-B gene, which is an MHC class I α chain. an HLA-B gene (including the promoter, exons and introns) and which span all regions in the sequence of the human genome (reference sequence: hg19).
    [00034] SEQ ID No. 4 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 31,325,796 to position 31,325,820 in a human genome sequence (reference sequence: hg19).
    [00035] SEQ ID No. 5 has a nucleotide sequence corresponding from position 31.321.212 to position 31.321.235 in a human genome sequence (reference sequence: hg19).
    [00036] The length of a PCR product obtained using these primer sets is estimated to be about 4,600 bases (bp).
    [00037] In Table 1, SEQ ID Nos. 6 to 8 represent a set of PCR primers specifically amplifying an HLA-C gene, which is an MHC class I α chain. an HLA-C gene (including the promoter, exons and introns) and which span all regions in the sequence of the human genome (reference sequence: hg19).
    [00038] SEQ ID No. 6 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 31,240,868 to position 31,240,892 in a human genome sequence (reference sequence: hg19).
    [00039] SEQ ID No. 7 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 31,240,868 to position 31,240,892 in a human genome sequence (reference sequence: hg19).
    [00040] SEQ ID No. 8 has a nucleotide sequence corresponding from position 31,236,991 to position 31,236,114 in a human genome sequence (reference sequence: hg19).
    [00041] The length of a PCR product obtained using these primer sets is estimated to be about 4,800 bases (bp). [Table 1]

    [00042] In Table 2, SEQ ID Nos. 9 to 11 depict a set of PCR primers specifically amplifying an HLA-DR1 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [00043] SEQ ID No. 9 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,131 to position 32,552,156 in a human genome sequence (reference sequence: hg19).
    [00044] SEQ ID No. 10 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,131 to position 32,552,156 in a human genome sequence (reference sequence: hg19).
    [00045] SEQ ID No. 11 has a nucleotide sequence corresponding from position 32,546,609 to position 32,546,629 in a human genome sequence (reference sequence: hg19).
    [00046] The length of a PCR product obtained using these primer sets is estimated to be about 5200 bases (bp).
    [00047] In Table 2, SEQ ID Nos. 31 and 32 depict a set of PCR primers specifically amplifying HLA-DR1, HLA-DR4, HLA-DR6 (DR13) and a gene of the HLA-DR10 subtype of an HLA-DRB1 gene, which is a class II MHC β chain . These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00048] SEQ ID No. 31 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,558,110 to position 32,558,133 in a human genome sequence (reference sequence: hg19).
    [00049] SEQ ID No. 32 has a nucleotide sequence that corresponds from position 32,551,974 to position 32,551,999 in a human genome sequence (reference sequence: hg19).
    [00050] The lengths of the PCR products obtained using these sets of primers are estimated to be about 6,100 bases (bp) in the case of an HLA-DR1 subtype, to about 9,100 bases (bp) in the case of an HLA- subtype DR4, of about 8,900 bases (bp) in the case of an HLA-DR6 (DR13) subtype and of about 8,900 bases (bp) in the case of an HLA-DR10 subtype.
    [00051] In Table 2, SEQ ID Nos. 11 and 12 depict a set of PCR primers specifically amplifying an HLA-DR2 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [00052] SEQ ID No. 11 is as defined above.
    [00053] SEQ ID No. 12 has a nucleotide sequence complementary to a nucleotide sequence corresponding from position 32,552,130 to position 32,552,151 in a human genome sequence (reference sequence: hg19).
    [00054] The length of a PCR product obtained using these primer sets is estimated to be about 5500 bases (bp).
    [00055] In Table 3, SEQ ID Nos. 31 and 33 depict a set of PCR primers specifically amplifying an HLA-DR2 (DR15) subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00056] SEQ ID No. 31 is as defined above.
    [00057] SEQ ID No. 33 has a nucleotide sequence that corresponds from position 32,551,974 to position 32,551,999 in a human genome sequence (reference sequence: hg19).
    [00058] The length of a PCR product obtained using these primer sets is estimated to be about 6100 bases (bp).
    [00059] In Table 2, SEQ ID Nos. 13 and 14 depict a set of PCR primers specifically amplifying an HLA-DR3, HLA-DR5, HLA-DR6 and HLA-DR8 subtype gene from the HLA-DRB1 gene, which is a class II MHC β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [00060] SEQ ID No. 13 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,137 to position 32,552,160 in a human genome sequence (reference sequence: hg19).
    [00061] SEQ ID No. 14 has a nucleotide sequence corresponding from position 32,546,609 to position 32,546,629 in a human genome sequence (reference sequence: hg19).
    [00062] The length of a PCR product obtained using these primer sets is estimated to be about 5100 bases (bp).
    [00063] In Table 2, SEQ ID Nos. 34 and 32 depict a set of PCR primers specifically amplifying an HLA-DR3 subtype gene from an HLA-DRB1 gene, which is a class II MHC β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00064] SEQ ID No. 34 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,558,110 to position 32,558,133 in a human genome sequence (reference sequence: hg19).
    [00065] SEQ ID No. 32 is as defined above.
    [00066] The length of a PCR product obtained using these primer sets is estimated to be about 8900 bases (bp).
    [00067] In Table 2, SEQ ID Nos. 15 and 16 depict a set of PCR primers specifically amplifying an HLA-DR4 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [00068] SEQ ID No. 15 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,131 to position 32,552,157 in a human genome sequence (reference sequence: hg19).
    [00069] SEQ ID No. 16 has a nucleotide sequence corresponding from position 32,546,609 to position 32,546,629 in a human genome sequence (reference sequence: hg19).
    [00070] The length of a PCR product obtained using these primer sets is estimated to be about 6200 bases (bp).
    [00071] In Table 2, SEQ ID Nos. 31 and 35 depict a set of PCR primers specifically amplifying an HLA-DR5 (DR11) subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00072] SEQ ID No. 31 is as defined above.
    [00073] SEQ ID No. 35 has a nucleotide sequence corresponding from position 32,551,974 to position 32,551,997 in a human genome sequence (reference sequence: hg19).
    [00074] The length of a PCR product obtained using these primer sets is estimated to be about 8900 bases (bp).
    [00075] In Table 2, SEQ ID Nos. 31 and 36 depict a set of PCR primers specifically amplifying an HLA-DR5 (DR12) subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00076] SEQ ID No. 31 is as defined above.
    [00077] SEQ ID No. 36 has a nucleotide sequence that corresponds from position 32,551,974 to position 32,551,999 in a human genome sequence (reference sequence: hg19).
    [00078] The length of a PCR product obtained using these primer sets is estimated to be about 8900 bases (bp). Table 2]


    [00079] In Table 3, SEQ ID Nos. 31 and 37 depict a set of PCR primers specifically amplifying an HLA-DR6 (DR14) subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00080] SEQ ID No. 31 is as defined above.
    [00081] SEQ ID No. 37 has a nucleotide sequence corresponding from position 32,551,974 to position 32,551,997 in a human genome sequence (reference sequence: hg19).
    [00082] The length of a PCR product obtained using these primer sets is estimated to be about 8900 bases (bp).
    [00083] In Table 3, SEQ ID Nos. 17 and 18 depict a set of PCR primers specifically amplifying the HLA-DR7 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [00084] SEQ ID No. 17 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,137 to position 32,552,160 in a human genome sequence (reference sequence: hg19).
    [00085] SEQ ID No. 18 has a nucleotide sequence corresponding from position 32,546,606 to position 32,546,629 in a human genome sequence (reference sequence: hg19).
    [00086] The length of a PCR product obtained using these primer sets is estimated to be about 5100 bases (bp).
    [00087] In Table 3, SEQ ID Nos. 38 and 36 depict a set of PCR primers specifically amplifying an HLA-DR7 and HLA-DR9 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00088] SEQ ID No. 38 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,558,110 to position 32,558,133 in a human genome sequence (reference sequence: hg19).
    [00089] SEQ ID No. 36 is as defined above.
    [00090] The length of a PCR product obtained using these primer sets is estimated to be about 11,400 bases (bp).
    [00091] In Table 3, SEQ ID Nos. 31 and 39 depict a set of PCR primers specifically amplifying an HLA-DR8 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [00092] SEQ ID No. 31 is as defined above.
    [00093] SEQ ID No. 39 has a nucleotide sequence corresponding from position 32,551,974 to position 32,551,994 in a human genome sequence (reference sequence: hg19).
    [00094] The length of a PCR product obtained using these primer sets is estimated to be about 8900 bases (bp).
    [00095] In Table 3, SEQ ID Nos. 19 and 20 depict a set of PCR primers specifically amplifying an HLA-DR9 subtype gene from an HLA-DRB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [00096] SEQ ID No. 19 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,137 to position 32,552,160 in a human genome sequence (reference sequence: hg19).
    [00097] SEQ ID No. 20 has a nucleotide sequence that corresponds from position 32,546,609 to position 32,546,629 in a human genome sequence (reference sequence: hg19).
    [00098] The length of a PCR product obtained using these primer sets is estimated to be about 5100 bases (bp).
    [00099] In Table 3, SEQ ID Nos. 21 and 22 depict a set of PCR primers specifically amplifying an HLA-DR10 subtype gene from an HLA-DRB1 gene, which is a class II MHC β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DRB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [000100] SEQ ID No. 21 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,552,137 to position 32,552,159 in a human genome sequence (reference sequence: hg19).
    [000101] SEQ ID No. 22 has a nucleotide sequence corresponding from position 32,546403 to position 32,546,435 in a human genome sequence (reference sequence: hg19).
    [000102] The length of a PCR product obtained using these primer sets is estimated to be about 5400 bases (bp). Table 3]


    [000103] In Table 4, SEQ ID Nos. 23 and 24 represent a set of PCR primers specifically amplifying an HLA-DPA1 gene, which is an α chain of MHC class II. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DPA1 gene (including the promoter, exons and introns) and that impact all regions in the human genome sequence (sequence of reference: hg19).
    [000104] SEQ ID No. 23 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 33,041,478 to position 33,041,502 in a human genome sequence (reference sequence: hg19).
    [000105] SEQ ID No. 24 has a nucleotide sequence corresponding from position 33,031,888 to position 33,031,911 in a human genome sequence (reference sequence: hg19).
    [000106] The length of a PCR product obtained using these primer sets is estimated to be about 9,600 bases (bp).
    [000107] In Table 4, SEQ ID Nos. 40 and 41 represent a set of PCR primers specifically amplifying an HLA-DPA1 gene, which is an MHC class II α chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DPA1 gene (including the promoter, exons and introns) and that impact all regions in the human genome sequence (sequence of reference: hg19).
    [000108] SEQ ID No. 40 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 33,041,573 to position 33,041,596 in a human genome sequence (reference sequence: hg19).
    [000109] SEQ ID No. 41 has a nucleotide sequence corresponding from position 33,031,888 to position 33,031,912 in a human genome sequence (reference sequence: hg19).
    [000110] The length of a PCR product obtained using these primer sets is estimated to be around 9,600 bases (bp).
    [000111] In Table 4, SEQ ID Nos. 25 and 26 represent a set of PCR primers specifically amplifying an HLA-DPB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DPB1 gene (including the promoter, exons and introns) and that impact all regions in the human genome sequence (sequence of reference: hg19).
    [000112] SEQ ID No. 25 has a nucleotide sequence corresponding from position 33,043,056 to position 33,043,079 in a human genome sequence (reference sequence: hg19).
    [000113] SEQ ID No. 26 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 33,055,476 to position 33,055,499 in a human genome sequence (reference sequence: hg19).
    [000114] The length of a PCR product obtained using these primer sets is estimated to be about 12,400 bases (bp).
    [000115] In Table 4, SEQ ID Nos. 42 and 43 represent a set of PCR primers specifically amplifying an HLA-DPB1 gene, which is an MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of a 5' untranslated region to exon 2 of an HLA-DRB1 gene and press the 5' untranslated region to exon 2 in the genome sequence human (reference sequence: hg19).
    [000116] SEQ ID No. 42 has a nucleotide sequence corresponding from position 33,043,168 to position 33,043191 in a human genome sequence (reference sequence: hg19).
    [000117] SEQ ID No. 43 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 33,049,084 to position 33,049,107 in a human genome sequence (reference sequence: hg19).
    [000118] The length of a PCR product obtained using these primer sets is estimated to be about 5900 bases (bp).
    [000119] In Table 4, SEQ ID Nos. 44 and 45 represent a set of PCR primers specifically amplifying an HLA-DPB1 gene, which is an MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream from exon 2 to a 3' untranslated region of an HLA-DPB1 gene and which span exon 2 to a 3' untranslated region in the genome sequence human (reference sequence: hg19).
    [000120] SEQ ID No. 44 has a nucleotide sequence that corresponds from position 33,048182 to position 33,048,207 in a human genome sequence (reference sequence: hg19).
    [000121] SEQ ID No. 45 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 33,055,428 to position 33,055,453 in a human genome sequence (reference sequence: hg19).
    [000122] The length of a PCR product obtained using these primer sets is estimated to be about 7,200 bases (bp).
    [000123] In Table 4, SEQ ID Nos. 27 and 28 represent a set of PCR primers specifically amplifying an HLA-DQA1 gene, which is an MHC class II α chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DQA1 gene (including the promoter, exons and introns) and that press all regions in the human genome sequence (sequence of reference: hg19).
    [000124] SEQ ID No. 27 has a nucleotide sequence corresponding from position 32,604,318 to position 32,604,338 in a human genome sequence (reference sequence: hg19).
    [000125] SEQ ID No. 28 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,611,681 to position 32,611,701 in a human genome sequence (reference sequence: hg19).
    [000126] The length of a PCR product obtained using these primer sets is estimated to be about 7,400 bases (bp).
    [000127] In Table 4, SEQ ID Nos. 46 and 47 represent a set of PCR primers specifically amplifying an HLA-DQA1 gene, which is an MHC class II α chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DQA1 gene (including the promoter, exons and introns) and that press all regions in the human genome sequence (sequence of reference: hg19).
    [000128] SEQ ID No. 46 has a nucleotide sequence corresponding from position 32,604,469 to position 32,604,488 in a human genome sequence (reference sequence: hg19).
    [000129] SEQ ID No. 47 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,611,936 to position 32,611,956 in a human genome sequence (reference sequence: hg19).
    [000130] The length of a PCR product obtained using these primer sets is estimated to be about 7,400 bases (bp).
    [000131] In Table 4, SEQ ID Nos. 29 and 30 represent a set of PCR primers specifically amplifying an HLA-DQB1 gene, which is an MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DQB1 gene (including the promoter, exons and introns) and that press all regions in the human genome sequence (sequence of reference: hg19).
    [000132] SEQ ID No. 29 has a nucleotide sequence corresponding from position 32,626,545 to position 32,626,568 in a human genome sequence (reference sequence: hg19).
    [000133] SEQ ID No. 30 has a nucleotide sequence complementary to a nucleotide sequence that corresponds from position 32,635,612 to position 32,635,637 in a human genome sequence (reference sequence: hg19).
    [000134] The length of a PCR product obtained using these primer sets is estimated to be about 9,100 bases (bp).
    [000135] In Table 4, SEQ ID Nos. 29, 30 and 48 to 50 represent a set of PCR primers specifically amplifying an HLA-DQB1 gene, which is a MHC class II β chain. These set primers are nucleotide sequences located at positions that correspond upstream and downstream of all regions of an HLA-DQB1 gene (including the promoter, exons and introns) and that press all regions in the human genome sequence (sequence of reference: hg19).
    [000136] SEQ ID Nos. 29 and 48 have a nucleotide sequence corresponding from position 32,626,545 to position 32,626,568 in a human genome sequence (reference sequence: hg19).
    [000137] SEQ ID Nos. 30, 49 and 50 have a nucleotide sequence complementary to a nucleotide sequence corresponding to position 32,635,612 to position 32,635,637 in a human genome sequence (reference sequence: hg19).
    [000138] The length of a PCR product obtained using these primer sets is estimated to be about 9,100 bases (bp). Table 4]

    [000139] These primers can be prepared by a method routinely used in this field. Furthermore, the sets of primers described in Table 1 and Table 2 are the most preferable examples. In the method of the present invention, any set of primers that can be used as the set of primers is a set of a normal primer and a reverse primer capable of ringing for positions, which correspond to the upstream and downstream of all regions of each. HLA gene and sandwich all regions. (2) PCR amplification step
    [000140] In the method of the present invention, a test sample (DNA) is amplified by PCR using the sets of primers prepared in the above step (1).
    [000141] The PCR amplification reaction is performed according to a general protocol and more specifically as follows. 1. DNA is extracted from a test sample depending on the shape of the sample. 2. Extracted DNA is quantified and primer concentrations are appropriately set to prepare the reaction solution. 3. Reaction conditions are defined and a PCR is performed.
    [000142] For example:
    [000143] Thermal denaturation step (generally 92 to 97°C)
    [000144] Annealing step (generally 55 to 72°C)
    [000145] Extension step (usually from 65 to 80 °C)
    [000146] In the method of the present invention, in the case of an HLA gene (except HLA-DRB1), the annealing temperature is preferably set at about 60°C. Due to annealing at about 60°C, the alleles can be produced in the equivalent ratio (evenly). In the case of an HLA-DRB1, the temperature of the annealing step is preferably set at about 70°C. Due to annealing at about 70°C, a desired DR subtype can only be specifically produced. 4. The PCR product obtained is purified and submitted to the nucleotide sequencing step below. (3) Nucleotide sequencing step
    [000147] Then, the nucleotide sequence of the PCR product (amplified DNA) produced in the above step (2) is determined. The step is preferably performed by a technique called next-generation sequencing (or ultra-high sequencing). Regarding next-generation sequencing, see, for example, “Experimental Medicine”, Vol. 27, No. 1, 2009 (Yodo-sha).
    [000148] The sequence of the present invention is determined by a method based on pyrosequencing, which is employed in a Roche FLX genome sequencer system. The sequencing method will be described below. 1. The PCR product obtained in step above (2) is broken up by a nebulizer into fragments of about 500 bases. 2. To one end of each of the DNA fragments, a DNA adapter is attached. 3. DNA fragments linked with a DNA adapter are dissociated into single-stranded DNA fragments, which are attached to the microspheres via the adapter. The obtained microspheres are enclosed and placed in a water-in-oil emulsion (a microreactor environment containing a single DNA fragment linked to a single microsphere is formed). 4. Emulsion PCR is performed to form copies of each DNA fragment on a microsphere (each DNA fragment is clonally amplified in each microreactor. In this way, many fragments can be simultaneously and parallel amplified without competing with other sequences). Afterwards, the emulsion is destroyed and the microspheres with amplified DNA fragments are collected. 5. The microspheres are concentrated and loaded into a picotitrator plate (a single well is large enough to place a single microsphere). 6. Pyrophosphoric acid produced by a polymerase during an enzymatic reaction is detected against each microsphere by a fluorescent luciferase reaction. Based on the intensity and pattern of fluorescence thus emitted, the nucleotide sequence of the DNA is determined. Four types of nucleic acids (A, C, G, T) are added in a predetermined order. The chemiluminescence pattern according to the added nucleic acid is recorded. Based on the signal strength and positional data in combination, the nucleotide sequence is determined. (4) DNA typing step
    [000149] Subsequently, the nucleotide sequence obtained in step (3) is compared with data from known HLA alleles in the nucleotide sequencing database. In this way, the allele type (up to 8 digits) contained in the test sample is determined.
    [000150] In the method of the present invention, typical sets of primers are listed in Table 1 (described above). The method of the present invention is characterized by the fact that the primers are designed to match all regions of each of the HLA class I and HLA class II genes, except HLA-DRB1, and the positions that sandwich the region between the 2 and 3' untranslated exon of HLA-DRB1, and the DNA sequence amplified to match almost all regions is determined. In this way, the phase ambiguity (uncertainty) is eliminated and information about a null allele can be obtained. Examples
    [000151] The present invention will be more specifically described by means of the examples below; however, the present invention is not limited to these examples. (Example 1) [Experimental Method] 1. Using the already extracted genomic DNA as a template and specific primer sets for the individual HLA class I genes (see Table 1: SEQ ID Nos. 1 to 8), a PCR it was made. The procedure is more specifically as follows. (1) PCR amplification was performed using the STAR GXL polymerase primer (TaKaRa). More specifically, at 50 ng of a genomic DNA solution, 4 mL of 5 x PrimeSTAR GXL buffer, 1.6 μL of a dNTP solution, PCR primers (4 μL (1 pmol/μL) each) and 0.8 µL of the STAR GXL polymerase primer was added. The total amount of the reaction solution was adjusted to 20 µL with sterilized water. (2) After holding at 94°C for 2 minutes, the reaction solution was subjected to a step consisting of a reaction at 98°C for 10 seconds, a reaction at 60°C for 20 seconds and a reaction at 68°C for 5 minutes. This step was repeated 30 times. Note that for PCR amplification, the Gene Amp PCR 9700 system (Applied Biosystems) was used. After PCR, the amplification states of the PCR products were verified by agarose gel electrophoresis. The electrophoretic patterns are shown in Figure 4. 2. The nucleotide sequences of the PCR products were specifically determined as follows. (1) A PCR product was purified by QIAquick PCR Purification Kit (QIAGEN) according to standard protocol. (2) The concentration of purified PCR product was measured by the PicoGreen dsDNA Quantitation Kit (Invitrogen) according to the standard protocol. (3) A solution of purified PCR product, a concentration of which was adjusted to be 500 ng/100 mL, was subjected to rapid library construction and then to emulsion PCR and sequencing by the Genomic Sequencer (GS). ) Junior (Roche) were performed according to the standard protocol to obtain the nucleotide sequences of 10,000 reads per sample. (4) These sequences have been linked and edited by the GS compiler again (Roche). Subsequently, a homology search with known nucleotide sequences in a DNA database was performed to identify the alleles in the HLA gene. [Discussion]
    [000152] In the PCR primers HLA-A, HLA-B and HLA-C, which specifically amplify 5.5 Kb, 4.6 Kb and 4.8 Kb, were designed. PCR conditions were studied and agarose gel electrophoresis of the resulting PCR products was performed. As a result, all HLA class I genes were found to provide a single amplified PCR product at a position corresponding to a desired molecular weight (Figure 4). Furthermore, the nucleotide sequences of the PCR products were determined by the Sanger method. As a result, the HLA alleles were obtained consistently with known documents. From this, it was confirmed that the PCR system of the invention can be used for HLA typing.
    [000153] Using three samples of HLA-B*40:02 homozygote and 17 samples of one HLA-B*40:02 heterozygote, including combinations of alleles (B*40 and B*55), in which phase ambiguity was observed in conventional DNA typing method, a PCR was performed. As a result of HLA typing of PCR products derived from the HLA-B gene by GS Junior, HLA-B*40:02:01:01 was detected from all samples. In the 17 heterozygous samples, 2 types of new alleles were detected in addition to the 15 already known alleles. In particular, in relation to a sample with a combination of alleles (B*40 and B*55), in which phase ambiguity was observed, HLA-B*40:02:01:01 and HLA-B*55:02 :01:01 have been identified by typing. From this, it was demonstrated that the method of the invention allows HLA typing at the 8-digit level, without phase ambiguity; and that the method of the invention is an excellent tool to efficiently detect a substitution, insertion and deletion of bases in a promoter and introns, which are causes of a null allele. (Example 2) [Experimental Method] 1. Using an already extracted genomic DNA as a template and the specific primer sets for the individual HLA class I and HLA class II genes (see Tables 1 to 4: SEQ ID Nos. 1 at 8, 9 to 22, 31 to 50), a PCR was performed. The procedure is more specifically as follows. (1) PCR amplification was performed using the STAR GXL polymerase primer (TaKaRa). More specifically, at 50 ng of a genomic DNA solution, 4 μL of 5 x PrimeSTAR GXL buffer, 1.6 μL of a dNTP solution, PCR primers (1 to 7 μL (4 pmol/μL)) and 0, 8 µl of the STAR GXL polymerase primer was added. The total amount of the reaction solution was adjusted to 20 µL with sterilized water. (2) After holding at 94°C for 2 minutes, the reaction solution was subjected to a step consisting of a reaction at 98°C for 10 seconds and a reaction at 70°C for 5 minutes. This step was repeated 30 times. Note that for PCR amplification, the Gene Amp PCR 9700 system (Applied Biosystems) was used. After PCR, the amplification states of the PCR products were verified by agarose gel electrophoresis. The electrophoretic patterns were shown in Figure 6. The nucleotide sequences of the PCR products were specifically determined as follows. (1) A PCR product was purified by QIAquick PCR Purification Kit (QIAGEN) according to standard protocol. (2) The concentration of purified PCR product was measured by the PicoGreen dsDNA Quantitation Kit (Invitrogen) according to the standard protocol. (3) The purified PCR product, the concentration of which was adjusted to be 100 ng, was subjected to the construction of a fragment library, and then to emulsion PCR and sequencing by the personal Ion genome machine (Ion PGM ) (Life Technologies) were performed according to standard protocol to obtain nucleotide sequences of 300,000 reads per sample. (4) These sequences have been linked and edited by the GS compiler again (Roche). Subsequently, a homology search with known nucleotide sequences in a DNA database was performed to identify the alleles in the HLA gene. [Results and discussion] 1. PCR primers, which specifically amplify 4kb to 12kb in the region of a 5' untranslated region up to exon 2 of HLA-A, HLA-B, HLA-C and HLA-DRB1, a region from exon 2 to a 3'untranslated region from HLA-DRB1, the region from an untranslated region to exon 2 from HLA-DQB1 and HLA-DPB1 and the region from exon 2 to a 3'untranslated region of HLA-DPB1 were designed. PCR conditions were studied and agarose gel electrophoresis of the resulting PCR products was performed. As a result, it was found that all HLA class I and HLA class II genes provide a single amplified product at a position corresponding to a desired molecular weight (Figure 6). Furthermore, the nucleotide sequences of the PCR products were determined by the Sanger method. As a result, the HLA alleles were obtained consistently with known documents. It has again been confirmed in the present invention that the PCR system of the invention can be used for HLA typing. 2. Using four samples containing a combination of alleles, where phase ambiguity is observed in a conventional DNA typing method, a PCR was performed. PCR products derived from the regions of a 5' untranslated region to exon 2 of the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes, the region from exon 2 to a 3' untranslated region of a gene HLA-DRB1, the region from a 5'untranslated region to exon 2 of the HLA-DQB1 and HLA-DPB1 genes and the region from exon 2 to a 3'untranslated region of an HLA-DPB1 gene were subjected to HLA-DPB1 gene typing. HLA by Ion PGM. As a result, typing of entire gene regions of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 was done successfully. Regarding HLA-DPB1, typing of only one exon was successful. Furthermore, in each of the HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genes, a new allele was detected. From this, it was demonstrated that the method of the invention allows HLA typing at the 8-digit level, without phase ambiguity; and that the method of the invention is an excellent tool to efficiently detect a substitution, insertion and deletion of bases in a promoter and introns, which are causes of a null allele. (Example 3) [Experimental method] 1. Genomic DNA was extracted using the Buccal Cellular DNA Extraction Kit, BuccillQuick (TRIMGEN). 2. Genomic DNA extracted using the Buccal BuccalQuick Cell DNA Extraction Kit (TRIMGEN) was further purified with isopropanol and ethanol. 3. Using a QIAamp Blood DNA Minikit (QIAGEN), genomic DNA was extracted. 4. Three of each of the genomic DNA samples extracted in items 1 to 3 above were subjected to PCR using specific primer sets for HLA-A, HLA-B, HLA-C and HLA-DQB1 performed in the same experimental method as in Example 1 and Example 2 (see Table 1 and Table 4: SEQ ID Nos. 1 to 8, 29, 30, 48 and 50). After PCR, the amplification states of the PCR products were verified by agarose gel electrophoresis. Electrophoretic patterns are shown in Figure 7. [Experimental results and discussion]
    [000154] In Figure 7, lanes 1 to 3 show the amplification states of the PCR products in the case where the extraction was done by experimental method 1, lanes 4 to 6 show the amplification states of the PCR products in the case where the extraction was done by experimental method 2 and lanes 7 to 9 show the state of amplification of the PCR products in the case where the extraction was done by the experimental method 3. The PCR amplification in the case where the genomic DNA was extracted by the Experimental method 1 was used as a template in any gene is equivalent to PCR amplification in the case where genomic DNA extracted by experimental method 3 was used, and a desired PCR product was obtained. In experimental method 3, blood must be aliquoted; however, in experimental method 1, cells can be taken from the oral mucous membrane. Therefore, it has been demonstrated that if the method of the present invention is used, HLA typing can be sufficiently performed even if blood cannot be sampled.
    权利要求:
    Claims (2)
    [0001]
    1. Method of typing HLA DNA up to an 8-digit level without phase ambiguity, characterized by the fact that it comprises the following steps: (1) a step of preparing a set of primers that respectively specifically anneale with a region to upstream and a downstream region of each of the HLA-A, HLA-B, HLA-C, HLADQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 genes in the human genome sequence, in which the primer set for the HLA-A gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 1, 2 and 3; the set of primers for the HLA-B gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 4 and 5; the set of primers for the HLA-C gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 6, 7 and 8; the set of primers for the HLA-DPA1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 23, 24, 40 and 41; the set of primers for the HLA-DPB1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 25, 26, 42, 43, 44 and 45; the set of primers for the HLA-DQA1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 27, 28, 46 and 47; the set of primers for the HLA-DQB1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 29, 30, 48, 49 and 50; the set of primers for the HLA-DRB1 gene comprise a set of primers selected from the group consisting of: (i) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID US. 9, 10, 11, 31 and 32; (ii) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 11, 12, 31 and 33; (iii) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 32 and 34; (iv) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 15, 16, 31 and 32; (v) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 35 and 36; (vi) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 32 and 37; (vii) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 17, 18, 36 and 38; (viii) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 31 and 39; (ix) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 19, 20, 36 and 38; (x) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 21, 22, 31 and 32; (2) a step of amplifying a test sample (DNA) by a PCR using the primer sets; (3) a step of determining the nucleotide sequences of the products amplified by PCR; and (4) a step of optionally performing a homology search in a database.
    [0002]
    2. Primer set for DNA typing of an HLA gene at up to an 8-digit level without phase ambiguity, characterized by the fact that it comprises a set of primers for at least one HLA gene selected from HLA-A genes, HLA-B, HLA-C, HLADQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1 and HLA-DRB1 in a human genome sequence, wherein the set of primers for the HLA-A gene comprise a set of primers consisting of in a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 1, 2 and 3; the set of primers for the HLA-B gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 4 and 5; the set of primers for the HLA-C gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 6, 7 and 8; the set of primers for the HLA-DPA1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 23, 24, 40 and 41; the set of primers for the HLA-DPB1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 25, 26, 42, 43, 44 and 45; the set of primers for the HLA-DQA1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 27, 28, 46 and 47; the set of primers for the HLA-DQB1 gene comprise a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 29, 30, 48, 49 and 50; the set of primers for the HLA-DRB1 gene comprise a set of primers selected from the group consisting of: (i) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID US. 9, 10, 11, 31 and 32; (ii) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 11, 12, 31 and 33; (iii) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 32 and 34; (iv) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 15, 16, 31 and 32; (v) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 35 and 36; (vi) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 31, 32 and 37; (vii) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 17, 18, 36 and 38; (viii) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 13, 14, 31 and 39; (ix) a primer set consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 19, 20, 36 and 38; (x) a set of primers consisting of a normal primer and a reverse primer primers selected from oligonucleotides with nucleotide sequences represented by SEQ ID Nos. 21, 22, 31 and 32.
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    同族专利:
    公开号 | 公开日
    CN106434865B|2021-05-18|
    EP2735617A1|2014-05-28|
    EP2735617B1|2018-02-21|
    AU2017261606A1|2018-02-15|
    JPWO2013011734A1|2015-02-23|
    JP2017158570A|2017-09-14|
    US20160208326A1|2016-07-21|
    US10704095B2|2020-07-07|
    CA2841060A1|2013-01-24|
    CN106434865A|2017-02-22|
    CA2841060C|2020-06-16|
    US20140206005A1|2014-07-24|
    JP6809977B2|2021-01-06|
    AU2012285223B2|2017-08-17|
    JP2020010698A|2020-01-23|
    EP2735617A4|2015-08-19|
    CN103890190B|2016-08-17|
    AU2012285223A1|2014-01-30|
    HK1199069A1|2015-06-19|
    BR112014001258A2|2017-02-21|
    CN103890190A|2014-06-25|
    EP3354749A1|2018-08-01|
    WO2013011734A1|2013-01-24|
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    法律状态:
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-04-07| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-06-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-07-13| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 18/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    JP2011159832|2011-07-21|
    JP2011-159832|2011-07-21|
    PCT/JP2012/062743|WO2013011734A1|2011-07-21|2012-05-18|Method and kit for dna typing of hla gene|
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