HK1235084A1

HK1235084A1 - Method and kit for dna typing of hla gene

Info

Publication number: HK1235084A1
Application number: HK17108314.3A
Authority: HK
Inventors: Shiina Takashi; Suzuki Shingo; Ozaki Yuki; Mitsunaga Shigeki; Inoko Hidetoshi
Original assignee: Genodive Pharma Inc.
Priority date: 2011-07-21
Filing date: 2017-08-21
Publication date: 2018-03-02

Description

DNA typing method and kit for HLA gene

The present application is a divisional application of PCT application having an international application date of 2012/5/18, international application number of PCT/JP2012/062743, application number of 201280036108.5 at the national stage of entry, and an invention name of "DNA typing method of HLA gene and kit".

Technical Field

The present invention relates to a method and a kit for DNA typing of HLA genes using a large-scale parallel sequence analyzer.

Background

Human Leukocyte Antigens (HLA), which are human Major Histocompatibility Complexes (MHC), are deeply involved in the induction of immune responses by presenting peptides derived from pathogen-derived foreign proteins as well as peptides derived from self-proteins to T cells, and 6 antigens are known as major HLA. That is, class I molecules (HLA-A, HLA-B, HLA-C) expressed in almost all cells and class II molecules (HLA-DR, HLA-DQ, HLA-DP) expressed mainly in cells of the immune system.

HLA class I antigens consist of an alpha chain exhibiting high polymorphism and beta 2-microglobulin with little polymorphism, and HLAII antigens consist of a beta chain with high polymorphism and an alpha chain with low polymorphism. The alpha chain of the I-class molecule is coded by HLA-A, HLA-B, HLA-C genes, the beta chain of the II-class antigen is coded by HLA-DRB1, HLA-DQB1 and HLA-DPB1 genes, and the alpha chain is coded by HLA-DRA1, HLA-DQA1 and HLA-DPA1 genes. At the gene level, in HLA class I antigens, exon 2 and exon 3 of the gene encoding the α chain show high polymorphism, and in HLA class II antigens, exon 2 of the gene encoding the β chain shows high polymorphism.

The HLA-encoding gene region is located in the short arm 6p21.3 of human chromosome 6 from the telomere side to the centromere side, and is arranged in the order of the class I region (HLA-A, HLA-C, HLA-B, etc.), the class III region, the class II region (HLA-DRA, HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, etc.), and many genes are encoded at very high density, and the association of these genes with blood transfusion, transplantation, and various diseases has been reported. In the class III region, HLA genes are not present, and genes of complement components, Tumor Necrosis Factor (TNF), and the like are present.

In the HLA-DRB gene region encoding the beta chain of HLA-DR antigen, 5 structural polymorphisms were confirmed. In DR1 type and DR10 type, pseudogenes such as HLA-DRB6 and HLA-DRB9 are located on the same chromosome in addition to HLA-DRB 1. In the DR2 type, HLA-DRB5 (DR 51) gene and pseudogenes such as HLA-DRB6 and HLA-DRB9 are located on the same chromosome in addition to HLA-DRB 1. In DR3, DR5 and DR6 types, HLA-DRB3 (DR 52) gene and pseudogenes such as HLA-DRB2 and HLA-DRB9 are located on the same chromosome in addition to HLA-DRB 1. In DR4, DR7 and DR9 types, HLA-DRB4 (DR 53) gene and HLA-DRB7, HLA-DRB8 and HLA-DRB9 pseudogenes are located on the same chromosome in addition to HLA-DRB 1. In contrast to these, in DR8 type, HLA-DRB genes other than HLA-DRB1 are not located on the same chromosome.

In the exons of the respective alleles, there are a plurality of regions showing polymorphisms, and in many cases, the base sequences (amino acid sequences) of specific polymorphic regions are common among the plurality of alleles. That is, each HLA allele is specified by a combination of a plurality of polymorphic regions. In HLA class I antigens, not only polymorphic regions within exons but also exon 2 or exon 3 having the same base sequence may be common in a plurality of alleles.

Since HLA is highly polymorphic, it is known that alleles are extremely abundant, and their expression is also determined. That is, the 1 st domain (2-digit level) for discriminating a serological HLA type, the 2 nd domain (4-digit level) for discriminating an allele with an amino acid substitution in the same serological HLA type, the 3 rd domain (6-digit level) for discriminating an allele with a base substitution not accompanied by an amino acid mutation, and the 4 th domain (8-digit level) for discriminating an allele with a base substitution in a region outside the gene region (intron) encoding an HLA molecule.

It is considered that in bone marrow transplantation, if it is desired that the HLA type of the transplant and the HLA type of the donor be perfectly matched at the 4-digit level, the success rate of transplantation is increased and the frequency of severe GVHD is reduced. In contrast, if the HLA types do not match at the 4-digit level, the risk of causing failure such as rejection increases. Therefore, accurate and highly precise HLA typing is extremely important clinically.

As DNA typing methods in HLA genes, an SBT (sequence based typing) method based on Polymerase Chain Reaction (PCR) and an SSO (sequence specific oligonucleotide) -Luminex method are mainstream.

These conventional DNA typing methods have an advantage that they can rapidly type many samples, but sometimes it is impossible to accurately determine the cis-trans positional relationship of exons on chromosomes in the case of polymorphic regions and class I genes, and therefore phase ambiguity (phase ambiguity) occurs, and it is sometimes difficult to perform HLA typing with high accuracy.

In addition, since the conventional method is a DNA typing method in which PCR is applied to each exon region as a center, base substitutions in the intron region and the promoter region are ignored, and as a result, there is a possibility that detection of a null allele whose expression is suppressed, which has the same gene structure as other expressed HLA genes, fails.

Documents of the prior art

Patent document

Patent document 1 Japanese unexamined patent application, first publication No. Hei 11-216000

Non-patent document

Non-patent document 1 Lind C, et al, Human Immunology, Vol.71, 1033-.

Disclosure of Invention

spool to which the invention is to be solved

The present invention addresses the problem of providing a highly accurate DNA typing method and a kit, which eliminate ambiguity caused by phase ambiguity.

Means for solving the problems

The present inventors have conducted extensive studies to solve the above-mentioned problems based on a novel idea of designing PCR primers capable of specifically amplifying HLA class I molecules HLA-A, HLA-B and HLA-C and HLA class II molecules HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1, setting suitable PCR conditions, and applying massively parallel sequencing technology.

That is, the present invention provides a method for typing DNA of HLA comprising the following steps.

(1) A step of preparing a primer set for annealing specifically to the upstream region and the downstream region of each of HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 genes in the base sequence of the human genome, respectively, and a primer set for annealing specifically to the untranslated regions on the exon 2 and 3' sides of HLA-DRB 1;

(2) a step of performing PCR amplification on a test sample (DNA) by using the primer set;

(3) a step of determining a base sequence of the PCR amplification product; and

(4) and (3) performing homology search with a database.

Effects of the invention

The method of the present invention obtains all base sequences necessary for DNA typing of HLA genes derived from 1 molecule, and thus is a final DNA typing method in which phase ambiguity in cis-trans positional relationship is eliminated. By this method, HLA matching between a donor candidate and a transplant recipient with high accuracy is realized.

Since all the nucleotide sequences of genes in the peripheral regions including the promoter region, exon region, intron region, and the like of the HLA gene are determined, null alleles and novel alleles that are not expressed or inhibited in expression at all can be detected.

Drawings

[ FIG. 1] (a) is a diagram showing the relationship between the HLA class I gene structure and the molecular structure. (b) A diagram showing the structure of the promoter region of an HLA class I gene. Reference is made to the editions from Transplantation/transfusion administration, Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juji, Kodan-sha Scientific, 2004, page 35.

[ FIG. 2] (a) is a diagram showing the correlation between the HLA class II gene structure and the molecular structure. (b) A diagram showing the structure of the promoter region of HLA class II gene. Reference is made to the editions from Transplantation/transfusion administration, Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juji, Kodan-sha Scientific, 2004, pages 46 to 47.

FIG. 3 shows a diagram of an HLA-DR gene region. Reference is made to the editions from Transplantation/transfusionevaluation, Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juji, Kodan-shaScientific, 2004, page 48.

FIG. 4 is an agarose gel electrophoresis chart showing the state of amplification of the PCR product amplified in example 1.

FIG. 5 is a schematic diagram showing the gene structure of HLA genes and the positions of primers for PCR design (the sequence numbers of primers designed in this region are indicated in parentheses).

FIG. 6 is an agarose gel electrophoresis chart showing the PCR amplification status of HLA genes amplified in example 2.

FIG. 7 shows agarose gel electrophoresis patterns of PCR products obtained by 3 DNA extraction methods in example 3.

Detailed Description

The DNA typing method of the present invention is explained in detail in the following steps.

(1) Preparation of primer set

In the DNA typing method of the present invention, first, a primer set specifically annealing to the upstream region and the downstream region of each of HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 in the base sequence of the human genome, respectively, and a primer set specifically annealing to the untranslated regions on the exon 2 and 3' sides of HLA-DRB1 are prepared.

The genomic base sequence of human chromosome 6 (6 p 21.3) including the region where an HLA gene is present has been elucidated, and the association of the gene structure thereof with the structure of an expression product (HLA molecule) is also known (refer to fig. 1 and 2).

That is, the genes of HLA-A, HLA-B and HLA-C, which are known as classical HLA class I molecules, contain 7 or 8 exons (FIG. 1 (a)), and 2 enhancer and promoter regions outside exon 1 regulate expression (FIG. 1 (B)).

Further, it is also known that a plurality of polymorphic regions exist in exons 2, 3 and 4, and therefore, in the conventional DNA typing method, PCR is carried out using primers prepared based on exons 2 and 3, in particular, with the attendant problem of phase ambiguity as described above.

In addition, HLA-DR, HLA-DQ and HLA-DP, which are known as classical HLA class II molecules, are composed of an alpha chain and a beta chain, and their respective genes contain 5 or 6 exons (FIG. 2 (a)), and the promoter region outside exon 1 regulates expression (FIG. 2 (b)).

Further, the presence of multiple polymorphic regions in exons 2 and 3 is also known, and therefore, in conventional DNA typing methods, PCR is performed using primers prepared based on, inter alia, exon 2, with the attendant phase ambiguity problem as described above.

In the present invention, a primer set capable of amplifying all of the HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 gene regions (including not only exons but also introns, 5' -and 3' -untranslated regions, and promoter regions) of the classical class I molecules (HLA-A, HLA-B, HLA-C) and the classical class II molecules by PCR and the gene region including the exon 2 to 3' -untranslated regions of HLA-DRB1 are prepared, and PCR products amplified by PCR using this primer set are subjected to next-generation sequencing (next-generation sequencing), whereby uncertainty such as phase ambiguity can be eliminated and the presence or absence of null alleles can be detected accurately.

Specifically, PCR primer sets listed in the following tables 1 to 4 were prepared.

Sequence numbers 1 to 3 in table 1 are PCR primer sets for specifically amplifying HLA-a gene as MHC class I α chain. These primer sets are nucleotide sequences present at positions sandwiching the entire HLA-A gene region (including promoter, exon, and intron) in the nucleotide sequence (reference sequence: hg 19) of the human genome from the upstream side and the downstream side (clamp み Write む).

SEQ ID No. 1 has a base sequence corresponding to No. 29,909,487 to No. 29,909,514 of the base sequence of human genome (reference sequence: hg 19).

SEQ ID No. 2 has a base sequence corresponding to No. 29,909,487 to No. 29,909,514 of the base sequence of human genome (reference sequence: hg 19).

SEQ ID No. 3 has a base sequence complementary to a base sequence corresponding to Nos. 29,914,925 to 29,914,952 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets is about 5,500 bases (bp).

Sequence numbers 4 to 5 in Table 1 are PCR primer sets for specifically amplifying HLA-B gene as MHC class I α chain. These primer sets are nucleotide sequences present at positions sandwiching the entire HLA-B gene region (including promoter, exon, and intron) in the nucleotide sequence of the human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID No. 4 has a base sequence complementary to a base sequence corresponding to Nos. 31,325,796 to 31,325,820 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID No. 5 has a base sequence corresponding to the base sequences No. 31,321,212 to No. 31,321,235 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 4,600 bases (bp).

Sequence numbers 6 to 8 in Table 1 are PCR primer sets for specifically amplifying HLA-C gene as MHC class I α chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region of the HLA-C gene (including a promoter, an exon, and an intron) in the nucleotide sequence of the human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID No. 6 has a base sequence complementary to a base sequence corresponding to Nos. 31,240,868 to 31,240,892 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID No. 7 has a base sequence complementary to a base sequence corresponding to Nos. 31,240,868 to 31,240,892 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID No. 8 has a base sequence corresponding to No. 31,236,991 to No. 31,236,114 of the base sequence of human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 4,800 bases (bp).

[ Table 1]

Sequence numbers 9 to 11 in Table 2 are PCR primer sets for specifically amplifying HLA-DR1 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID No. 9 has a base sequence complementary to a base sequence corresponding to Nos. 32,552,131 to 32,552,156 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID No. 10 has a base sequence complementary to a base sequence corresponding to Nos. 32,552,131 to 32,552,156 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 11 has a base sequence corresponding to Nos. 32,546,609 to 32,546,629 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets is about 5,200 bases (bp).

In Table 2, SEQ ID Nos. 31 and 32 show PCR primer sets for specifically amplifying HLA-DR1, HLA-DR4, HLA-DR6 (DR 13) and HLA-DR10 subtype genes which are MHC class II beta-chain HLA-DRB1 genes. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 31 has a base sequence complementary to the base sequence corresponding to Nos. 32,558,110 to 32,558,133 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 32 has a base sequence corresponding to Nos. 32,551,974 to 32,551,999 of the base sequence of the human genome (reference sequence: hg 19).

The predicted lengths of PCR products obtained by using these primer sets were about 6,100 bases (bp) for the HLA-DR1 subtype, about 9,100 bases (bp) for the HLA-DR4 subtype, about 8,900 bases (bp) for the HLA-DR6 (DR 13) subtype, and about 8,900 bases (bp) for the HLA-DR10 subtype.

Sequence numbers 11 and 12 in Table 2 are PCR primer sets for specifically amplifying HLA-DR2 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

Seq id No. 11 is as described above.

SEQ ID NO. 12 has a base sequence complementary to the base sequence corresponding to Nos. 32,552,130 to 32,552,151 of the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 31 and 33 in Table 3 are PCR primer sets for specifically amplifying HLA-DR2 (DR 15) subtype gene of HLA-DRB1 gene as MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

Seq id No. 31 is as defined above.

SEQ ID NO. 33 has a base sequence corresponding to Nos. 32,551,974 to 32,551,999 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 6,100 bases (bp).

In Table 2, SEQ ID Nos. 13 and 14 represent PCR primer sets for specifically amplifying HLA-DR3, HLA-DR5, HLA-DR6 and HLA-DR8 subtype genes as MHC class II beta-chain HLA-DRB1 genes. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID No. 13 has a base sequence complementary to a base sequence corresponding to Nos. 32,552,137 to 32,552,160 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 14 has a base sequence corresponding to Nos. 32,546,609 to 32,546,629 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets is about 5,100 bases (bp).

Sequence numbers 34 and 32 in Table 2 are PCR primer sets for specifically amplifying HLA-DR3 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 34 has a base sequence complementary to the base sequence corresponding to Nos. 32,558,110 to 32,558,133 of the base sequence of the human genome (reference sequence: hg 19).

Seq id No. 32 is as described above.

The predicted length of the PCR product obtained using these primer sets was about 8,900 bases (bp).

Sequence numbers 15 and 16 in Table 2 are PCR primer sets for specifically amplifying HLA-DR4 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 15 has a base sequence complementary to the base sequence corresponding to Nos. 32,552,131 to 32,552,157 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 16 has a base sequence corresponding to Nos. 32,546,609 to 32,546,629 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 6,200 bases (bp).

Sequence numbers 31 and 35 in Table 2 are PCR primer sets for specifically amplifying HLA-DR5 (DR 11) subtype gene of HLA-DRB1 gene as MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

Seq id No. 31 is as defined above.

SEQ ID NO. 35 has a base sequence corresponding to Nos. 32,551,974 to 32,551,999 of the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 31 and 36 in Table 2 are PCR primer sets for specifically amplifying HLA-DR5 (DR 12) subtype gene of HLA-DRB1 gene as MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

Seq id No. 31 is as defined above.

SEQ ID NO. 36 has a base sequence corresponding to Nos. 32,551,974 to 32,551,999 of the base sequence of the human genome (reference sequence: hg 19).

[ Table 2]

Sequence numbers 31 and 37 in Table 3 are PCR primer sets for specifically amplifying HLA-DR6 (DR 14) subtype gene of HLA-DRB1 gene as MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

Seq id No. 31 is as defined above.

SEQ ID NO. 37 has a base sequence corresponding to Nos. 32,551,974 to 32,551,999 of the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 17 and 18 in Table 3 are PCR primer sets for specifically amplifying HLA-DR7 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 17 has a base sequence complementary to the base sequence corresponding to Nos. 32,552,137 to 32,552,160 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 18 has a base sequence corresponding to Nos. 32,546,606 to 32,546,629 of the base sequence of the human genome (reference sequence: hg 19).

In Table 3, SEQ ID Nos. 38 and 36 are PCR primer sets for specifically amplifying HLA-DR7 and HLA-DR9 subtype genes of HLA-DRB1 gene which is an MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 38 has a complementary base sequence corresponding to Nos. 32,558,110 to 32,558,133 of the base sequence of the human genome (reference sequence: hg 19).

Seq id No. 36 is as described above.

The predicted length of the PCR product obtained using these primer sets was about 11,400 bases (bp).

Sequence numbers 31 and 39 in Table 3 are PCR primer sets for specifically amplifying HLA-DR8 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

Seq id No. 31 is as defined above.

SEQ ID NO. 39 has a base sequence corresponding to Nos. 32,551,974 to 32,551,999 of the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 19 and 20 in Table 3 are PCR primer sets for specifically amplifying HLA-DR9 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 19 has a base sequence complementary to the base sequence corresponding to Nos. 32,552,137 to 32,552,160 of the base sequence of the human genome (reference sequence: hg 19).

The sequence No. 20 had a base sequence corresponding to the base sequences No. 32,546,609 to No. 32,546,629 in the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 21 and 22 in Table 3 are PCR primer sets for specifically amplifying HLA-DR10 subtype gene as HLA-DRB1 gene of MHC class II beta chain. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DRB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID No. 21 has a base sequence complementary to a base sequence corresponding to Nos. 32,552,137 to 32,552,159 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 22 has a base sequence corresponding to Nos. 32,546,403 to 32,546,435 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 5,400 bases (bp).

[ Table 3]

In Table 4, SEQ ID Nos. 23 and 24 are PCR primer sets for specifically amplifying the HLA-DPA1 gene as the MHC class II a chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including a promoter, an exon, and an intron) of HLA-DPA1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID No. 23 has a base sequence complementary to the base sequence corresponding to Nos. 33,041,478 to 33,041,502 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 24 has a base sequence corresponding to Nos. 33,031,888 to 33,031,911 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 9,600 bases (bp).

In Table 4, SEQ ID Nos. 40 and 41 are PCR primer sets for specifically amplifying the HLA-DPA1 gene as the MHC class II a chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including a promoter, an exon, and an intron) of HLA-DPA1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 40 has a base sequence complementary to the base sequence corresponding to Nos. 33,041,573 to 33,041,596 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 41 has a base sequence corresponding to Nos. 33,031,888 to 33,031,912 of the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 25 and 26 in Table 4 are PCR primer sets for specifically amplifying the HLA-DPB1 gene as MHC class II beta strands. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including promoter, exon and intron) of HLA-DPB1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 25 has a base sequence corresponding to Nos. 33,043,056 to 33,043,079 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 26 has a base sequence complementary to a base sequence corresponding to Nos. 33,055,476 to 33,055,499 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 12,400 bases (bp).

Sequence numbers 42 and 43 in Table 4 are PCR primer sets for specifically amplifying the HLA-DPB1 gene as MHC class II beta strands. These primer sets are nucleotide sequences present at positions sandwiching the 5' untranslated region to exon 2 of HLA-DPB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 42 has a base sequence corresponding to Nos. 33,043,168 to 33,043,191 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 43 has a base sequence complementary to the base sequence corresponding to Nos. 33,049,084 to 33,049,107 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets is about 5,900 bases (bp).

Sequence numbers 44 and 45 in Table 4 are PCR primer sets for specifically amplifying the HLA-DPB1 gene as MHC class II beta strands. These primer sets are nucleotide sequences present at positions sandwiching the exon 2 to 3' untranslated region of HLA-DPB1 gene in the human genome nucleotide sequence (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 44 has a base sequence corresponding to Nos. 33,048,182 to 33,048,207 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID No. 45 has a base sequence complementary to a base sequence corresponding to Nos. 33,055,428 to 33,055,453 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 7,200 bases (bp).

Sequence numbers 27 and 28 in table 4 are PCR primer sets for specifically amplifying HLA-DQA1 gene as MHC class II α chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including the promoter, exon and intron) of HLA-DQA1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 27 has a base sequence corresponding to Nos. 32,604,318 to 32,604,338 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 28 has a base sequence complementary to the base sequence corresponding to Nos. 32,611,681 to 32,611,701 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 7,400 bases (bp).

Sequence numbers 46 and 47 in table 4 are PCR primer sets for specifically amplifying HLA-DQA1 gene as MHC class II α chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including the promoter, exon and intron) of HLA-DQA1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 46 has a base sequence corresponding to Nos. 32,604,469 to 32,604,488 of the base sequence of the human genome (reference sequence: hg 19).

SEQ ID NO. 47 has a base sequence complementary to the base sequence corresponding to Nos. 32,611,936 to 32,611,956 of the base sequence of the human genome (reference sequence: hg 19).

Sequence numbers 29 and 30 in table 4 are PCR primer sets for specifically amplifying HLA-DQB1 gene as MHC class II β chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including the promoter, exon and intron) of HLA-DQB1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID NO. 29 has a base sequence corresponding to Nos. 32,626,545 to 32,626,568 of the base sequence of the human genome (reference sequence: hg 19).

The sequence No. 30 has a base sequence complementary to a base sequence corresponding to No. 32,635,612 to No. 32,635,637 of the base sequence of the human genome (reference sequence: hg 19).

The predicted length of the PCR product obtained using these primer sets was about 9,100 bases (bp).

In Table 4, SEQ ID Nos. 29, 30 and 48 to 50 are PCR primer sets for specifically amplifying the HLA-DQB1 gene as the MHC class II β chain. These primer sets are nucleotide sequences present at positions sandwiching the entire region (including the promoter, exon and intron) of HLA-DQB1 gene in the nucleotide sequence of human genome (reference sequence: hg 19) from the upstream side and the downstream side.

SEQ ID Nos. 29 and 48 have base sequences corresponding to the base sequences No. 32,626,545 to No. 32,626,568 in the base sequence of the human genome (reference sequence: hg 19).

SEQ ID Nos. 30, 49 and 50 have complementary nucleotide sequences corresponding to nucleotide sequences 32,635,612 to 32,635,637 in the human genome (reference sequence: hg 19).

[ Table 4]

These primers can be prepared by methods commonly used in the art. The primer sets shown in tables 1 and 2 are the most preferable examples, and any primer set can be used in the method of the present invention as long as it is a set of a forward primer and a reverse primer that can anneal at positions sandwiching the entire region of each HLA gene from the upstream side and the downstream side.

(2) PCR amplification procedure

In the method of the present invention, the test sample (DNA) is subjected to PCR amplification using the primer set prepared in the aforementioned step (1).

The PCR amplification reaction is carried out according to a usual protocol. The details are as follows.

1. Depending on the form of the sample to be tested, DNA is extracted from the sample.

2. The extracted DNA was quantified, and a reaction solution was prepared by setting an appropriate primer concentration.

3. Setting reaction conditions to carry out PCR reaction.

For example: thermal denaturation step (usually 92 to 97 ℃ C.)

Annealing step (typically 55 to 72 deg.C)

Extension step (typically 65 to 80 ℃)

In the method of the present invention, in the case of an HLA gene other than HLA-DRB1, the temperature in the annealing step is preferably set to about 60 ℃. Due to annealing at about 60 ℃, alleles can be generated at equal rates (uniformly). In addition, the temperature in the annealing step is preferably set to about 70 ℃ for HLA-DRB 1. Due to annealing at about 70 ℃, only the DR subtype of interest can be specifically produced.

4. The resulting PCR product was purified and subjected to the subsequent base sequence determination step.

(3) A base sequence determination step.

Subsequently, the nucleotide sequence of the PCR product (amplified DNA) produced in the aforementioned step (2) is determined. This step is preferably performed using a method known as next generation sequencing (or superspeed sequencing). For next generation sequencing, reference is made to, for example, ExperimentalMedicine, vol. 27, number 1, 2009 (Yodo-sha), and the like.

In this specification, a method of sequencing based on pyrophosphate sequencing by the FLX system of genome sequence analyzer using Roche is described below.

1. The PCR product obtained in the step (2) is fragmented into about 500 bases by a nebulizer.

2. DNA adapters are attached to the ends of the fragmented DNA fragments.

3. After the DNA fragments to which the DNA adapters are added are made into single-stranded DNA fragments, they are bound to beads via the adapters, and the resulting beads are embedded in a water-in-oil emulsion (forming a microreactor environment in which 1 bead binds to 1 DNA fragment).

4. Emulsion PCR reactions were performed to form copies of each DNA fragment on each bead (since each DNA fragment was clonally amplified in each microreactor, amplification of many fragments could be performed simultaneously and in parallel without competing with other sequences). Subsequently, the emulsion is broken and the beads with amplified DNA fragments are recovered.

5. The beads were concentrated and loaded into a picotiter plate (1 well size to fit 1 bead).

6. The base sequence of DNA was determined from the intensity and pattern of luminescence emitted by detecting pyrophosphate generated by the polymerase upon the enzymatic reaction by the luciferase fluorescence reaction on each bead. 4 kinds of nucleic acids are added in this order (A, C, G, T), the chemiluminescence patterns corresponding to the added nucleic acids are recorded, and the base sequences are determined based on the combination of the signal intensities and positional information.

(4) DNA typing step

Then, the base sequence obtained in the aforementioned step (3) is compared with the data of the base sequence database of known HLA alleles, thereby determining the allele type (up to 8 digits) of the DNA contained in the test sample.

The method of the present invention is exemplified by the primer sets shown in Table 1. It is characterized in that primers are set at positions of all regions of each gene sandwiching HLA class I and HLA class II except HLA-DRB1 and exon 2 to 3' untranslated region of HLA-DRB1, and the sequence of DNA amplified across almost all regions is determined, thereby eliminating phase ambiguity (uncertainty) and enabling information on null alleles to be obtained.

Examples

The present invention will be described in more detail below with reference to specific examples, but the present invention is not limited to these examples.

(example 1)

[ Experimental method ]

1. Using the extracted genomic DNA as a template, PCR reactions were carried out using respective HLA class I gene-specific primer sets (refer to Table 1: SEQ ID NOS: 1 to 8). The specific sequence is as follows.

(1) PCR amplification was performed using Prime STAR GXL polymerase (TaKaRa). Specifically, to 50 ng of the genomic DNA solution, 4. mu.L of 5 XPrimeSTAR GXL buffer solution, 1.6. mu.L of dNTP solution, 4. mu.L (1 pmol/. mu.L) of each PCR primer, and 0.8. mu.L of Prime STAR GXL polymerase were added, and the total amount of the reaction solution was adjusted to 20. mu.L with sterile water.

(2) After 2 minutes of incubation at 94 degrees, 3 steps of 98 degrees 10 seconds, 60 degrees 20 seconds, 68 degrees 5 minutes were subsequently repeated 30 times as 1 sequence. In addition, GeneAmp PCR System 9700 (applied biosystems) was used for the PCR amplification. After the PCR, the amplification of the PCR product was confirmed by agarose gel electrophoresis. The electrophoretogram is shown in FIG. 4.

2. The base sequence of the PCR product was determined. Specifically, this is performed as follows.

(1) The PCR product was purified according to the standard protocol of QIAquick PCR Purification Kit (QIAGEN).

(2) Concentrations were determined according to the standard protocol of PicoGreen dsDNA quantification Kit (Invitrogen).

(3) The purified PCR products were adjusted to a concentration of 500 ng/100. mu.L, and preparation, emulsion PCR, and sequencing of a rapid library (rapid library) were performed according to the standard protocol of Genome Sequence (GS) Junior (Roche) to obtain 1 sample of 1 ten thousand read-out (reads) base sequences.

(4) These sequences were combined and edited by GS de novo Assembler (Roche), followed by identity search with known base sequences on DNA databases to identify alleles of HLA genes.

[ discussion ]

In HLA-A, HLA-B and HLA-C, PCR primers were designed to specifically amplify 5.5kb, 4.6kb and 4.8kb, respectively. According to the study of PCR conditions and agarose gel electrophoresis of these PCR products, PCR products were obtained for each HLA class I gene, and a single amplification product was obtained at the desired molecular weight position (FIG. 4). In addition, since the nucleotide sequence of the PCR product was determined by Sanger method, HLA alleles that are not inconsistent with the known nucleotide sequence were obtained, it was confirmed that the present PCR system can be used for HLA typing.

HLA-B40: 02 homozygotes and 17 samples of HLA-B40: 02 heterozygote, HLA-typing the PCR product from HLA-B gene by GS Junior, resulting in detection of HLA-B40: 02: 01: 01, in a heterozygote of 17 samples, 2 new alleles were detected in addition to 15 known alleles. In particular, 1 sample of combinations of alleles where phase ambiguity was observed (B × 40 and B × 55) could be typed as HLA-B × 40: 02: 01: 01 and HLA-B55: 02: 01: 01. the method of the present invention is therefore capable of HLA typing at the 8-digit level without phase ambiguity, and is a good means for efficiently detecting base substitutions, insertions and deletions in promoters and introns which are responsible for null alleles.

(example 2)

[ Experimental method ]

1. PCR was carried out using the extracted genomic DNA as a template, and HLA class I and HLA class II gene-specific primer sets (refer to tables 1 to 4: SEQ ID Nos. 1 to 8,9 to 22, and 31 to 50) were used. The specific sequence is as follows.

(1) PCR amplification was performed using Prime STAR GXL polymerase (TaKaRa). That is, to 50 ng of the genomic DNA solution, 4. mu.L of 5 XPrimeSTAR GXL buffer, 1.6. mu.L of dNTP solution, 1 to 7. mu.L (4 pmol/. mu.L) of PCR primers, and 0.8. mu.L of Prime STAR GXL polymerase were added, and the total amount of the reaction solution was adjusted to 20. mu.L with sterilized water.

(2) After 2 minutes at 94 degrees, the 2 steps of 98 degrees for 10 seconds and 70 degrees for 5 minutes were then repeated 30 times as 1 sequence. In addition, GeneAmp PCR System 9700 (Applied Biosystems) was used for the PCR amplification. After the PCR, the amplification of the PCR product was confirmed by agarose gel electrophoresis. The electrophoretogram is shown in FIG. 6.

(3) The purified PCR product was adjusted to a concentration of 100 ng, and preparation of a fragment library, emulsion PCR, sequencing were performed according to the standard protocol of Ion Personal Genome Machine (Ion PGM) (Life technologies) to obtain 30 ten thousand read base sequences for 1 sample.

[ results and discussion ]

1. PCR primers specifically amplifying 4kb to 12kb of each of them were designed in the 5' untranslated region to exon 2 of HLA-A, HLA-B, HLA-C, HLA-DRB1, in the exon 2 to 3' untranslated region of HLA-DRB1, in the 5' untranslated region to exon 2 of HLA-DQB1, in the 5' untranslated region to exon 1 of HLA-DPB1, and in the exon 2 to 3' untranslated region of HLA-DPB 1. According to the study of PCR conditions and agarose gel electrophoresis of these PCR products, PCR products were obtained for each of HLA class I and HLA class II genes, and a single amplification product was obtained at the desired molecular weight position (FIG. 6). In addition, since the nucleotide sequence of the PCR product was determined by Sanger method, HLA alleles not inconsistent with the known one were obtained, and it was confirmed again that the present PCR system can be used for HLA typing.

2. The PCR products of each gene from the 5 'untranslated region to exon 2 of HLA-A, HLA-B, HLA-C, HLA-DRB1, exon 2 to 3' untranslated region of HLA-DRB1, HLA-DQB1, 5 'untranslated region to exon 2 of HLA-DPB1, and exon 2 to 3' untranslated region of HLA-DPB1 were HLA-typed by Ion PGM using 4 samples containing combinations of alleles with phase ambiguity observed in the conventional DNA typing method, and as a result, typing of the entire gene region was possible for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB 1. For HLA-DPB1, typing of only portions of exons can be performed. Furthermore, novel alleles were detected for HLA-B, HLA-C, HLA-DRB1 and HLA-DQB 1. The method of the present invention is therefore capable of HLA typing at the 8-digit level without phase ambiguity, and is a good means for efficiently detecting base substitutions, insertions and deletions in promoters and introns which are responsible for null alleles.

(example 3)

[ Experimental method ]

1. Genomic DNA was extracted using Buccal Cell DNA Extraction Kit, Buccal quick (TRIMGEN).

2. Genomic DNA extracted using Buccal Cell DNA Extraction Kit, Buccal quick (TRIMGEN) was further purified by isopropanol and ethanol.

3. Genomic DNA was extracted using QIAamp DNA Blood Mini Kit (QIAGEN).

4. PCR was carried out using 3 samples each of the genomic DNAs extracted in items 1 to 3 by the same experimental method as in example 1 and example 2 using HLA-A, HLA-B, HLA-C, HLA-DQB 1-specific primer sets (refer to tables 1 and 4: SEQ ID Nos. 1 to 8, 29, 30, 48 to 50). After the PCR, the amplification of the PCR product was confirmed by agarose gel electrophoresis. The electrophoretogram is shown in FIG. 7.

[ Experimental results and discussion ]

Lanes 1 to 3 of FIG. 7 show the amplification of the PCR product in the case of extraction by Experimental method 1, lanes 4 to 6 show the amplification of the PCR product in the case of extraction by Experimental method 2, and lanes 7 to 9 show the amplification of the PCR product in the case of extraction by Experimental method 3. For any gene, PCR amplification using the genomic DNA extracted by Experimental method 1 as a template yielded the objective PCR product equivalent to the genomic DNA extracted by Experimental method 3. Since blood collection was carried out in experiment 3, but cells were collected from the oral mucosa in experiment 1, it was confirmed that sufficient HLA typing could be carried out even when blood collection was impossible by applying the method of the present invention.

Sequence listing

<110>TOKAI University Educational System

<120>Method and Kit for DNA typing of HLA genes

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<150>JP 2011-159832

<151>2011-07-21

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AACTC AGAGC TAAGG AATGA TGGCA AAT 28

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AACTC AGAGC TATGG AATGA TGGTA AAT 28

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ATATA ACCAT CATCG TGTCC CAAGG TTC 28

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CCCGG TTGCA ATAGA CAGTA ACAAA 25

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GGGTC CAATT TCACA GACAA ATGT 24

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TGCTT AGATG TGCAT AGTTC ACGAA 25

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TGCTT AGATG TGCAT AGTTC CGGAA 25

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TGGAC CCAAT TTTAC AAACA AATA 24

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GCACG TTTCT TGTGG CAGCT TAAGT T 26

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GCACG TTTCT TGTGG CAGCT AAAGT T 26

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ATGCA CGGGA GGCCA TACGG T 21

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TTTCC TGTGG CAGCC TAAGA GG 22

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CACAG CACGT TTCTT GGAGT ACTC 24

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ATGCA CAGGA GGCCA TAGGG T 21

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AGCAC GTTTC TTGGA GCAGG TTAAA CA 27

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ATGCA TGGGA GGCAG GAAGC A 21

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CACAG CACGT TTCCT GTGGC AGGG 24

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CAGAT GCATG GGAGG CAGGA AGCG 24

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CACAG CACGT TTCTT GAAGC AGGA 24

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ATGCA TGGGA GGCAG GAAGC G 21

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ACAGC ACGTT TCTTG GAGGA GGT 23

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TGGAA TGTCT AAAGC AAGCT ATTTA ACATA TGT 33

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TGATT TCTCT GATAG GTGAA TCCCA 25

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TTGGC CTCTT GGCTA TACCT CTTT 24

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ATTGA AGACA AGGAA TCGAA GTCC 24

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TCCCC CGATG GAAGA TATTA TTTG 24

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GCAAA GGTAT TGCTT GGGCT A 21

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CAGAC TGCGC CTCTA TTCAG G 21

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AAGAA ACAAA CTGCC CCTTA CACC 24

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TAGTA TTGCC CCTAG TCACT GTCAA G 26

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CTGCT GCTCC TTGAG GCATC CACA 24

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CTTCT GGCTG TTCCA GTACT CGGCA T 26

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CTTCT GGCTG TTCCA GTACT CAGCG T 26

<210>34

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CTGCT GCTCC CTGAG GCATC CACA 24

<210>35

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CTTCT GGCTG TTCCA GTACT CCTCA T 26

<210>36

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CTTCT GGCTG TTCCA GGACT CGGCG A 26

<210>37

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CTTCT GGCTG TTCCA GTGCT CCGCA G 26

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CTGCT ACTCC TTGAG GCATC CACA 24

<210>39

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CTTCT GGCTG TTCCA GTACT CGGCG C 26

<210>40

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CTCTC TTGAC CACGC TGGTA CCTA 24

<210>41

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TTGGC CTCTT GGCTA TACCT CTTTT 25

<210>42

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CCTCC TGACC CTGAT GACAG TCCT 24

<210>43

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CCATC TGCCC CTCAA GCACC TCAA 24

<210>44

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CTCAG TGCTC GCCCC TCCCT AGTGA T 26

<210>45

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GCACA GTAGC TTTCG GGAAT TGACC A 26

<210>46

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GCCAG GGAGG GAAAT CAACT 20

<210>47

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<400>47

ATCCA GTGGA GGACA CAGCA C 21

<210>48

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<400>48

AAGAA ACAAA CTGCC CCTTA TACC 24

<210>49

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TAGTA CTGCC CCTAG TCACT GCCAA G 26

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TAGTA CTGTC CCTAG TCACT GCCAA G 26

Claims

A method for typing DNA of HLA, characterized by comprising the steps of:

(1) a step of preparing a primer set for annealing specifically to the upstream region and the downstream region of each of HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 genes in the base sequence of the human genome, respectively, and a primer set for annealing specifically to the untranslated regions on the exon 2 and 3' sides of HLA-DRB 1;

(2) a step of performing PCR amplification on a test sample (DNA) using the primer set;

(3) a step of determining a base sequence of the PCR amplification product; and

(4) optionally, a step of homology search with a database is performed.
2. The method of claim 1, wherein the gene is an HLA-a gene and the primer set is selected from the group consisting of seq id nos: 1. 2 and 3, or a pharmaceutically acceptable salt thereof.
3. The method of claim 1, wherein the gene is an HLA-B gene and the primer set is selected from the group consisting of seq id nos: 4 and 5, respectively.
4. The method of claim 1, wherein the gene is an HLA-C gene and the primer set is selected from the group consisting of seq id nos: 6. 7 and 8.
5. The method of claim 1, wherein the gene is the DR1 type of HLA-DRB1 gene, and the primer set is selected from the group consisting of primers having the sequence numbers: 9. 10, 11, 31 and 32.
6. The method of claim 1, wherein the gene is the DR2 type of HLA-DRB1 gene, and the primer set is selected from the group consisting of primers having the sequence numbers: 11. 12, 31 and 33.
7. The method of claim 1, wherein the gene is the DR3 type of HLA-DRB1 gene, and the primer set is selected from the group consisting of primers having the sequence numbers: 13. 14, 32 and 34.
8. The method of claim 1, wherein the gene is the DR4 type of HLA-DRB1 gene, and the primer set is selected from the group consisting of primers having the sequence numbers: 15. 16, 31 and 32.
9. The method of claim 1, wherein the gene is the DR5 type of HLA-DRB1 gene, and the primer set is selected from the group consisting of primers having the sequence numbers: 13. 14, 31, 35 and 36.
10. The method of claim 1, wherein the gene is the DR6 type of HLA-DRB1 gene, and the primer set is selected from the group consisting of primers having the sequence numbers: 13. 14, 31,32 and 37.