US20140206005A1 - Method and Kit for DNA Typing of HLA Gene - Google Patents

Method and Kit for DNA Typing of HLA Gene Download PDF

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US20140206005A1
US20140206005A1 US14/233,909 US201214233909A US2014206005A1 US 20140206005 A1 US20140206005 A1 US 20140206005A1 US 201214233909 A US201214233909 A US 201214233909A US 2014206005 A1 US2014206005 A1 US 2014206005A1
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hla
gene
seq
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nucleotide sequences
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Takashi Shiina
Shingo Suzuki
Yuki Ozaki
Shigeki Mitsunaga
Hidetoshi Inoko
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GENODIVE PHARMA Inc
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method and a kit for DNA typing of a HLA gene using a massive parallel sequencer.
  • HLA human leucocyte antigen
  • MHC human histocompatibility complex
  • HLA-A, HLA-B, HLA-C class I molecules
  • HLA-DR, HLA-DQ, HLA-DP class II molecules
  • the HLA class I antigen consists of a highly polymorphic ⁇ chain and a substantially non-polymorphic ⁇ 2-microglobulin; whereas the HLA class II antigen consists of a highly polymorphic ⁇ chain and a less polymorphic ⁇ chain.
  • the ⁇ chains of class I molecules are encoded by HLA-A, HLA-B and HLA-C genes.
  • the ⁇ chains of class II antigens are encoded by HLA-DRB1, HLA-DQB1 and HLA-DPB1 genes, whereas the ⁇ chains are encoded by HLA-DRA1, HLA-DQA1 and HLA-DPA1 genes.
  • exon 2 and exon 3 of a gene encoding an ⁇ chain are highly polymorphic; whereas, in HLA class II antigens, exon 2 of a gene encoding a ⁇ chain is highly polymorphic.
  • a gene region encoding a HLA is located on 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 toward the centromere side.
  • Many genes are encoded at an extremely high density and association of these genes with transfusion, transplantation and various diseases have been reported.
  • In the class III region no HLA genes are present and genes of complement components and tumor necrosis factors (TNF), etc. are present.
  • HLA-DRB gene region encoding a ⁇ chain of a HLA-DR antigen
  • 5 types of structural polymorphisms are present.
  • pseudogenes such as HLA-DRB6 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome.
  • DR2 type a HLA-DRB5 (DR51) gene and pseudogenes such as HLA-DRB6 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome.
  • a HLA-DRB3 (DR52) gene and pseudogenes such as HLA-DRB2 and HLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome.
  • a 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.
  • no HLA-DRB genes except HLA-DRB1 are located on the same chromosome.
  • each allele In the exon of each allele, a plurality of regions exhibiting polymorphism are present. In many cases, a nucleotide sequence (amino acid sequence) present in a certain 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 a 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.
  • a first field (two-digit level) is for discrimination of serologic HLA types
  • a second field (4-digit level) is for discrimination of alleles having an amino acid substitution in the same serologic HLA type
  • a third field (6-digit level) is for discrimination of alleles having a base substitution not accompanying an amino acid mutation
  • a fourth field (8-digit level) is for discrimination of alleles having a base substitution in an intron, which is out of the genetic region encoding a HLA molecule.
  • a SBT (sequence based typing) method and a SSO (Sequence Specific Oligonucleotide)-Luminex method based on a polymerase chain reaction (PCR) are in mainstream.
  • An object of the present invention is to provide a method and a kit for highly precise DNA typing in which ambiguity derived from phase ambiguity is eliminated.
  • the present inventors newly conceived an idea of newly designing a PCR primer capable of specifically amplifying genes of HLAs such as HLA class I molecules including HLA-A, HLA-B and HLA-C and HLA class II molecules including HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1, setting suitable PCR conditions and applying a massive parallel sequencing technique.
  • HLAs such as HLA class I molecules including HLA-A, HLA-B and HLA-C and HLA class II molecules including HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1
  • the present invention provides a method for DNA typing of HLA, including the following steps:
  • the method of the present invention since it provides all nucleotide sequences required for DNA typing of a HLA gene from a single molecule, is an ultimate DNA typing method in which phase ambiguity due to unclear cis/trans positional relationship is eliminated. Owing to this, highly precise matching of HLAs between a patient seeking to receive a transplant and a donor candidate upon transplantation is realized.
  • nucleotide sequences of a 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 at all or suppressed in expression, and a novel allele can be detected.
  • FIG. 1( a ) A diagram showing the relationship between the structure of a HLA class I gene and the structure of HLA class I molecule; and ( b ) A diagram showing the structure of a promoter region of a HLA class I gene, cited from “Transplantation/transfusion Examination”, supervised by Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juuji, Kodan-sha Scientific, 2004, page 35.
  • FIG. 2( a ) A diagram showing the relationship between the structure of a HLA class II gene and the structure of HLA class II molecule; and ( b ) A diagram showing the structure of a promoter region of a 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.
  • FIG. 3 A diagram showing a HLA-DR gene region, cited from “Transplantation/transfusion Examination”, supervised by Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juuji, Kodan-sha Scientific, 2004, page 48.
  • FIG. 4 An agarose gel electrophoretic pattern showing amplification states of PCR products amplified in Example 1.
  • FIG. 5 A diagram schematically showing the structure of a HLA gene and the position to which a PCR primer is designed to bind (SEQ ID No. of the primer designed in the indicated region is indicated within parentheses).
  • FIG. 6 An agarose gel electrophoretic pattern showing the amplification states of amplified PCR products of a HLA gene in Example 2.
  • FIG. 7 An agarose gel electrophoretic pattern of amplified PCR products obtained by three types of DNA extraction methods in Example 3.
  • a set of primers which respectively anneal specifically to an upstream region and a downstream region of each of 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 which respectively anneal specifically to exon 2 and a 3′ untranslated region of HLA-DRB1 are prepared.
  • genes of HLA-A, HLA-B and HLA-C which are called classic HLA class I molecules, each contain 7 or 8 exons ( FIG. 1( a )). Outside of exon 1, two types of enhancers and a promoter region are present to control expression ( FIG. 1( b )).
  • HLA-DR HLA-DR
  • HLA-DQ HLA-DP
  • HLA-DP which are called classic HLA class II molecules, consist of ⁇ chains and ⁇ chains, whose genes each contain 5 to 6 exons ( FIG. 2( a )). Outside of exon 1, a promoter region is present to control expression ( FIG. 2( b )).
  • a set of primers which 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 classic class I molecules (HLA-A, HLA-B, HLA-C) and classic class II molecules (HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1); and a set of primers which can amplify (by PCR) the gene regions of HLA-DRB1 including exon 2 to a 3′ untranslated region are prepared, and PCR products obtained by PCR amplification using the sets of primers are subjected to next-generation sequencing (described later). Therefore, uncertainty such as phase ambiguity can be eliminated and the presence or absence of a null allele can be accurately detected.
  • PCR primer sets listed in Table 1 to Table 4 below are prepared.
  • SEQ ID Nos. 1 to 3 represent a set of PCR primers specifically amplifying a HLA-A gene, which is an a chain of MHC class I. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-A gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 1 has a nucleotide sequence corresponding to the 29,909,487th position to the 29,909,514th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 2 has a nucleotide sequence corresponding to the 29,909,487th position to the 29,909,514th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 3 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 29,914,925th position to the 29,914,952nd position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,500 bases (bp).
  • SEQ ID Nos. 4 and 5 represent a set of PCR primers specifically amplifying a HLA-B gene, which is an ⁇ chain of MHC class I. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-B gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 4 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 31,325,796th position to the 31,325,820th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 5 has a nucleotide sequence corresponding to the 31,321,212nd position to the 31,321,235th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 4,600 bases (bp).
  • SEQ ID Nos. 6 to 8 represent a set of PCR primers specifically amplifying a HLA-C gene, which is an ⁇ chain of MHC class I. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-C gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 6 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 31,240,868th position to the 31,240,892nd position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 7 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 31,240,868th position to the 31,240,892nd position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 8 has a nucleotide sequence corresponding to the 31,236,991st position to the 31,236,114th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 4,800 bases (bp).
  • SEQ ID Nos. 9 to 11 represent a set of PCR primers of specifically amplifying a HLA-DR1 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 9 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,131st position to the 32,552,156th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 10 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,131st position to the 32,552,156th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 11 has a nucleotide sequence corresponding to the 32,546,609th position to the 32,546,629th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,200 bases (bp).
  • SEQ ID Nos. 31 and 32 represent a set of PCR primers of specifically amplifying HLA-DR1, HLA-DR4, HLA-DR6 (DR13) and a HLA-DR10 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 31 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,558,110th position to the 32,558,133rd position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 32 has a nucleotide sequence corresponding to the 32,551,974th position to the 32,551,999th position in a human genome sequence (Reference sequence: hg19).
  • the lengths of PCR products obtained by using these primer sets are estimated as about 6,100 bases (bp) in the case of a HLA-DR1 subtype, about 9,100 bases (bp) in the case of a HLA-DR4 subtype, about 8,900 bases (bp) in the case of a HLA-DR6 (DR13) subtype and about 8,900 bases (bp) in the case of a HLA-DR10 subtype.
  • SEQ ID Nos. 11 and 12 represent a set of PCR primers of specifically amplifying a HLA-DR2 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 11 is as defined above.
  • SEQ ID No. 12 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,130th position to the 32,552,151st position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,500 bases (bp).
  • SEQ ID Nos. 31 and 33 represent a set of PCR primers of specifically amplifying a HLA-DR2 (DR15) subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 31 is as defined above.
  • SEQ ID No. 33 has a nucleotide sequence corresponding to the 32,551,974th position to the 32,551,999th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 6,100 bases (bp).
  • SEQ ID Nos. 13 and 14 represent a set of PCR primers of specifically amplifying a HLA-DR3, HLA-DR5, HLA-DR6 and HLA-DR8 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 13 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,137th position to the 32,552,160th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 14 has a nucleotide sequence corresponding to the 32,546,609th position to the 32,546,629th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,100 bases (bp).
  • SEQ ID Nos. 34 and 32 represent a set of PCR primers of specifically amplifying a HLA-DR3 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 34 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,558,110th position to the 32,558,133rd position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 32 is as defined above.
  • the length of a PCR product obtained by using these primer sets is estimated as about 8,900 bases (bp).
  • SEQ ID Nos. 15 and 16 represent a set of PCR primers of specifically amplifying a HLA-DR4 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 15 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,131st position to the 32,552,157th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 16 has a nucleotide sequence corresponding to the 32,546,609th position to the 32,546,629th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 6,200 bases (bp).
  • SEQ ID Nos. 31 and 35 represent a set of PCR primers of specifically amplifying a HLA-DR5 (DR11) subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 31 is as defined above.
  • SEQ ID No. 35 has a nucleotide sequence corresponding to the 32,551,974th position to the 32,551,999th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 8,900 bases (bp).
  • SEQ ID Nos. 31 and 36 represent a set of PCR primers of specifically amplifying a HLA-DR5 (DR12) subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 31 is as defined above.
  • SEQ ID No. 36 has a nucleotide sequence corresponding to the 32,551,974th position to the 32,551,999th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 8,900 bases (bp).
  • SEQ ID Nos. 31 and 37 represent a set of PCR primers of specifically amplifying a HLA-DR6 (DR14) subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 31 is as defined above.
  • SEQ ID No. 37 has a nucleotide sequence corresponding to the 32,551,974th position to the 32,551,999th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 8,900 bases (bp).
  • SEQ ID Nos. 17 and 18 represent a set of PCR primers of specifically amplifying a HLA-DR7 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 17 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,137th position to the 32,552,160th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 18 has a nucleotide sequence corresponding to the 32,546,606th position to the 32,546,629th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,100 bases (bp).
  • SEQ ID Nos. 38 and 36 represent a set of PCR primers of specifically amplifying a HLA-DR7 and HLA-DR9 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II.
  • These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 38 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,558,110th position to the 32,558,133rd position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 36 is as defined above.
  • the length of a PCR product obtained by using these primer sets is estimated as about 11,400 bases (bp).
  • SEQ ID Nos. 31 and 39 represent a set of PCR primers of specifically amplifying a HLA-DR8 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DRB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 31 is as defined above.
  • SEQ ID No. 39 has a nucleotide sequence corresponding to the 32,551,974th position to the 32,551,999th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 8,900 bases (bp).
  • SEQ ID Nos. 19 and 20 represent a set of PCR primers of specifically amplifying a HLA-DR9 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 19 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,137th position to the 32,552,160th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 20 has a nucleotide sequence corresponding to the 32,546,609th position to the 32,546,629th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,100 bases (bp).
  • SEQ ID Nos. 21 and 22 represent a set of PCR primers of specifically amplifying a HLA-DR10 subtype gene of a HLA-DRB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DRB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 21 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,552,137th position to the 32,552,159th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 22 has a nucleotide sequence corresponding to the 32,546,403rd position to the 32,546,435th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,400 bases (bp).
  • DRB_PE2-R2 26 CTTCTGGCTGTTCCAGG 36 ACTCGGCGA HLA-DR10 DR-E2-10-F 23 ACAGCACGTTTCTTGG 21 5,354 AGGAGGT DR-E2-10-R 33 TGGAATGTCTAAAGCA 22 AGCTATTTAACATATGT HLA-DR10 DRB_PE2-F1 24 CTGCTGCTCCTTGAGGC 31 8,888 ATCCACA DRB_PE2-R1 26 CTTCTGGCTGTTCCAGT 32 ACTCGGCAT
  • SEQ ID Nos. 23 and 24 represent a set of PCR primers specifically amplifying a HLA-DPA1 gene, which is an ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DPA1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 23 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 33,041,478th position to the 33,041,502nd position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 24 has a nucleotide sequence corresponding to the 33,031,888th position to the 33,031,911st position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 9,600 bases (bp).
  • SEQ ID Nos. 40 and 41 represent a set of PCR primers specifically amplifying a HLA-DPA1 gene, which is an ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DPA1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 40 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 33,041,573rd position to the 33,041,596th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 41 has a nucleotide sequence corresponding to the 33,031,888th position to the 33,031,912nd position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 9,600 bases (bp).
  • SEQ ID Nos. 25 and 26 represent a set of PCR primers specifically amplifying a HLA-DPB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DPB1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 25 has a nucleotide sequence corresponding to the 33,043,056th position to the 33,043,079th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 26 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 33,055,476th position to the 33,055,499th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 12,400 bases (bp).
  • SEQ ID Nos. 42 and 43 represent a set of PCR primers of specifically amplifying a HLA-DPB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of a 5′ untranslated region to exon 2 of a HLA-DPB1 gene and sandwich the 5′ untranslated region to exon 2 in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 42 has a nucleotide sequence corresponding to the 33,043,168th position to the 33,043,191st position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 43 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 33,049,084th position to the 33,049,107th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 5,900 bases (bp).
  • SEQ ID Nos. 44 and 45 represent a set of PCR primers of specifically amplifying a HLA-DPB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of exon 2 to a 3′ untranslated region of a HLA-DPB1 gene and sandwich the exon 2 to a 3′ untranslated region in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 44 has a nucleotide sequence corresponding to the 33,048,182nd position to the 33,048,207th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 45 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 33,055,428th position to the 33,055,453rd position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 7,200 bases (bp).
  • SEQ ID Nos. 27 and 28 represent a set of PCR primers specifically amplifying a HLA-DQA1 gene, which is an ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DQA1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 27 has a nucleotide sequence corresponding to the 32,604,318th position to the 32,604,338th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 28 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,611,681st position to the 32,611,701st position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 7,400 bases (bp).
  • SEQ ID Nos. 46 and 47 represent a set of PCR primers specifically amplifying a HLA-DQA1 gene, which is an ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DQA1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 46 has a nucleotide sequence corresponding to the 32,604,469th position to the 32,604,488th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 47 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,611,936th position to the 32,611,956th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 7,400 bases (bp).
  • SEQ ID Nos. 29 and 30 represent a set of PCR primers specifically amplifying a HLA-DQB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DQB1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 29 has a nucleotide sequence corresponding to the 32,626,545th position to the 32,626,568th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID No. 30 has a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,635,612nd position to the 32,635,637th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 9,100 bases (bp).
  • SEQ ID Nos. 29, 30 and 48 to 50 represent a set of PCR primers specifically amplifying a HLA-DQB1 gene, which is a ⁇ chain of MHC class II. These primers of the set are nucleotide sequences located at positions, which correspond to the upstream and downstream of all regions of a HLA-DQB1 gene (including promoter, exons and introns), and sandwich the all regions, in the human genome sequence (Reference sequence: hg19).
  • SEQ ID Nos. 29 and 48 have a nucleotide sequence corresponding to the 32,626,545th position to the 32,626,568th position in a human genome sequence (Reference sequence: hg19).
  • SEQ ID Nos. 30, 49 and 50 have a complementary nucleotide sequence to a nucleotide sequence corresponding to the 32,635,612nd position to the 32,635,637th position in a human genome sequence (Reference sequence: hg19).
  • the length of a PCR product obtained by using these primer sets is estimated as about 9,100 bases (bp).
  • 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 can be used as long as the set of primers is a set of a forward primer and a reverse primer capable of annealing to the positions, which correspond to the upstream and downstream of all regions of each HLA gene and sandwich the all regions.
  • a test sample (DNA) is amplified by PCR using the sets of primers prepared in the above step (1).
  • the PCR amplification reaction is performed in accordance with a general protocol and more specifically, as follows.
  • DNA is extracted from a test sample depending upon the form of the sample.
  • the DNA extracted is quantified and the concentrations of primers are appropriately set to prepare the reaction solution.
  • the temperature of the annealing step is preferably set at about 60° C. Owing to the annealing at about 60° C., alleles can be produced at the equivalent ratio (uniformly).
  • the temperature of the annealing step is preferably set at about 70° C. Owing to the annealing at about 70° C., a desired DR subtype alone can be specifically produced.
  • the obtained PCR product is purified and subjected to the following nucleotide sequencing step.
  • next-generation sequencing or ultrahigh sequencing.
  • next-generation sequencing see, for example, “Experimental Medicine”, Vol. 27, No. 1, 2009 (Yodo-sha).
  • the sequence herein is determined by a method based on pyro-sequencing, which is employed in a genome sequencer FLX system of Roche. The sequencing method will be described below.
  • the PCR product obtained in the above step (2) is broken up by a nebulizer into fragments of about 500 bases.
  • a DNA adaptor is attached to an end of each of the DNA fragments.
  • DNA fragments attached with a DNA adaptor are dissociated into single stranded DNA fragments, which are allowed to bind to beads via the adaptor.
  • the obtained beads are encompassed and taken in a water-in-oil emulsion (a micro-reactor environment containing a single DNA fragment bound to a single bead is formed).
  • Emulsion PCR is performed to form copies of each DNA fragment on a bead (Each DNA fragment is clonally amplified in each micro reactor. In this manner, many fragments can be simultaneously and in parallel amplified without competition with other sequences). Subsequently, the emulsion is destroyed and beads having amplified DNA fragments are collected.
  • the beads are concentrated and loaded in a pico-titer plate (a single well has a size enough to place a single bead).
  • the nucleotide sequence obtained in step (3) is compared with data of known HLA alleles within the nucleotide sequencing database. In this manner, the allele type (up to 8 digits) contained in the test sample is determined.
  • primers are designed so as to correspond to all regions of each of the genes of HLA class I and HLA class II except HLA-DRB1 and the positions sandwiching exon 2 to 3′ untranslated region of HLA-DRB1 and the sequence of the DNA amplified so as to correspond to almost all regions is determined. In this manner, phase ambiguity (uncertainty) is eliminated and information on a null allele can be obtained.
  • PCR amplification was performed by use of Prime STAR GXL polymerase (TaKaRa). More specifically, to 50 ng of a genomic DNA solution, 4 ⁇ L of 5 ⁇ PrimeSTAR GXL buffer, 1.6 ⁇ L of a dNTP solution, PCR primers (4 ⁇ L (1 pmol/ ⁇ L) for each) and 0.8 ⁇ L of Prime STAR GXL polymerase were added. The whole amount of the reaction solution was adjusted to be 20 ⁇ L with sterilized water.
  • Prime STAR GXL polymerase TaKaRa
  • 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.
  • Gene Amp PCR System 9700 (Applied Biosystems) was used. After the PCR, the amplification states of PCR products were checked by agarose gel electrophoresis. The electrophoretic patterns were shown in FIG. 4 .
  • the nucleotide sequences of the PCR products were determined specifically as follows.
  • a PCR product was purified by QIAquick PCR Purification Kit (QIAGEN) in accordance with the standard protocol.
  • HLA-A, HLA-B and HLA-C PCR primers, which specifically amplify 5.5 kb, 4.6 kb and 4.8 kb, respectively, were designed. PCR conditions were studied and agarose gel electrophoresis of the resultant PCR products was performed. As a result, it was found that HLA class I genes all provide a single PCR amplified product at a position corresponding to a desired molecular weight ( FIG. 4 ). Furthermore, the nucleotide sequences of the PCR products were determined by the Sanger method. As a result, HLA alleles were obtained in consistent with known documents. From this, it was confirmed that the PCR system of the invention can be used for HLA typing.
  • HLA-B*40:02 homozygote Using three specimens of a HLA-B*40:02 homozygote and 17 specimens of a HLA-B*40:02 heterozygote including combinations of alleles (B*40 and B*55), in which phase ambiguity was observed in a conventional DNA typing method, a PCR was performed. As the result of HLA typing of the PCR products derived from the HLA-B gene by GS Junior, HLA-B*40: 02: 01: 01 was detected from all specimens. In the 17 heterozygote specimens, 2 types of novel alleles were detected in addition to 15 alleles already known.
  • HLA-B*40: 02: 01: 01 and HLA-B*55: 02: 01: 01 were identified by typing. From this, it was demonstrated that the method of the invention enables HLA typing at a 8-digit level without phase ambiguity; and that the method of the invention is an excellent tool for efficiently detecting a substitution, an insertion and a deletion of bases in a promoter and introns, which are causes of a null allele.
  • PCR amplification was performed by use of Prime STAR GXL polymerase (TaKaRa). More specifically, to 50 ng of a genomic DNA solution, 4 ⁇ L of 5 ⁇ PrimeSTAR GXL buffer, 1.6 ⁇ L of a dNTP solution, PCR primers (1 to 7 ⁇ L (4 pmol/ ⁇ L)) and 0.8 ⁇ L of Prime STAR GXL polymerase were added. The whole amount of the reaction solution was adjusted to be 20 ⁇ L with sterilized water.
  • Prime STAR GXL polymerase TaKaRa
  • 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.
  • Gene Amp PCR System 9700 (Applied Biosystems) was used. After the PCR, the amplification states of PCR products were checked by agarose gel electrophoresis. The electrophoretic patterns were shown in FIG. 6 .
  • the nucleotide sequences of the PCR products were determined specifically as follows.
  • a PCR product was purified by QIAquick PCR Purification Kit (QIAGEN) in accordance with the standard protocol.
  • PCR primers which specifically amplify 4 kb to 12 kb in the region from a 5′ untranslated region to exon 2 of HLA-A, HLA-B, HLA-C and HLA-DRB1, the region from exon 2 to a 3′ untranslated region of HLA-DRB1, the region from a 5′ untranslated region to exon 2 of 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 resultant PCR products was performed.
  • HLA class I and HLA class II genes all provide a single amplified product at a position corresponding to a desired molecular weight ( FIG. 6 ). Furthermore, the nucleotide sequences of the PCR products were determined by the Sanger method. As a result, HLA alleles were obtained in consistent with known documents. It was confirmed herein again that the PCR system of the invention can be used for HLA typing.
  • HLA-A As a result, typing of whole gene regions of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 were successfully made. With respect to HLA-DPB1, typing of an exon alone was successfully made. Furthermore, in each of the HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genes, a novel allele was detected. From this, it was demonstrated that the method of the invention enables HLA typing at a 8-digit level without phase ambiguity; and that the method of the invention is an excellent tool for efficiently detecting a substitution, an insertion and a deletion of bases in a promoter and introns, which are causes of a null allele.
  • Genomic DNA was extracted by using Buccal Cell DNA Extraction Kit, BuccalQuick (TRIMGEN).
  • lanes 1 to 3 show the amplification states of PCR products in the case where extraction was made by Experimental method 1
  • lanes 4 to 6 show the amplification states of PCR products in the case where extraction was made by Experimental method 2
  • lanes 7 to 9 show the amplification state of PCR products in the case where extraction was made by Experimental method 3.
  • PCR amplification in the case where genomic DNA extracted by 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.
  • blood must be taken; however in Experimental method 1, cells can be taken from the oral mucous membrane. Therefore, it was demonstrated that if the method of the present invention is employed, HLA typing can be sufficiently performed even if blood cannot be taken.

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WO2022050721A1 (ko) * 2020-09-03 2022-03-10 주식회사 엔젠바이오 Hla 유전자 증폭용 조성물 및 이의 용도

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