WO2005095648A1 - PROCEDE POUR DETERMINER L’ALLELE DU GENE DU COMPLEXE MAJEUR D’HISTOCOMPATIBILITE BOVIN (BoLA) DQA CLASSE II - Google Patents

PROCEDE POUR DETERMINER L’ALLELE DU GENE DU COMPLEXE MAJEUR D’HISTOCOMPATIBILITE BOVIN (BoLA) DQA CLASSE II Download PDF

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WO2005095648A1
WO2005095648A1 PCT/JP2005/006465 JP2005006465W WO2005095648A1 WO 2005095648 A1 WO2005095648 A1 WO 2005095648A1 JP 2005006465 W JP2005006465 W JP 2005006465W WO 2005095648 A1 WO2005095648 A1 WO 2005095648A1
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primer
seq
nucleotide sequence
bola
dqa2
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PCT/JP2005/006465
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English (en)
Japanese (ja)
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Yoko Aida
Shinnosuke Takeshima
Akimi Miki
Mayu Kado
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Riken
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Priority to JP2006511839A priority Critical patent/JP4662929B2/ja
Publication of WO2005095648A1 publication Critical patent/WO2005095648A1/fr

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    • 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
    • C12Q1/6881—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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for determining alleles of BoLA-DQA, and a set of primers and a kit therefor.
  • a major histocompatibity complex (MHC) molecule is a membrane protein that binds to a foreign antigen peptide such as a graft ⁇ bacterium or virus and presents it to T cells.
  • MHC molecules are broadly divided into class I and class II.
  • MHC is called H-2 in mice and HLA in humans, whereas MHC is called BoLA (Bovine Leukocyte Antigen) in mice.
  • the BoLA gene region is located on chromosome 23 and contains class I, class II and class III genes that are highly homologous to humans and mice.
  • the BOLA class II gene differs greatly from that of human and mouse in that it is divided into two subregions, class Ila and class lib, with at least a 15cM region! The DR and DQ gene subregions are mapped to the class lib subregion.
  • class Ila regions include DMA and DMB that promote the binding of MHC to antigen, LMP2 and LMP7 that are involved in antigen processing, transporter gene TAP, and DOA and DOB whose protein expression and function are unknown. DIB, DYA, and DYB have been identified.
  • each gene is located in the order of DRB1-DRB2-DRB3-DRA from the centromere side, and the ⁇ -chain encoded by DRA and the ⁇ -chain encoded by DRB3 A potent heterodimer is expressed as a DR molecule on the cell surface and induces strong V ⁇ antigen presenting ability.
  • DRB3 is the most functional and polymorphic of the BoLA genes, with 104 alleles identified.
  • five DQA and five DQB genes have been identified in the DQ gene region to date.
  • the DQA and DQB genes differ in number and composition depending on the haplotype, and are thought to form one or two DQ molecules and induce antigen presentation ability, albeit weakly (Schneider S., Roessli D., and Excoffier L., A software for population genetics data analysis, rlequin, Genetics and Biometry Laboratory, University of Geneva, Switzerland, 2000; Aida Y.,
  • Bovine MHC Class II Genes Bulletin de la Societe Franco- Japonaise des Sciences Veterinaires, Brother b, p.17-24, 1995; Burke MG, Stone RT, and Muggli- Cockett NE, Nucleotide- sequence and northern analysis of a bovine major histocompatibility class II DR— beta— like cDNA, Animal genetics, Vol. 22, p. 343-352, 1991; Fraser DC, Craigmile S., Campbell JDM, Oliver RA, Brown DJ, Russell GC, Spooner R ⁇ ., And Glass EJ, Functional expression of a cattle MHC class II DR—like antigen on mouse L—cells, Immunogenetics, Vol. 43, pp. 296-303, 1996).
  • BLV Bovine leukemia virus
  • HTLV-1 human T-cell leukemia virus
  • EBL endemic bovine leukemia
  • BLV-infected cattle exhibit three pathologies: unhealthy health, persistent lymphocytosis (PL), and leukemia after a longer incubation period.
  • Investigation of alleles in BLV-infected cattle showed that the BoLA-DRB3 gene may regulate the susceptibility of endemic bovine leukemia (Yoko Ma, Genome analysis and disease resistance [2], agriculture and horticulture) 76, p.1289-1294, 2001).
  • Yoko Ma Genome analysis and disease resistance [2], agriculture and horticulture
  • PCR-SBT PCR-based sequence-based typing
  • an object of the present invention is to provide a method for determining an allele of BoLA-DQA.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found a primer set capable of specifically amplifying or sequencing the second exon of BoLA-DQAl or BoLA-DQA2. After successful design, it was used to specifically amplify the nucleotide sequence of the second exon and sequenced the amplified nucleic acid fragment to identify the BoLA-D QA1 and BoLA-DQA2 alleles. The knowledge that the determination can be made simply and reliably has been completed, and the present invention has been completed.
  • the present invention provides the following (1) to (8).
  • BoLA a method for determining an allele of DQA1
  • amplification can be performed using the following primer set (a) or (b):
  • step (ii) the nucleotide sequence can be determined using the following primer set (c):
  • the above method may further include the step of amplifying a nucleic acid fragment containing the base sequence of the second exon of BoLA-DQA1, using the nucleic acid fragment amplified in step (i) as type III.
  • a genomic DNA derived from a test shell is used as a type II
  • a nucleic acid fragment is amplified using the following set of primers (a)
  • the amplified nucleic acid fragment is used as a type III. It is preferable to amplify a nucleic acid fragment using the following primer set (b):
  • BoLA a method for determining an allele of DQA2
  • step (i) of the above method the following primer sets (d) to (g) Amplification can be performed using the following:
  • step (ii) the base sequence can be determined using the following primer set (e) or (g):
  • the above method may further include a step of amplifying a nucleic acid fragment containing the base sequence of the second exon of BoLA-DQA2, using the nucleic acid fragment amplified in step (i) as type III.
  • a genomic DNA derived from a test shell is used as a type II
  • a nucleic acid fragment is amplified using the following set of primers (d)
  • the amplified nucleic acid fragment is used as a type III. It is preferable to amplify a nucleic acid fragment using the following primer set (e).
  • a step of treating the amplified nucleic acid fragment with exonuclease I and alkaline phosphatase may be further performed before determining the nucleotide sequence of the nucleic acid fragment.
  • a primer set for amplifying a nucleic acid fragment containing the base sequence of the second exon of BoLA-DQAl comprising at least one of the following primer sets (a) and (b):
  • a primer set for determining the base sequence of the second exon of BoLA-DQAl including the following primer set (c):
  • the above-described kit for determining an allele may further include the primer set of (4).
  • a primer set for amplifying a nucleic acid fragment containing the base sequence of the second exon of BoLA-DQA2, comprising at least one of the following primer sets (d) to (g):
  • the kit for determining an allele may further include the primer set of (7).
  • the present invention provides a method for determining the allele of BoLA-DQA. This method is useful for studies on the relationship between MHC and disease susceptibility, since the alleles of BoLA-DQAl and BoLA-DQA2 can be determined simply, quickly and reliably. Brief Description of Drawings
  • FIG. 1 is a diagram showing an outline of a PCR-SBT method of a PHC MHC (BoLA) -DQAl and DQA2 according to the present invention.
  • the arrow indicates the position where the primer binds.
  • FIG. 2 shows the results of sequencing of MHC (BoLA)-second intron of DQAl and DQA2 (A), sequencing of third intron of DQA1 (B), and the second PCR of DQA2 by inverse PCR.
  • FIG. 2 shows the outline of a method for performing intron sequencing (C). Arrows indicate where the primers bind. Hindlll in (C) of FIG. 2 indicates a site considered to be recognized by Hindlll.
  • FIG. 3A is a view showing primers used for PCR-SBT method for determination of BoLA-DQA1 allele.
  • Figure 3B is a diagram showing the primers used for 0 ⁇ trioctahedral 1 1 3 0 ⁇ -38 chome method for allele determination.
  • FIG. 1 A first figure.
  • FIG. 5 is a diagram showing alleles amplified by the PCR-SBT method of BoLA-DQA1.
  • FIG. 6A is a view showing primers used for PCR-SBT method for determining BoLA-DQA2 allele.
  • Figure 6B is a diagram showing the primers used for 0 ⁇ trioctahedral 2 1 3 0 ⁇ -38 chome method for allele determination.
  • FIG. 7 is a diagram showing combinations of primers used for the PCR-SBT method of BoLA-DQA2.
  • FIG. 8 is a diagram showing alleles amplified by the PCR-SBT method of BoLA-DQA2.
  • FIG. 9 is a diagram showing primers used for closing introns of BoLA-DQA1 and DQA2.
  • FIG. 10A is a view showing an arrangement of a boundary portion between a first exon and a second intron of BoLA-DQA1.
  • W1 indicates the sequence on the database, and A100, WK10 and WK15 were sequenced.
  • FIG. 10B is a view showing an arrangement of a boundary portion between the second intron and the second exon of BoLA-DQA1.
  • FIG. 10C is a view showing a sequence of a boundary sequence between the second exon and the third intron and a sequence of a boundary sequence between the third intron and the third exon of BoLA-DQA1.
  • FIG. 11A is a diagram showing an arrangement of a boundary portion between the first exon and the second intron of BoLA-DQA2. SheepDQA shows the sequence on the database and Ul, A100 and Ml2 were sequenced.
  • Fig. 11B is a diagram showing an arrangement of a boundary portion between the second intron and the second exon of BoLA-DQA2.
  • FIG. 12A is a diagram showing the names of samples obtained by performing PCR-SBT on DQA1 and the primers used.
  • FIG. 12B is a diagram showing the names of samples obtained by performing PCR-SBT on DQA1 and the primers used.
  • Figure 12C shows the names of samples obtained by PCR-SBT for DQA1 It is a figure which shows a primer.
  • FIG. 12D is a diagram showing a sample name obtained by performing PCR-SBT method on DQA1 and primers used.
  • FIG. 13A is a diagram showing the names of samples obtained by performing PCR-SBT on DQA2 and the primers used.
  • FIG. 13B is a diagram showing the names of samples obtained by performing PCR-SBT on DQA2 and the primers used.
  • FIG. 13C is a diagram showing the names of samples obtained by performing PCR-SBT on DQA2 and the primers used.
  • FIG. 14A is a diagram showing the typing results of 52 samples of workshop standard DNA.
  • FIG. 14B is a diagram showing typing results of 52 samples of workshop standard DNA.
  • Fig. 14C is a diagram showing typing results of 52 samples of workshop standard DNA.
  • the present invention is a method for determining the alleles of BoLA-DQAl and DQA2 in the Psi MHC class II gene region.
  • the second exon having the largest allelic variation among the first and fourth exons present in BoLA-DQAl and DQA2 is specifically amplified by PCR, followed by sequencing. It is based on the PCR-based sequence-based typing method (PCR-SBT method) for determining allelic variants.
  • PCR-SBT method PCR-based sequence-based typing method
  • FIG. 1 (A) and (B) after specifically amplifying the second exon by performing the amplification reaction twice or more (FIG. 1 (A) and (B)). ), And specifically sequencing only the second exon (FIG. 1 (C)).
  • BoLA-DQAl and BoLA-DQA2 alleles can be identified simply, promptly and reliably. You can really decide.
  • genomic DNA is prepared from a mosquito (test mosquito) whose allele is to be determined.
  • the genomic DNA can be prepared by a known method, for example, phenol Z-cloth form method, cetyltrimethylammonium-bromobromide (CTAB) method, and the like.
  • CTLAB cetyltrimethylammonium-bromobromide
  • Many kits for preparing genomic DNA are commercially available, and for example, a Wizard Genomic DNA Purification Kit (Promega) can be used.
  • the species of the test pesticide is not particularly limited as long as it belongs to a ruminant (Bos Taurus, Bos indicus). For example, Ayrshire, British Friesian, Danish Black Pied, Danish Red, Hereford, Holstein Friesian, Jersey , Limousin, Japanese black cattle, Japanese short angle.
  • the genomic DNA prepared as described in the previous section “1.
  • the amplification reaction thus performed is performed to amplify a nucleic acid fragment having the base sequence of the second exon.
  • primers capable of amplifying the second exon of BoLA-DQAl or BoLA-DQA2 can amplify the entire region of the second exon. Therefore, as shown in FIG. 1 (A), primers can be designed based on the sequences of introns located on both sides of the second exon. Alternatively, as shown in FIG. 1 (B), primers can be designed based on the arrangement of the boundary region between the intron and etason.
  • Primer design techniques are well known in the art, and primers that can be used in the present invention satisfy conditions that allow specific annealing, for example, a length and a base that allow specific annealing.
  • a composition melting temperature
  • the length having a function as a primer is preferably 10 to 50 bases, more preferably 16 to 50 bases, and further preferably 20 to 30 bases.
  • Tm melting temperature
  • Tm is 50% of any nucleic acid strand This means the temperature at which the complementary strand forms a hybrid with the complementary strand.
  • annealing is necessary to optimize the annealing temperature. On the other hand, if the temperature is lowered too much, a non-specific reaction occurs, so it is desirable that the temperature be as high as possible.
  • known primer design software can be used for confirmation of Tm.
  • Primer sets that can amplify the second exon of BoLA-DQAl that can be used in the present invention include, but are not limited to, the following (a) and (b):
  • R represents A or G
  • primer set capable of amplifying the second exon of BoLA-DQA2 usable in the present invention include, but are not limited to, the following (d) and (e):
  • the amplification reaction is not particularly limited !, for example, a polymerase chain reaction (PCR) method, a LAMP (Loop-mediated isothermal AMP lincation method, an ICAN (Isothermal and Cnimenc primer- initiated Amplification of Nucleic acids). And the SDA (Strand Displacement Amplification) method, etc. Amplification is performed until the amplification product reaches a detectable level.
  • PCR polymerase chain reaction
  • LAMP Loop-mediated isothermal AMP lincation method
  • ICAN Isothermal and Cnimenc primer- initiated Amplification of Nucleic acids
  • SDA Strand Displacement Amplification
  • a genomic DNA is used as a type II, and a DNA polymerase is used to synthesize a base sequence between a pair of primers.
  • a nucleic acid fragment can be exponentially amplified by repeating a cycle in which denaturation, annealing, and synthetic power are repeated.
  • the optimal conditions for PCR can be easily determined by those skilled in the art. Specifically, the optimal conditions for PCR differ depending on the melting temperature (Tm) of the primer considered in the above-mentioned primer design.
  • the primers designed as described above are used to convert the genomic DNA derived from the test specimen into type II, and the base of the second exon of BoLA-DQA1 or BoLA-DQA2. Amplify the nucleic acid fragment containing the sequence.
  • the amplification reaction twice or more using two or more sets of primers. Specifically, first, as shown in FIG. 1 (A), the genomic DNA derived from the test specimen is used as a type II, and the second step of BoLA-DQA1 or BoLA-DQA2 is performed. A nucleic acid fragment containing the exon base sequence is amplified. Next, as shown in FIG. 1 (B), the amplified nucleic acid fragment is used as a type II, and a nucleic acid fragment containing the base sequence of the second exon of BoLA-DQAl or BoLA-DQA2 is further amplified.
  • the primers used in each amplification reaction may be designed at the same position, or the positions of the primers in the first amplification as shown in FIG. Design inside more than a little.
  • BoLA examples of primer sets when performing two amplification reactions to determine DQA1 alleles include, but are not limited to, genomic DNA derived from test mice
  • the following primer set (a) can be used in the amplification reaction
  • the following primer set (b) can be used in an amplification reaction in which the amplified nucleic acid fragment is type III:
  • BoLA examples of primer sets for performing two amplification reactions to determine the DQA2 allele include, but are not limited to, genomic DNA derived from the test fish
  • the following primer set (d) can be used in the amplification reaction
  • the following primer set (e) can be used in an amplification reaction in which the amplified nucleic acid fragment is type III:
  • primer set (g) can be used in an amplification reaction using the primer set of (f) and the amplified nucleic acid fragment as type III:
  • nucleotide sequence of the nucleic acid fragment amplified as described in the previous section “2. Specific amplification of second exon” is determined, and the determined nucleotide sequence is compared with the nucleotide sequence of a known allele. As a result, it is possible to determine what alleles of BoLA-DQA1 and BoLA-DQA2 of the test mice.
  • Sequencing methods are well known in the art and any method can be used. Examples include, but are not limited to, a direct 'sequence method in which the sequence can be determined without cloning the amplified nucleic acid fragment into a vector. Powerful sequencing can be conveniently performed using commercially available kits such as the CEQ TM DTCS Quick Start Kit (BECKMAN), BigDye Terminator and Ycle Sequencing Ready Reaction Kit ABI310 (Applied Biosystems).
  • Exonuclease I is preferably derived from Escherichia coli
  • alkaline phosphatase is preferably derived from yeast (SAP).
  • SAP yeast
  • ExoSAP-IT Amersham
  • primers that can be used to determine the nucleotide sequence of BoLA-DQA1 second exon include, but are not limited to, the following set of primers (c): (c) a primer having the nucleotide sequence shown in SEQ ID NO: 5 and a primer having the nucleotide sequence shown in SEQ ID NO: 6
  • R represents A or G
  • primers that can be used to determine the nucleotide sequence of BoLA-DQA2 second exon are not limited, but the following primer set (e) or (g) Examples include:
  • nucleotide sequence of the nucleic acid fragment ie, the nucleotide sequence of the second exon
  • the nucleotide sequence is compared with the nucleotide sequence of a known allele. Allele information on BoLA-DQA1 and BoLA-DQA2 can be found in, for example, the DDBjZEMBLZGenBank International Nucleotide Sequence Database (Internet site).
  • known alleles include not only alleles that have already been identified but also alleles that will be elucidated in the future, and alleles that are known when the method of the present invention is performed. Including. [0056] In addition, by designing a primer capable of selectively amplifying two types of alleles in an exon or the ability to close an amplified nucleic acid fragment, unknown alleles can be identified. Become.
  • BoLA-DQAl and BoLA-DQA2 of the test pod and determine the allele.
  • novel alleles of BoLA-DQA1 and BoLA-DQA2 can be identified. This also makes it easier to find alleles associated with a particular disease, for example, by determining and comparing alleles in pesticides affected by the particular disease and normal mice.
  • the kit of the present invention can be used for determining an allele of BoLA-DQA1 or BoLA-DQA2, and specifically amplifies a nucleic acid fragment containing the nucleotide sequence of the second exon of BoLA-DQA1 or BoLA-DQA2. It contains at least a primer set that can be used.
  • the kit of the present invention may further comprise a primer set for determining the base sequence of the second exon of BoLA-DQA1 or BoLA-DQA2.
  • the kit of the present invention comprises at least one of the following primers (a) or (b) as a primer set for amplifying the second exon: It is preferred to include a set, and more preferably to include both primer sets (a) and (b):
  • kit of the present invention preferably contains the following primer set (c) as a primer set for sequencing the second exon:
  • the kit of the present invention comprises at least one of the following (d) to (g) as a primer set for amplifying the second exon: It is preferable to include one primer set. Further, it is more preferable to include both primer sets (d) and (e), or both primer sets (f) and (g).
  • kit of the present invention preferably contains the following primer set (e) or (g) as a primer set for sequencing the second exon:
  • the kit of the present invention may further contain, in addition to the above primers, a buffer constituting a reaction solution, a dNTP mixture, enzymes (such as a polymerase), and the like.
  • peripheral blood of the pea 200 heads; see M series, see FIGS. 12A-D and 13A-C
  • the power of the NOSAI Yamagata Okita livestock clinic that were distributed by the Livestock Improvement Corporation were also distributed.
  • Peripheral blood of the pear, Kitamurayama clinic's peripheral blood, and 52 standard DNAs typed at the 5th International Bo LA Workshop WK series, Figures 12A-D and 13A- Genomic DNA was extracted using C).
  • the PCR method is used to amplify BoLA-D QA1 between the first and second exons (hereinafter referred to as the second intron), and the base sequence is obtained by closing.
  • the genomic DNA isolated from the blood of the northeastern clinic which was prepared in Example 1 and which was supplied to the Kitamurayama clinic, was used.
  • 117 ng of the genome was dissolved in 501 X1 KOD-Plus-buffer containing KOD-Plus- (TOYOBO) and 5% DMSO. After denaturation treatment at 94 ° C for 4 minutes, 35 cycles of treatment were performed at 94 ° C for 20 seconds; 68 ° C for 5 minutes as one cycle, and elongation was performed at 68 ° C for 10 minutes.
  • the primer used was DQAexonlF and DQAlexon2R4 which can specifically amplify the BoLA-DQAl second intron by PCR (SEQ ID NOs: 27 and 28; FIG. 9). As a result, a DNA fragment of about 1300 bp was obtained.
  • the PCR product was electrophoresed on 0.8% agarose gel containing ethidium bromide, only the target band was cut out, and the DNA was purified using Mag Extractor (TOYOBO). Furthermore, ethanol precipitation was performed and phosphorylated with T4 Polynucleotide Kinase (TOYOBO).
  • pBluescript II SK (+) (Stratagene) was digested with EcoRV, and ligated to a vector treated with pup small intestinal alkaline phosphatase (CIAP), and transformed into a competent cell XL10 Gold (Stratagene).
  • Luria-Belta 1- (hereinafter, referred to as “ampicillin”, isopropylthio- ⁇ -D-galactoside (hereinafter, IPTG), 5-bromo-4-chloro-3-indori ⁇ -D-galactoside (hereinafter, X-gal) LB) plate, select colonies into which DNA was inserted by color selection, and use 2 ml of 2X YT medium (Molecular Cloning- A Laooratory Manual, second edition, Cold spring harbor laboratory press (1989), Vol. 3, Cultured in Appendix A.3). This was mi-prepped by the alkali method and purified by Mag Extractor (TOYOBO).
  • T3 and T7 were used as sequencing primers (SEQ ID NOS: 25 and 26; FIG. 9).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, and then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER). Then, sequencing was performed using the CEQ TM 2000XL DNA Analysis System (BECKMAN COULTER). The determined base sequence, vector IJ, was deleted from position No. 15 and had a partial sequence of exon 1 or exon 2 using BLAST, a homology search program on the National Center for Biotechnology Information. And the upstream or downstream sequence was designated as the second intron.
  • FIG. 10A shows the sequence of the boundary between the first exon and the second intron
  • FIG. 10B shows the sequence of the boundary between the second intron and the second intron.
  • a primer as shown in FIG. in order to design a primer capable of specifically amplifying the second exon of BoLA-DQA1, a primer as shown in FIG.
  • primers are designed for the second and third exons
  • BoLA-D QA1 between the second and third exons (hereinafter, third intron) is amplified by PCR, and the nucleotide sequence is determined by direct sequencing. It was determined. For samples that could not be sequenced by direct 'sequencing, the PCR products were cloned and sequenced.
  • genomic DNA isolated from the blood of the sea lion which was prepared in Example 1 and which was given to the Kitamurayama clinic, was used.
  • DQA1522F and DQA1-1084R were used as sequence primers (SEQ ID NOS: 32 and 34; FIG. 9).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • Mix 1 ⁇ l PCR product, 2 ⁇ l lOpmolZ ⁇ 1 primer, 4 ⁇ l DTCS Quick Start Mix (BECKMAN), 4 ⁇ l 2.5X Sequencing Buffer (Edge Biosystems), and 9 ⁇ l sterile water Then, 30 cycles of treatment were performed at 95 ° C for 20 seconds; 50 ° C for 20 seconds; and 60 ° C for 4 minutes as one cycle.
  • Sequence was performed by Analysis System (BECKMAN COULTER). The primer site is removed from the determined base sequence, and a homologous search program on the National Center for Biotechnology Information, BLAST, is used to select one having a partial sequence of the second or third exon, and to select the upstream one Alternatively, the downstream sequence was designated as the third intron.
  • BLAST National Center for Biotechnology Information
  • the PCR product was ligated into the SK-T vector (see below). This was transformed into Combinent Senor XL10 Gold (Stratagene), cultured on an LB plate containing ampicillin, IPTG, and X-gal. The cells were cultured in 2 XYT medium (Molecular Cloning-A Laboratory Manual, supra). This was mini-prepped by the alkali method and purified by Mag Extractor (TOYOBO).
  • SK-T vector 10 ⁇ g of pBluescript II SK (+) (Stratagene) plasmid was used with EcoRV37. After C and ⁇ reactions, follow the instructions for CENTRI- SEP COLUMNS (PRINCETON
  • reaction product and 40 ⁇ l of 1 ⁇ rTaq buffer containing 2 units of rTaq DNA elongase (TOYOBO) were reacted at 70 ° C. for 3 hours.
  • TOYOBO rTaq DNA elongase
  • T3 was used as a sequencing primer (SEQ ID NO: 25; FIG. 9).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • Fig. 10C shows the sequence of the boundary between the second exon and the third intron, and the sequence of the boundary between the third intron and the third exon.
  • a PCR primer capable of specifically amplifying DQA1 second exon was designed by the method shown in Fig. 1. Then, these primers were combined to design seven PCR primer sets, namely DQA1-1, 2, 3, 4, 5, 6, and -7 (FIGS. 3A and B). The experiment was carried out using the genome (M series) purified from the peripheral blood of Pepsi which was provided by the Livestock Improvement Corporation prepared in Example 1.
  • the primers shown in FIGS. 3A and 3B were used in the combination shown in FIG. CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • the treatment was performed at 95 ° C. for 20 seconds; at the Tm value shown in FIG. 4 for 20 seconds; and at 60 ° C. for 4 minutes, as one cycle, and the number of cycles shown in FIG. 4 was performed.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, and then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for the CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER). And CEQ TM 2000XL DNA
  • Sequence was performed by Analysis System (BECKMAN COULTER). The primer site is removed from the determined base sequence, and the allele base sequence is registered in the database using the homology search program BLAST on the National Center for Biotechnology Information, and compared with the base sequence. And the allele was determined.
  • the primers shown in FIGS. 3A and 3B were used in the combination shown in FIG.
  • 1 ⁇ l of the IstPCR product is mixed with 24 ⁇ l of the reagent under the same conditions, denatured at 95 ° C for 10 minutes, and then at 95 ° C for 1 minute; the Tm value shown in Figure 4 is 30 seconds and The cycle of the treatment at 72 ° C for 40 seconds was regarded as one cycle, and after the treatment with the number of cycles shown in FIG.
  • the primers shown in FIGS. 3A and 3B were used in the combination shown in FIG. 4 so that the inside could be specifically amplified.
  • the PCR product was electrophoresed on a 2% agarose gel containing ethidium bromide to confirm that the DNA was amplified!
  • the primer shown in FIG. 3 was used in the combination shown in FIG. CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • 1 ⁇ l ⁇ CR product, 2 ⁇ l lOpmolZ ⁇ 1 primer, 4 ⁇ l DTCS Quick Start Mix (BECKMAN) was used for the sequencing reaction.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, and then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for the CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER). Then, sequencing was performed using the CEQ TM 2000XL DNA Analysis System (BECKMAN COULTER). The primer site is removed from the determined base sequence, and the allele base sequence is registered in the database using the homology search program BLAST on the National Center for Biotechnology Information, and compared with the base sequence. And the allele was determined.
  • CENTRI-SEP COLUMNS PRINCETON SEPARATIONS
  • FIG. 5 shows the results.
  • DQA1-3 both heterologous signals were obtained favorably, and it was considered that the ease of amplification for each allele was the same.
  • DQA1-4 could be sequenced except for WK10, 15, 32 and 38.
  • DQA1-5 the sequencing results with the 3 'primer improved.
  • DQA1-6 the sequence results were good with primers on both sides of 3, and 5.
  • salt Sufficient signals were obtained for the determination of the base sequence, and V and shifted alleles could be amplified evenly.
  • RTaq DNA elongase (TOYOBO), 25 j WIX rTaq buffer containing 1 ⁇ l of genome after denaturation at 94 ° C for 2 minutes, 94 ° C for 20 seconds; 60 ° C for 20 seconds, and The treatment was performed at 72 ° C for 40 seconds as one cycle, followed by 15 cycles of treatment, followed by extension at 72 ° C for 4 minutes.
  • primers DQAlintL2 and DQA1-677R capable of specifically amplifying BoLA-DQA1 second exon by PCR were used (SEQ ID NOS: 1 and 2; FIG. 3B). As a result, a 426 bp DNA fragment was obtained.
  • DQAintL3.1 and DQAlex2REVverl.1 were used as sequence primers (SEQ ID NOS: 5 and 6; FIG. 3B).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction. 1 ⁇ l PCR product, 2 ⁇ l lOpmolZ ⁇ 1 primer, 4 ⁇ l DTCS Quick Start Mix (BECKMAN), 4 ⁇ l 2.5X Sequencing Buffer (Edge Biosystems), Then, 9 ⁇ l of sterilized water and 9 ⁇ l of sterilized water were mixed, and the treatment was performed at 95 ° C for 20 seconds; 50 ° C for 20 seconds; Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions, then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER), and CEQ TM 2000XL DNA
  • Sequence was performed by Analysis System (BECKMAN COULTER). The primer site is removed from the determined base sequence, and the allele base sequence is registered in the database using the homology search program BLAST on the National Center for Biotechnology Information, and compared with the base sequence. And the allele was determined.
  • FIG. 5 shows the results.
  • BoLA In order to design primers that can specifically amplify DQA2 second exon, as shown in Fig. 2 (A), based on the published sequence information of DQA2 mRNA, the first etason and second exoson Primers were designed, and the BoLA-DQA2 second intron of the three DNAs (U1, A100 and M12) was amplified by PCR and cloned to determine the nucleotide sequence.
  • 117 ng of the genome was dissolved in 50 ⁇ l of XlExTaq buffer (TaKaRa) containing 150 ⁇ l of each dNTP, 300 nM primer, and 1.5 units of ExTaq (TaKaRa). After denaturation at 94 ° C for 4 minutes, 20 cycles at 94 ° C; 20 seconds at 68 ° C; 5 cycles at 72 ° C for 40 cycles, followed by 10 minutes at 72 ° C Extended.
  • DQAexonlF and DQA2exon2R capable of specifically amplifying the BoLA-DQA2 second intron by PCR were used (SEQ ID NOS: 27 and 29; FIG. 9). As a result, a DNA fragment of about 4000 bp was obtained.
  • the PCR product is electrophoresed on a 0.8% agarose gel containing ethyl bromide, And DNA was purified using MagExtractor (TOYOBO). 40 ⁇ of each dNTP ⁇ 500 ⁇ MOMgSO, containing 2.5 units of KOD (TOYOBO) and purified DNA
  • T3 and T7 were used as sequence primers (SEQ ID NOS: 25 and 26; FIG. 9).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, and then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER). Then, sequencing was performed using the CEQ TM 2000XL DNA Analysis System (BECKMAN COULTER). The determined base sequence, vector IJ, was deleted from position No. 15 and had a partial sequence of exon 1 or exon 2 using BLAST, a homology search program on the National Center for Biotechnology Information. And the upstream or downstream sequence was designated as the second intron.
  • 200 ⁇ l of Medium Buffer (TOYOBO) containing 30 to 200 units of HindIII and 10 ⁇ l of DNA solution was treated overnight at 37 ° C, and purified with CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions.
  • the colonies were lysed in 25 ⁇ l of XlEx Taq buffer (TaKaRa) containing 150 ⁇ M of each dNTP, 400 nM of primer and 0.5 units of ExTaq (TaKaRa). After denaturation at 94 ° C for 2 minutes, 20 cycles at 94 ° C; 30 seconds at 60 ° C; 1 minute 30 seconds at 72 ° C, 35 cycles of treatment, followed by 72 ° C Extended for 4 minutes.
  • the primers used were T3 and T7 (SEQ ID NOS: 25 and 26; FIG. 9).
  • the PCR product was electrophoresed on a 0.8% agarose gel containing ethidium bromide, and from 0.9 to 6 units of a PCR product, which was considered to have obtained the same fragment as the inserted DNA fragment of approximately 100 One hundred and one Medium Buffer (TOYOBO) containing HindIII and 5 ⁇ l of PCR product is treated at 37 ° C for 1 hour and 30 minutes, electrophoresed on 0.8% agarose gel, and the DNA is cleaved! And the corresponding colonies were cultured in 2 ml of 2XYT medium (Molecular Cloning-A Laboratory Manual, supra). This was mini-prepped by the alkali method and purified by Mag Extractor (TOYOBO).
  • T3 and T7 were used as sequence primers (SEQ ID NOS: 25 and 26; FIG. 9).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, and then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER). Then, sequencing was performed using the CEQ TM 2000XL DNA Analysis System (BECKMAN COULTER). The determined base sequence, vector IJ, was deleted from position No. 15 and had a partial sequence of exon 1 or exon 2 using BLAST, a homology search program on the National Center for Biotechnology Information. And the upstream or downstream sequence was designated as the second intron. [0104] FIG. 11A shows the arrangement of the boundary between the first exon and the second intron, and FIG.
  • FIG. 11B shows the arrangement of the boundary portion with 2 exons.
  • a PCR primer capable of specifically amplifying DQA2 second exon was designed by the method shown in Fig. 1. These primers were combined to design five types of PCR primer sets, DQA2-1, 2, 3, 4, and 5 (FIGS. 6A and B). The experiment was carried out using the genome (M series) purified from the peripheral blood of Pepsi which was distributed by the Livestock Improvement Corporation prepared in Example 1.
  • the primers shown in FIGS. 6A and 6B were used in combination shown in FIG. CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • the treatment was performed at 95 ° C for 20 seconds; the Tm value shown in FIG. 7 was 20 seconds; and the treatment at 60 ° C for 4 minutes was regarded as one cycle, and the number of cycles shown in FIG. 7 was performed.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, and then purified using the CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER). Dissolve in 20 ⁇ l of Sample Loading Solution (BECKMAN) and CEQ TM 2000XL DNA
  • Sequence was performed by Analysis System (BECKMAN COULTER). The primer site is removed from the determined base sequence, and the allele base sequence is registered in the database using the homology search program BLAST on the National Center for Biotechnology Information, and compared with the base sequence. And the allele was determined.
  • FIG. 8 shows the results. When 30 samples were typed using DQA2-2, more than 5 samples could be amplified. DQA2-3 was able to determine alleles that could not be amplified with DQA2-2. DQA2-4 was also able to determine the alleles of the vigorous sample that could not be amplified with DQA2-3. DQA2-5 was able to obtain a good signal.
  • the primers shown in FIGS. 6A and 6B were used in the combination shown in FIG.
  • Sequence primers were used in the combinations shown in FIG. CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction. 1 ⁇ l PCR product, 2 ⁇ l lOpmo ⁇ / ⁇ 1 primer, 4 ⁇ l DTCS Quick Start Mix (BECKMAN), 4 ⁇ l 2.5 X
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions, then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER), and CEQ Sequences were performed using the 2000XL DNA Analysis System (BECKMAN COULTER). Determined nucleotide sequence Remove the primer site, compare the nucleotide sequence of the allele with the nucleotide sequence registered in the database using the homology search program on the National Center for Biotechnology Information, BLAST It was determined.
  • CENTRI-SEP COLUMNS PRINCETON SEPARATIONS
  • RTaq DNA elongase (TOYOBO), 25 ⁇ l of 1XrTaq buffer containing 1 ⁇ l genome for 2 minutes at 94 ° C, 20 seconds at 94 ° C; 20 seconds at 55 ° C , And 15 cycles of the treatment at 72 ° C for 20 seconds as one cycle, followed by extension at 72 ° C for 4 minutes.
  • DQA2intL06 and QA006 capable of specifically amplifying BoLA-DQA2 second exon by PCR were used (SEQ ID NOs: 7 and 8; FIG. 6B). As a result, a 292 bp DNA fragment was obtained.
  • DQA2intL06 and DQA2exon2-1 were used as sequence primers (SEQ ID NOS: 7 and 9; FIG. 6B).
  • the CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequence reaction.
  • the treatment was performed at 95 ° C for 20 seconds; at 50 ° C for 20 seconds; at 60 ° C for 4 minutes as one cycle, and 30 cycles of treatment were performed.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions, then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER), and CEQ Sequences were performed using the 2000XL DNA Analysis System (BECKMAN COULTER).
  • BECKMAN Sample Loading Solution
  • CEQ TM 2000XL DNA Analysis System sequencer BECKMAN COULTER
  • CEQ Sequences were performed using the 2000XL DNA Analysis System (BECKMAN COULTER).
  • the primer site is removed from the determined base sequence, and the allele base sequence is registered in the database using the homology search program BLAST on the National Center for Biotechnology Information, and compared with the base sequence. And the allele was determined.
  • each dNTP at 150 / zM, 400 nM primer, 0.5 units of Ex Taq elongase, and 25 ⁇ l of 1X Ex Taq buffer containing 1 ⁇ l genome For 20 cycles at 94 ° C for 20 seconds; 56 ° C for 20 seconds, and 72 ° C for 20 seconds, the cells were elongated at 72 ° C for 5 minutes.
  • the primer used was DQA2intron2F and QA006, which can specifically amplify the BoLA-DQA2 second intron by PCR (SEQ ID NOS: 35 and 8; FIG. 6B). As a result, a 283 bp DNA fragment was obtained.
  • EXOSAP Escherichia coli-derived exonuclease
  • SAP shrimp-derived alkaline phosphatase
  • DQA2intL06ver2 and QA006 were used as sequence primers (SEQ ID NOs: 36 and 8; FIG. 6B).
  • the CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • the treatment was performed at 96 ° C for 20 seconds; at 50 ° C for 20 seconds; at 60 ° C for 4 minutes as one cycle, and 30 cycles of treatment were performed.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the manufacturer's instructions, followed by CEQ TM 2000XL It was dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for DNA Analysis System sequencer (BECKMAN COULTER) and sequenced using CEQ TM 2000XL DNA Analysis System (BECKMAN COULTER).
  • BECKMAN Sample Loading Solution
  • the primer site is removed from the determined base sequence, and the base sequence of the allele is compared with the base sequence registered in the database using BLAST, a homology search program on the National Center for Biotechnology Information, and the allele is determined. It was determined.
  • Fig. 8 shows the results.
  • the primer set of DQA2-7 was able to determine a total of eight alleles.
  • RTaq DNA elongase (TOYOBO), 25 ⁇ l of 1X rTaq buffer containing 1 ⁇ l genome, denatured at 94 ° C for 2 minutes, then at 94 ° C for 20 seconds; at 60 ° C for 20 seconds , And a cycle of 40 seconds at 72 ° C., followed by 15 cycles of treatment, followed by extension at 72 ° C. for 4 minutes.
  • DQAlintL2 and DQA1-677R capable of specifically amplifying BoLA-DQA1 second exon by PCR were used (SEQ ID NOS: 1 and 2; FIG. 3B). This resulted in a 426 bp DNA fragment.
  • AmpliTaq Gold TM DNA extension enzyme Gene in 24 mu 1 containing (Applied Biosystems) Amp R Gold buffer for 10 minutes at 95 ° C of 20 seconds at 94 ° C; 60 After 30 cycles of processing at 20 ° C for 20 seconds and 40 seconds at 72 ° C as one cycle Was extended at 72 ° C for 4 minutes.
  • DQAintL3 and DQAlex2REVver2.1 which can specifically amplify the inside further, were used (SEQ ID NOS: 3 and 4; FIG. 3B). As a result, a 355 bp DNA fragment was obtained.
  • the PCR product was electrophoresed on a 2% agarose gel containing ethidium bromide to confirm that the DNA was amplified!
  • DQAintL3.1 and DQAlex2REVverl.1 were used as sequence primers (SEQ ID NOS: 5 and 6; FIG. 3B).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • the treatment was performed at 95 ° C for 20 seconds; at 50 ° C for 20 seconds; at 60 ° C for 4 minutes as one cycle, and 30 cycles of treatment were performed.
  • sequence PCR product is purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions, then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER), and CEQ TM 2000XL DNA
  • Sequence was performed by Analysis System (BECKMAN COULTER). The primer site is removed from the determined base sequence, and the allele base sequence is registered in the database using the homology search program BLAST on the National Center for Biotechnology Information, and compared with the base sequence. And the allele was determined.
  • PCR-SBT was performed on 52 standard DNA samples typed at the 5th International BoLA Workshop under the conditions of DQA2-6 or DQA2-7.
  • Figures 13A-C the base sequence of BoLA-DQA2 second exon amplified by the PCR method using the PCR primers shown in FIGS. 6A and B was determined by the direct 'sequence method, and then based on the homology of the base sequences. Then, the allele was determined.
  • DQA2intL06 and DQA2exon2-1 were used as sequence primers (SEQ ID NOS: 7 and 9; FIG. 6B).
  • the CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequence reaction.
  • the treatment was performed at 95 ° C for 20 seconds; at 50 ° C for 20 seconds; at 60 ° C for 4 minutes as one cycle, and 30 cycles of treatment were performed.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions, then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER), and CEQ TM 2000XL DNA
  • Sequence was performed by Analysis System (BECKMAN COULTER). Remove the primer site from the determined base sequence, and Using a homology search program, BLAST, the nucleotide sequence of the allele was registered in the database and collated with the nucleotide sequence to determine the allele.
  • EXOSAP Escherichia coli-derived exonuclease
  • SAP shrimp-derived alkaline phosphatase
  • DQA2intL06ver2 and QA006 were used as sequence primers (SEQ ID NOs: 36 and 8; FIG. 6B).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) for sequencing reactions was.
  • the treatment was performed at 96 ° C for 20 seconds; at 50 ° C for 20 seconds; at 60 ° C for 4 minutes as one cycle, and 30 cycles of treatment were performed.
  • Sequence PCR products are purified using CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) according to the instructions, then dissolved in 20 ⁇ l of Sample Loading Solution (BECKMAN) for CEQ TM 2000XL DNA Analysis System sequencer (BECKMAN COULTER), and CEQ Sequencing was performed using the 2000XL DNA Analysis System (BECKMAN COULTER).
  • BECKMAN Sample Loading Solution
  • CEQ TM 2000XL DNA Analysis System sequencer BECKMAN COULTER
  • the primer site is removed from the determined base sequence, and the base sequence of the allele is compared with the base sequence registered in the database using BLAST, a homology search program on the National Center for Biotechnology Information, and the allele is determined. It was determined.
  • rTaq DNA elongase (TOYOBO), 50 ⁇ l of 1X rTaq buffer containing 1 ⁇ l genome, denatured at 94 ° C for 2 minutes, 94 ° C for 1 minute; 55 ° C for 30 seconds, After 1 cycle of treatment at 72 ° C. and 1 cycle, 15 cycles of treatment were performed, followed by extension at 72 ° C. for 4 minutes.
  • primers QA05 and QA010 capable of specifically amplifying the second exon of BoLA-DQAl and DQA2 by PCR were used (SEQ ID NOS: 21 and 8; FIGS. 6A and B).
  • the primer is the second exo of DQA2 QA005 and QA006 capable of specifically amplifying the inside of the protein were used (SEQ ID NOS: 21 and 8; FIGS. 6A and B). As a result, a 263 bp DNA fragment was obtained.
  • the PCR product was subjected to electrophoresis on a 2% agarose gel containing ethambromide to confirm that the DNA was amplified.
  • the colonies were lysed in 25 ⁇ l of 1X rTaq buffer containing rTaq DNA elongase (TOYOBO). After a denaturation treatment at 94 ° C for 2 minutes, a cycle of 94 ° C for 20 seconds; 55 ° C for 30 seconds; 72 ° C for 30 seconds as one cycle, followed by 35 cycles of treatment, followed by 72 ° C for 2 minutes Extended.
  • the primers used were T3 and T7 (SEQ ID NOS: 25 and 26; FIG. 6B).
  • the PCR product was electrophoresed on a 2% agarose gel containing ethidium bromide, and from the size of the band, the same fragment as the inserted 263 bp DNA fragment could be obtained.
  • the cells were cultured in a medium (Molecular Cloning-A Laboratory Manual, supra). This was mini-prepped by the alkali method and purified by Mag Extractor (TOYOBO).
  • T7 was used as a sequencing primer (SEQ ID NOS: 25 and 26; FIG. 6B).
  • CEQ TM DTCS Quick Start Kit (BECKMAN) was used for the sequencing reaction.
  • One 2.5 X Sequencing Buffer (Edge Biosystems) was mixed and subjected to 40 cycles of processing at 95 ° C for 20 seconds; 55 ° C for 20 seconds; and 60 ° C for 4 minutes as one cycle.
  • BECKMAN Sample Loading Solution
  • BECKMAN COULTER CEQ TM 2000XL DNA Analysis System sequencer
  • BECKMAN COULTER CEQ TM 2000XL DNA Analysis System
  • PCR was performed by the method of Ballingall et al. (1997) on 6 of 11 samples for which amplification was not confirmed in the electrophoresis image of the 2nd PCR product, and it was confirmed that DQA2 was not amplified.
  • PCR was performed by the method of Ballingall et al. (1997), the product was cloned, and the nucleotide sequence was determined. This proved that there was one allele.
  • the present invention provides a method for determining the allele of BoLA-DQA. This method is useful for studies on the association between MHC and disease susceptibility, since the BoLA-DQA1 and BoLA-DQA2 alleles can be determined easily, quickly and reliably. Sequence listing free text
  • SEQ ID NOS: 1 to 36 Synthetic oligonucleotide

Abstract

Il est prévu un procédé de détermination d’un allèle de BoLA-DQA bovin ; et un ensemble et kit initiateurs correspondants. Il devient possible de déterminer un allèle de BoLA-DQA1 ou BoLA-DQA2 facilement, rapidement et en toute sécurité.
PCT/JP2005/006465 2004-04-01 2005-04-01 PROCEDE POUR DETERMINER L’ALLELE DU GENE DU COMPLEXE MAJEUR D’HISTOCOMPATIBILITE BOVIN (BoLA) DQA CLASSE II WO2005095648A1 (fr)

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DAVIES C.J. ET AL: "Polymorphism of bovine MHC class II genes. Joint report of the Fifth International Bovine Lymphocyte Antigen (BoLA) Workshop, Interlaken, Switserland, 01 August 1992", EUR.J.IMMUNOGENET, vol. 21, no. 4, 1994, pages 259 - 289, XP002992617 *
HAZAMA Y. ET AL: "Nihon Nohaku Taikai Symposium Bovine Genome Kaiseki to Kobyosei", AGRICULTURE AND HORTICULTURE, vol. 76, no. 12, 2001, pages 1289 - 1294, XP002992615 *
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