JP3192128B2 - Genetic diagnostic method for bovine claudin-16 deficiency - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a bovine claudin-16.
The present invention relates to a genetic diagnosis method (or detection method) for a deficiency.

[0002]

Definition of Terms Related to the Invention In this specification, "Clau
"Din-16" means a protein produced by transcription and translation of Claudin-16 gene, and "Claudin-16 gene" is Claudin
In addition to the exon portion of the translation region encoding din-16, the exon portion of the adjacent untranslated region, their introns, and regions involved in controlling the expression of the gene, the mutated portion associated with the disease is It means the region of DNA contained.

[0003]

2. Description of the Related Art Claudin-16 deficiency is an autosomal recessive hereditary disease characterized by congenital renal tubular dysplasia, and clinically presents with poor growth, dorsi-posture, and overlong hooves. The disease has high BUN (urea nitrogen) level and creatinine level in blood as biochemical characteristics. This disease occurs in Japanese beef
Outbreaks have been noted since 1991, but no similar disease has been reported in humans. Cows that have developed Claudin-16 deficiency can be detected as stunted with abnormally high BUN and creatinine levels in the blood. However, it is difficult to know the abnormality of a cow that is a heterozygote having a gene associated with an abnormality on only one chromosome, that is, a cow that is genetically a carrier of Claudin-16 deficiency. Therefore, even when the apparently abnormal cows are bred to each other, abnormalities may appear in the calves born, which is problematic in preventing the occurrence of the disease.

[0004]

[Problems to be Solved by the Invention] Bovine Claudin-16
One method for preventing deficiency is to avoid crosses between heterozygotes. To do this, a diagnosis of cattle claudin-16 deficiency is made at the genetic level,
It is necessary to find a carrier for the disease gene as soon as possible. If it is possible to clarify how a disease gene in a bovine with an abnormality is mutated compared to a normal one and obtain a means to quickly detect the mutated gene by various genetic engineering techniques, such a gene could be used. Diagnostic methods can be established. Therefore, an object of the present invention is to provide bovine Cl
It is to provide a genetic diagnosis method (gene detection method) for audin-16 deficiency. Thus, by screening carriers of Claudin-16 deficiency, it is possible to prevent the occurrence of the present disease in the future.

[0005]

Means for Solving the Problems As a result of repeated studies to achieve the above object, the present inventors have found a DNA deficiency of about 30 kb which is closely related to this disease, Successfully identified a new gene. The structure of this gene has recently been reported in mice
Gene family (M. Furuse et al., J. Cell Biol.,
141, 1539-1550 (1998); K. Morita et al., Proc. Nat.
L. Acad. Sci. USA, 96, 511-516 (1999)) and was named Claudin-16. Furthermore, the present inventors have found that the absence of the Claudin-16 gene due to a mutation called DNA deletion is the cause of the disease,
PCR using oligonucleotide primers containing specific oligonucleotide primers set on the gene
It has been found that such a mutation is detected by the (polymerase chain reaction) method, and the present invention has been completed.

Namely, the gist of the present invention, (1) step (a): as engineering to obtain the bovine nucleic acid sample, step (b): urging step resulting nucleic acid sample in (a) to gene amplification reaction A step of obtaining a nucleic acid fragment in which a region containing a mutation site that may be present in the bovine Claudin-16 gene is amplified.
And step (c): the step of determining the presence of mutations on nucleic acid fragments of step (b), a genetic diagnosis of Claudin-16 deficiency in cattle including, region containing the mutation site of bovine Claudin-16 gene In the nucleotide sequence , bovine claudin -16, which is a region including positions 1 to 629 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing
Genetic diagnosis of deficiency . (2) those gene amplification reaction carried out by the PCR method
The genetic diagnostic method according to the above (1) , wherein (3) characterized by examining the presence of the mutation by PCR amplification
Gene diagnosis according to any one of the above (1) and (2)
Law. ( 4 ) The method according to any one of (1) to ( 3 ) above, wherein the nucleic acid sample is a sample containing genomic DNA, cDNA, or mRNA.

In another aspect, the present invention provides (5) a kit for detecting bovine claudin-16 deficiency, which comprises amplifying a region containing a mutation site which may be present in bovine claudin-16 gene. Contains oligonucleotide primers used to amplify by reaction,
In addition, the oligonucleotide primer is (a) the 5 ' end of the base sequence shown in SEQ ID NO: 1 in the sequence listing.
Selected from the group consisting of: an oligonucleotide having a base sequence corresponding to the terminal region; and (b) an oligonucleotide having a complementary base sequence to the 3 ′ terminal region of the base sequence shown in SEQ ID NO: 1 in the sequence listing. A kit for detecting bovine claudin-16 deficiency, which is characterized in that it has been isolated. ( 6 ) A kit for detecting bovine claudin-16 deficiency, which is an oligonucleotide primer used for amplifying a region containing a mutation site possibly present in bovine claudin-16 gene by a gene amplification reaction. Contains
The oligonucleotide primer comprises: (1) an oligonucleotide having a base sequence corresponding to the 5′- terminal region of the base sequence shown in SEQ ID NO: 1 in the sequence listing; and (2)
3 'of the base sequence shown in SEQ ID NO: 1 in the sequence listing
Bovine Clau, which is selected from the group consisting of oligonucleotides having a complementary base sequence to the terminal region, and wherein the oligonucleotide primers are composed of 15 to 35 nucleotides.
Kit for detecting din-16 deficiency. ( 7 ) A kit for detecting bovine claudin-16 deficiency, which is an oligonucleotide primer used for amplifying a region containing a mutation site which may be present in bovine claudin-16 gene by a gene amplification reaction. Contains
Moreover, the oligonucleotide primer is selected from the group consisting of those shown in SEQ ID NOs: 2 to 7 in the sequence listing, a kit for detecting bovine claudin-16 deficiency .

( 8 ) Bovine Claudin-16 gene and bovine Clau
The nucleotide sequence corresponding to the din-16 deficiency gene or the whole or a part of the complementary chain thereof, and (a) the nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing or the whole of the complementary chain or the Partly, (b) any sequence that hybridizes with the sequence (a) and can be used to amplify a region containing a mutation site that may be present in the bovine Claudin-16 gene by PCR, (c) the gene code A sequence selected from the group consisting of sequences derived from the sequences (a) and (b) for the degeneration of ( 9 ) A nucleotide sequence comprising the nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing or the whole or a part of the complementary strand thereof. ( 10 ) The above (8) to (8), which are selected from the group consisting of genomic DNA sequences, cDNA sequences, RNA sequences, hybrid sequences, synthetic sequences, and semi-synthetic sequences.
The nucleotide sequence according to any one of (9) .

( 11 ) A nucleotide probe capable of hybridizing with the sequence according to any one of the above (8) to (9) or the corresponding mRNA. ( 12 ) To detect bovine claudin-16 deficiency,
( 11 ) The nucleotide probe according to the above ( 11 ), for use in elucidating and / or isolating a sequence containing a mutation site that may be present in the Claudin-16 gene. ( 13 ) A step of clarifying and / or isolating a sequence containing a mutation site that may be present in the bovine claudin-16 gene using the nucleotide probe according to ( 11 ). For detecting bovine claudin-16 deficiency. ( 14 ) The oligonucleotide primer according to any one of the above (5) to (7) . ( 15 ) A genetic diagnostic reagent for bovine claudin-16 deficiency, comprising the oligonucleotide primer according to (14) .

The objects, features, advantages and ideas of the present invention will be apparent to those skilled in the art from the description herein. It is understood that the description of the following embodiments of the invention and the description of specific examples show preferred embodiments of the present invention, and are shown here only for explanation. And various alterations and modifications within the spirit and scope of the invention disclosed herein will be readily apparent to those skilled in the art based on their description.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Establishment of a genetic diagnosis method (detection method) for bovine claudin-16 deficiency: As described below, the mutation in the gene responsible for bovine claudin-16 deficiency was elucidated (Fig. 1), and this mutation was utilized. The disease can be detected. Concrete Bovine Clau
Examples of the method for genetic diagnosis (detection method) of din-16 deficiency include the following steps. Step (a): a step of obtaining a bovine nucleic acid sample; Step (b): subjecting the nucleic acid sample obtained in the step (a) to a gene amplification reaction to be present in the bovine Claudin-16 gene Step of obtaining a nucleic acid fragment in which the region containing the mutation site has been amplified,
And step (c): examining the nucleic acid fragment of step (b) for the presence of a mutation.

First, the step (a) will be described. The bovine nucleic acid sample used in the present invention is not particularly limited as long as it has a nucleotide sequence encoding Claudin-16, and nucleic acids derived from appropriate cells or tissues (whole genomic DNA and total cells And cDNA transcribed from RNA), for example, genomic DNA, cDNA, mRNA and the like. Preparation of a bovine nucleic acid sample can be performed by a known method, for example, Molecular cloning, a laboratory manual (2nd editi).
on) (J. Sambrook, EF Fritsch & T. Maniatis (E
d.), Cold Spring Harbor Press, Cold Spring Harbor,
New York (1989)).

Second, the step (b) will be described. Using the nucleic acid sample obtained in step (a) and appropriate primers, a region containing a displacement site that may be present in the bovine Claudin-16 gene is amplified, and a desired nucleic acid fragment can be obtained.
The method of the gene amplification reaction used in this step is not particularly limited as long as the method can amplify the region.
Nucleic acid amplification methods such as the R method, a nucleic acid amplification method using RNA polymerase, and a strand displacement amplification method can be used. Among them, the PCR method is preferably used. Subject to amplification,
The region containing the mutation site is not particularly limited as long as it is a region containing a mutation that causes bovine Claudin-16 deficiency in the nucleotide sequence of the bovine Claudin-16 gene.
1 to 629 in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing
Region including the position.

[0014] As used herein, the term "polymerase chain reaction" or "PCR" generally refers to a method as described in US Patent No. 4,683,195. Refers to a method for enzymatic amplification. In general, the PCR method involves repeating a cycle of performing primer extension synthesis using two oligonucleotide primers capable of hybridizing preferentially to a template nucleic acid. Typically, primers used in the PCR method can use primers complementary to the nucleotide sequence to be propagated inside the template, for example, complementary to the nucleotide sequence to be amplified at both ends. Those which are targeted or adjacent to the nucleotide sequence to be amplified can be preferably used.

The PCR method is based on MA Innis, DM Gelfaud,
JJ Snindky, & TJ White (Ed.), PCR protocols:
a guide to methods and applications, Academic Pre
ss, Inc., New York (1990); MJ McPherson, P. Qui
rke & GR Taylor (Ed.), PCR: a practical approac
h, IRL Press, Oxford (1991); RK Saiki et al., Sc
ience, 239, 487-491 (1988); MA Frohman et al.,
Natl. Acad. Sci. USA, 85, 8998-9002 (1988), or a method modified or modified. The PCR method can be carried out using a commercially available kit suitable for the PCR method, and can also be carried out according to a protocol specified by a kit manufacturer or a kit seller.

As used herein, the term "oligonucleotide" refers to a relatively short single-stranded or double-stranded polynucleotide, preferably a polydeoxynucleotide.
Agnew. Chem. Int. Ed. Engl., Vol. 28, p. 716-734
(1989), for example, the triester method, the phosphite method, the phosphoramidite method,
It can be chemically synthesized by a method such as a phosphonate method. It is known that conventional synthesis can conveniently be performed on a modified solid support, for example,
This can be done using an automated synthesizer, which is commercially available. The oligonucleotide may contain one or more modified bases, for example, an unnatural base such as inosine or a tritylated base.

As a primer used in the PCR method, any primer capable of amplifying a DNA fragment containing the above mutation site may be used.
There is no particular limitation. Typically, the primer is (a) an oligonucleotide having a nucleotide sequence corresponding to an arbitrary region of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing;
(b) An oligonucleotide having a complementary nucleotide sequence to any region of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing can be used, and more preferably (1) SEQ ID NO: 1 in the sequence listing And an oligonucleotide having a base sequence corresponding to an arbitrary region at the 5 'end of the base sequence shown in (2) and (2) the 3' end of the base sequence shown in SEQ ID NO: 1 in the sequence listing Oligonucleotides having a complementary base sequence to any region of, for example, 3
~ 100, preferably 10-50, more preferably 15-3
And those containing 5 nucleotides. In addition, the PCR conditions are not particularly limited, and may be well-known conditions that are usually performed. In PCR, one cycle consisting of heat denaturation of a DNA strand, annealing of a primer and synthesis of a complementary strand by a polymerase is repeated, for example, 10 to 50 times, preferably 20 to 35 times, more preferably 25 to 30 times. Done.

Third, the step (c) will be described. In this step, the nucleic acid fragment obtained in step (b) is examined for the presence of a mutation. Methods for detecting the presence of a mutation include:
Although not particularly limited, it is detected by examining the length of the DNA fragment obtained by the PCR method. The method for examining the DNA fragment length is not particularly limited, and the DNA fragments are separated by electrophoresis on polyacrylamide or agarose gel, for example, based on the mobility thereof relative to the mobility of a marker DNA fragment of a known molecular weight. Preferred is a method for identifying a target DNA fragment.

As a detection method other than the above, there is a method in which the step (c) of the above exemplified embodiment is changed to another mutation detection method. Known mutation detection methods such as a hybridization method using a suitable DNA fragment containing a mutation site as a probe can be used for mutation detection. Furthermore, mutation can also be detected by cloning the amplified DNA into an appropriate vector and determining the nucleotide sequence, or by determining the nucleotide sequence using the amplified fragment itself as a template.

Oligonucleotides and probes can be labeled with a label component to facilitate detection. The label component comprises spectroscopic means, optical means, biochemical means, immunological means, enzymatic chemical means,
It can be detected by radiochemical means or the like. Examples of label components include peroxidase,
Examples include enzymes such as alkaline phosphatase, radioactive labels such as ³² P, isotopes, biotin, fluorescent dyes, luminescent substances, and chromogenic substances.

According to the genetic diagnosis method (detection method) for bovine claudin-16 deficiency of the present invention, not only cattle having claudin-16 deficiency but also cattle as a carrier of claudin-16 deficiency can be detected. Can be diagnosed. Therefore, the term "bovine claudin-16 deficiency" as used in the present invention means genetically abnormal, and is to be interpreted in a broad sense regardless of the presence or absence of symptoms and including carriers.

Normal cattle and cattle with Claudin-16 deficiency
Analysis of Claudin-16 gene: In order to investigate the relationship between mutations in the gene and this disease, first, the gene (cDNA) encoding normal bovine claudin-16 is isolated and its nucleotide sequence is determined. Although no isolated cases of the gene have been reported so far, the present inventors have performed genomic linkage analysis of a family including the cattle with the disease, and have used a method called positional cloning to identify bovine Claudin-16. The gene was isolated.

Next, the entire nucleotide sequence of the cDNA encoding the obtained normal bovine Claudin-16 is shown in SEQ ID NO: 1 in the sequence listing. Determination of base sequence of DNA fragment (sequencing)
Can be carried out by a chemical decomposition method (Maxam & Gilbert method), a chain terminator method (Sanger dideoxy method), or the like.

Mutations in the gene causing Claudin-16 deficiency can be identified by comparing the nucleotide sequences of the Claudin-16 gene in normal and affected cattle. That is, the mutation causing the disease can be confirmed by examining the nucleotide sequence of the claudin-16 gene of the onset cattle and comparing it with the normal bovine gene in the same manner as in the case of the normal cattle described above.

In the present invention, Claudi of a Claudin-16 deficient cow is used.
On the n-16 gene, a mutation in which the positions 1 to 629 are deleted in the base sequence of the translation region encoding Claudin-16 on the normal bovine Claudin-16 gene shown in SEQ ID NO: 1 in the sequence listing is shown. (Figure 1).

[0026]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto. In the following description, unless otherwise specified, DM Glover & BD Hames (Ed.), DNA Clonin
g 1, Core Techniques (2nd edition), A Practical Ap
proach, Oxford University Press, Oxford (1995);
Sambrook, EF Fritsch & T. Maniatis (Ed.), Mole
cular cloning, a laboratory manual (2nd edition),
Cold Spring Harbor Press, Cold Spring Harbor, New
York (1989); MA Innis, DM Gelfaud, JJ Sni
ndky & TJWhite (Ed.), PCR protocols: a guide to
methods and applications, Academic Press, Inc., N
ew York (1990); MJ McPherson, P. Quirke & GR
Taylor (Ed.), PCR: a practical approach, IRL Pres
s, Oxford (1991), or a modification or modification thereof. When a commercially available reagent kit or measuring device is used, unless otherwise specified, the measurement is performed according to the protocol attached thereto.

[Experimental Example 1] (1) Determination of base sequence of normal bovine Claudin-16 gene First, fresh kidney collected from normal bovine (Japanese beef) was used as a material, and Trizol (Gibco-BRL, Inc.) was used as an RNA preparation reagent. Made)
Was used to prepare total RNA. Oligo (d
T) n Cellulose (manufactured by Sharp) was added, and the RNA fraction containing polyA was separated to obtain an mRNA fraction. Using this mRNA fraction as a template,
Superscript cDNA synthesis kit (Gibco-BRL)
Was used to synthesize cDNA. From the above cDNA, Uni-ZAP c
A cDNA library was synthesized using a DNA library synthesis kit (STRATAGENE).

On the other hand, disease cows with claudin-16 deficiency (29 cows)
And polymorphic DNA for families of both parents
Markers were used to determine the region of the bovine chromosome linked to Claudin-16 deficiency. A separately prepared bovine artificial yeast chromosome (YAC) library [Reference H. Takeda et al., Animal
Genetics, 29, 216-219 (1998)], the YAC clone present in this region was isolated, and the gene-like structural portion contained in the corresponding cosmid clone was found. The cDNA corresponding to this site was cloned from the above cDNA library, and BigDye Terminator Cycle Sequen
377 fluorescent DN with cing reagent (PE Biosystems)
The nucleotide sequence was determined using an A sequencer (manufactured by PE Biosystems). The sequence of the translation region among the obtained base sequences is shown in SEQ ID NO: 1 in the sequence listing. When the nucleotide sequence of this part was compared with other known genes, Claudi
It turned out to be a novel gene belonging to the n gene family, and was named Claudin-16.

(2) Expression of Claudin-16 gene According to the method described in Experimental Example 1- (1), an mRNA fraction was prepared from fresh kidney of the diseased cow, and the expression of Claudin-16 gene was determined according to a conventional method. Examined. The mRNA fraction was electrophoresed on a 1% agarose gel containing formaldehyde, blotted on a nylon membrane, and Northern blotting was performed to examine the expression of the Claudin-16 gene using a Claudin-16 gene fragment as a probe. It was revealed that the gene was specifically expressed and hardly expressed in the diseased cow compared with the normal cow.

(3) Determination of the base sequence of the Claudin-16 deficient bovine Claudin-16 gene As described in Experimental Example 1- (2), C
Since no expression of the laudin-16 gene was observed, the nucleotide sequence of the Claudin-16 gene on the genomic DNA was determined. Cosmid clone containing the Claudin-16 gene described in Experimental Example 1- (1), and the genomic DN of the diseased cow
The cosmid clone in the corresponding region from A was subjected to shotgun nucleotide sequencing. First, the DNA of the cosmid clone was physically shredded to a size of about 700 bp with a nebulizer, and both ends were blunted with DNA polymerase, and cloned into a plasmid. We randomly selected from these plasmid clones and read the DNA base sequence from both ends. The DNA sequence of the Claudin-16 gene in normal cattle and Claudin-16 deficient cattle was determined by connecting the nucleotide sequences with reference to the sequence of SEQ ID NO: 1 in the sequence listing. As a result of comparing the determined nucleotide sequence with that determined for the normal cattle, the claudin-16 gene of the Claudin-16 deficient cattle was found to have 1 to 1 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. Approximately 30 kb deletion including position 629 was observed. For this reason, it is considered that the expression of the claudin-16 gene is not observed in the diseased cow.

[Experimental Example 2] Confirmation of absence of mutation in the Claudin-16 gene on bovine genomic DNA: Primer DN- for amplifying a DNA fragment containing a defective site observed in Claudin-16 deficient cattle F1,
DN-F2 and DN-R were synthesized. The nucleotide sequences of primers DN-F1, DN-F2, and DN-R are shown in SEQ ID NOs: 2, 3, and 4 in the sequence listing, respectively.

DN-F1: tatgctgttg atgtttatgt ag [SEQ ID NO: 2] DN-F2: tatgtagaac gttcttctctg g [SEQ ID NO: 3] DN-R: cccccccccg cctttttc [SEQ ID NO: 4]

Blood of normal cows, cows with Claudin-16 deficiency, and their cows (including EDTA and heparin as anti-pseudosolid agents)
Genomic DNA was prepared using an automatic nucleic acid separator NA-1000 (manufactured by Kurabo Industries, Ltd.). These genomic DNA
With the primers as primers DN-F1 and DN-R, and / or DN-F2
And DN-R-based PCR (using TAKARA Taq, a 40-cycle reaction consisting of a cycle consisting of 94 ° C for 1 minute, 57 ° C for 1 minute, and 72 ° C for 1 minute). The gel was subjected to electrophoresis using a% gel (0.5 × TBE), and the gel after swimming was stained with ethidium bromide to confirm an amplified DNA fragment. When genomic DNAs of normal cows and cows of Claudin-16 deficient cows were used as templates, amplification of DNA fragments of 375 bp and / or 360 bp was observed as shown in FIG. 2a. But C
No amplification was observed when genomic DNA of laudin-16 deficient cattle was used as a template.

[Experimental Example 3] Confirmation of the presence of a mutation in the claudin-16 gene on bovine genomic DNA: genomic DNA of bovine with claudin-16 deficiency
Only to amplify the DNA fragment, primers DA-F1 and DA-F were added to both ends of the approximately 30 kb deletion portion described in Experimental Example 1- (3).
2. DA-R was synthesized. The nucleotide sequences of the primers DA-F1, DA-F2, and DA-R are shown in SEQ ID NOs: 5, 6, and 7 in the sequence listing, respectively.

DA-F1: attgtatttt taggagtgac tc [SEQ ID NO: 5] DA-F2: tgattccata aaccatacag g [SEQ ID NO: 6] DA-R: ccccccccca ctctatac [SEQ ID NO: 7]

Using the genomic DNA of a normal cow, a claudin-16 deficient cow, and its mother cow as a template, primer DA-F1 was used.
PCR using DA-R and / or DA-F2 and DA-R (TAKARA Taq
Reaction at 94 ° C for 1 minute, 57 ° C for 1 minute, and 72 ° C for 1 minute in a 40-cycle reaction), and the reaction was performed using 1.5% gel (0.5 x TBE). The gel was subjected to electrophoresis, and the gel after electrophoresis was stained with ethidium bromide to confirm an amplified DNA fragment. When genomic DNA of a Claudin-16 deficient cow and its cow was used as a template, amplification of a DNA fragment of 722 bp and / or 753 bp was observed as shown in FIG. Did not.

From these results, for a deletion of about 30 kb including the positions 1 to 629 of the translation region of the Claudin-16 gene, the cow of the affected cow was heterozygous for this mutation,
It was confirmed that Claudin-16 deficiency was a hereditary disease with chromosomal recessive inheritance.

[0038]

Industrial Applicability According to the present invention, bovine claudin-16 deficiency and its carrier can be easily and quickly detected and diagnosed.

[0039]

[Sequence List] SEQUENCE LISTING <110> Japan Livestock Technology Association <120> Gene Diagnosis for Bovine Claudin-16 Deficiency <130> Co990106 (reference number) <140> <141> <160> 7 <170> PatentIn Ver.2.0 < 210> 1 <211> 765 <212> DNA <213> Bovine <400> 1 ATGGGCCCAG GGCTGGCAGC TTCCCATGTG TCCTTTCCAG ACAGTCTGCT CGCTAAGATG 60 AGGGATCTTC TTCAGTATGT TGCCTGCTTC TTTGCTTTTT TCTCCGCTGG GTTCTTGGTT 120 GTGGCCACCT GGACAGACTG TTGGATGGTG AATGCAGATG ACTCCCTGGA GGTGAGCACA 180 AAATGCCGAG GCCTCTGGTG GGAGTGTGTC ACAAATGCTT TTGATGGAAT TCGCACCTGT 240 GATGAGTATG ATTCTATACT TGCTGAGCAC TCCTTGAAGC TGGTGGTAAC TCGGGCGCTG 300 ATGATTACTG CAGATATTCT AGCTGGATTT GGATTTATTA CTCTGCTCCT TGGACTTGAC 360 TGCGTGAAAT TCCTCCCTGA TGAGCCATAC ATCAAAGTCC GCATCAGCTT TGTTGCTGGA 420 ACCACGTTAC TAATAGCAGG TGCCCCAGGA ATAATTGGCT CTGTTTGGTA TGCTGTTGAT 480 GTTTATGTAG AACGTTCTTC TCTGGTTTTG CACAATATAT TTCTTGGCAT CCAATATAAA 540 TTTGGTTGGT CCTGTTGGCT TGGAATGGCT GGTTCTTTGG GTTGTTTTTT GGCTGGTGCT 600 ATCCTCACAT GCTGCTTATA CCTGTTCAAA GATGTTGGAC CTGAGAGGAG CTATCCTTAT 660 TCCACAAGGA AAGCCTATTC AACAACAGCT GTTTCCATGC CCCGGTCACA TGCGATCCCT 720 CGAACACAGA CTGCCAAAAT GTATGCTGTC GACACCAGGG TGTGA 765 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> nucleotide sequence for PCR <400> 2 tatgctgttg atgtttatgt ag 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 3 tatgtagaac gttcttctct g 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 4 cccccccccg cctttttc 18 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 5 attgtatttt taggagtgac tc 22 <210> 6 <211> 21 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 6 tgattccata aaccatacag g 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide to act as a primer for PCR <400> 7 ccccccccca ctctatac 18

[Brief description of the drawings]

FIG. 1. (a) Bovine Claudi derived from a cow with Claudin-16 deficiency
It is a figure which shows the deletion part of n-16 gene. (B) Position of PCR primer for detecting a defective portion.

FIG. 2 (a) is an electrophoresis pattern diagram showing that amplification is performed by PCR using genomic DNA having no mutation in the Claudin-16 gene as a template. (B) Electrophoresis pattern diagram showing that PCR is performed using genomic DNA having a mutation in the Claudin-16 gene as a template.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takashi Hirano 1 Odakura, Ogokura, Nishigo-mura, Nishishirakawa-gun, Fukushima Prefecture 1 Animal Research Institute, Animal Husbandry Technology Association (58) Fields surveyed (Int.Cl. ⁷ , DB Name) C12N 15/10-15/28 C12Q 1/68 BIOSIS (DIALOG) GenBank / EMBL / DDBJ / Geneseq JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (7)

(57) [Claims] Step (a): a step of obtaining a bovine nucleic acid sample; Step (b): subjecting the nucleic acid sample obtained in the step (a) to a gene amplification reaction to obtain a bovine Claudin-16 gene. A step of obtaining a nucleic acid fragment in which a region containing a mutation site that may exist is amplified,
And step (c): a step of examining the presence of a mutation in the nucleic acid fragment of step (b), wherein the method comprises the step of: diagnosing the bovine Claudin-16 deficiency in the bovine Claudin-16 gene. In the nucleotide sequence, 1 to 1 of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing
Gene diagnosis for bovine claudin-16 deficiency, which is a region containing position 629. 2. The method according to claim 1, wherein the gene amplification reaction is performed by a PCR (polymerase chain reaction) method. 3. The method according to claim 1, wherein the presence of the mutation is examined by PCR amplification. 4. The method according to claim 1, wherein the nucleic acid sample is genomic DNA, cDNA, or m.
The method for gene diagnosis according to any one of claims 1 to 3, which is a sample containing RNA. 5. A kit for detecting bovine claudin-16 deficiency, which is used for amplifying a region containing a mutation site which may be present in bovine claudin-16 gene by a gene amplification reaction. An oligonucleotide containing a nucleotide primer, and wherein the oligonucleotide primer has a base sequence corresponding to the 5′- terminal region of the base sequence shown in (1) SEQ ID NO: 1 in the sequence listing; and (2) a bovine bovine, which is selected from the group consisting of oligonucleotides having a complementary base sequence to the 3′- terminal region of the base sequence shown in SEQ ID NO: 1 in the sequence listing. Kit for detecting Claudin-16 deficiency. 6. A kit for detecting bovine claudin-16 deficiency, which is used for amplifying a region containing a mutation site which may be present in bovine claudin-16 gene by a gene amplification reaction. An oligonucleotide having a nucleotide sequence corresponding to a region at the 5 ' end of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing (1). Nucleotides and (2) selected from the group consisting of oligonucleotides having a complementary base sequence to the 3′- terminal region of the base sequence shown in SEQ ID NO: 1 of the sequence listing,
In addition, the kit for detecting a bovine claudin-16 deficiency, wherein the oligonucleotide primer comprises 15 to 35 nucleotides. 7. A kit for detecting bovine claudin-16 deficiency, which is used for amplifying a region containing a mutation site which may be present in bovine claudin-16 gene by a gene amplification reaction. A bovine Claudi, comprising a nucleotide primer, wherein the oligonucleotide primer is selected from the group consisting of those shown in SEQ ID NOs: 2 to 7 in the sequence listing.
Kit for detecting n-16 deficiency.