JP4129546B2 - Genetic diagnosis of bovine cartilage dysplasia dwarfism - Google Patents

Description

【００３９】
変異１型検出用のプライマーでＰＣＲを行った場合、291 bpのＤＮＡ断片のみが検出されれば正常ウシであり、291 bpと295 bpの両方のＤＮＡ断片が検出されれば変異１型のヘテロ接合ウシであり、291 bpのＤＮＡ断片のみが検出されれば変異１型による軟骨異形成性矮小体躯症ウシである。
【００４０】
変異２型検出用のプライマーでＰＣＲを行った場合、168 bpのＤＮＡ断片のみが検出されれば正常ウシであり、166 bpと168 bpの両方のＤＮＡ断片が検出されれば変異２型のヘテロ接合ウシであり、166 bpのＤＮＡ断片のみが検出されれば変異２型による軟骨異形成性矮小体躯症ウシである。
【００４１】
[その他の実施例]
本発明に用いられる核酸試料の由来は、血液に限らず、例えばそれ以外の適当な細胞又は組織由来でもよい。核酸としては、ゲノムＤＮＡに限らず、例えばｍＲＮＡ等でもよい。ｍＲＮＡの場合は、逆転写酵素によって、ｃＤＮＡが合成され、その塩基配列が調べられることが好ましいが、ｍＲＮＡの塩基配列が直接解析されてもよい。ウシの核酸試料の調整は、例えばMolecular cloning, a laboratory manual (2nd edition)（J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)）に記載の方法により行うことができる。
【００４２】
ウシＬＩＭＢＩＮ遺伝子中で変異部位を含むと考えられる領域が増幅される際、遺伝子増幅反応としてＰＣＲ法を用いたが、それに限らず、例えばＲＮＡポリメラーゼを利用した核酸増幅法や鎖置換増幅法のような核酸増幅法を利用してもよい。
【００４３】
増幅の対象となる領域としては、ウシＬＩＭＢＩＮ遺伝子の塩基配列のうち、ウシの軟骨異形成性矮小体躯症の原因となる変異を含んでいる領域であれば特に限定されず、例えば、配列表の配列番号：1に示される塩基配列の中の全体でも構わない。
【００４４】
検出する変異として、１３５６位のＣからＴへの変異あるいは２０５４〜２０５５位のＣＡからＧへの変異を対象としたが、軟骨異形成性矮小体躯症を引きおこす変異であれば、これらに限らない。
【００４５】
ＰＣＲ法で用いるプライマーとしては、上記のプライマーに限定されず、（ａ）配列表の配列番号：１に示された塩基配列のうちの連続した塩基配列を有するオリゴヌクレオチド及び（ｂ）配列表の配列番号：１に示された塩基配列のうちの連続した相補塩基配列を有するオリゴヌクレオチドで、配列番号：１に示された塩基配列において、ａのオリゴヌクレオチドに対応する塩基配列の３’末端の塩基が、ｂのオリゴヌクレオチドに相補的な配列に対応する塩基配列の５’末端の塩基より、５’末端側に存在すれば使用することができる。（ａ）配列表の配列番号：１に示された塩基配列のうちの５’端に近い領域に相当する塩基配列を有するオリゴヌクレオチド及び（２）配列表の配列番号：１に示された塩基配列のうちの３’端に近い領域に対する相補塩基配列を有するオリゴヌクレオチドを使用してもよい。プライマーの長さは、好ましくは１０〜５０個のヌクレオチドであり、より好ましくは１５〜３５個のヌクレオチドであるのがよい。また、オリゴヌクレオチドは、一つ又はそれ以上の修飾された塩基を含有していてもよく、例えば、イノシンなどの天然には存在しない塩基あるいはトリチル化された塩基などを含有していてもよい。
【００４６】
ＰＣＲの反応条件は上記の条件に限定されず、上記した文献の記載を参考に反応条件を選択することができる。ＰＣＲにおいては、ＤＮＡ鎖の熱変性、プライマーのアニーリング及びポリメラーゼによる相補鎖の合成からなる一つのサイクルが、例えば、１０〜５０回、好ましくは２０〜３５回、より好ましくは２５〜３０回繰り返して行われる。
【００４７】
軟骨異形成性矮小体躯症の原因となる変異の存在の検出方法としては、ＰＣＲ法により得られたＤＮＡ断片長を調べることにより検出されるとしたが、ハイブリダイゼーションや塩基配列の決定やＲＦＬＰ（restriction fragment length polymorphism）などによって検出してもよい。ＤＮＡ断片の塩基配列の決定は、化学分解法（Maxam & Gilbert法）、チェーンターミネーター法（Sangerジデオキシ法）などにより行うことができる。また、軟骨異形成性矮小体躯症の原因となる塩基を含む遺伝子断片を一旦ＰＣＲで増幅した後、別のプライマーを用いてＰＣＲを行うことにより、変異の有無を検出してもよい。
【００４８】
プライマーのラベル成分は、分光学的手段、光学的手段、生化学的手段、免疫学的手段、酵素化学的手段、放射化学的手段などにより検出できるものであってもよい。例えば、ペルオキシダーゼ、アルカリ性ホスファターゼなどの酵素、^３２Pなどの放射性ラベル、アイソトープ、ビオチン、ディゴキシジェニン、蛍光色素、発光物質、発色物質などが挙げられる。
【００４９】
本発明の目的、特徴、利点及びそのアイデアは、本明細書の記載により当業者には明らかであろう。以下の発明の実施の形態の項の記載及び具体的な実施例などの記載は、本発明の好ましい態様を示すものであり、説明のためにのみここにおいて示されているものであることは理解されるべきであり、本明細書で開示されている本発明の意図並びに範囲内で様々な改変並びに修飾が、それらの記載に基づいて当業者に容易に明らかになるであろう。
【００５０】
【発明の効果】
本発明により軟骨異形成性矮小体躯症ウシおよびそのキャリアを簡便かつ迅速に検出し、診断することが可能となる。
【００５１】
【配列表】

【図面の簡単な説明】
【図１】軟骨異形成性矮小体躯症ウシ由来のウシＬＩＭＢＩＮ遺伝子の1356位のCからTへの変異（変異１：1356位のCを四角形で囲んでいる）および2054〜2055位の塩基配列がCAからGへの変異（変異２：2054〜2055位のCAを四角形で囲んでいる）を示す図である。→は、変異を検出するためのＰＣＲプライマーの位置を示す。
【図２】 ＬＩＭＢＩＮ遺伝子の変異１型および変異２型を検出するための検査法を示す。変異１型では、正常型は291 bpのＰＣＲ生成物が得られるが、変異型では295 bpのＰＣＲ生成物が得られる。また、変異２型では、正常型は168 bpのＰＣＲ生成物が得られるが、変異型では166 bpのＰＣＲ生成物が得られる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a genetic diagnosis method for bovine cartilage dysplasia dwarfism.
[0002]
[Definition of terms related to the invention]
In the present specification, “LIMBIN” means a protein produced by transcription and translation of the LIMBIN gene, and “LIMBIN gene” is an exon including a translation region encoding LIMBIN and an untranslated region, and intervening between them. It means a region of DNA containing a transcription region containing an intron and a control region involved in controlling the expression of this transcription region. Cartilage dysplastic dwarfism refers to a phenotypic abnormality resulting from a mutation in the LIMBIN gene. A “mutant gene” means an allele that has undergone a mutation. “Polymerase chain reaction” or “PCR” generally refers to a method as described in US Pat. No. 4,683,195, which refers to a method for enzymatically amplifying a nucleotide sequence of interest in vitro. ing. That is, the PCR method includes repeating the process of primer extension synthesis using two oligonucleotide primers that can preferentially hybridize with a template nucleic acid.
[0003]
[Background technology]
Cartilage dysplasia dwarfism has been observed in Japanese brown cattle since 1983, and is an autosomal recessive genetic disease whose main symptom is chondrogenic dysfunction. That is, in the cattle homozygous for the causative mutant gene, short limbs are observed at 0 to 6 months of age, but the heterozygous bovine of the mutant gene has no apparent abnormality and has cartilage dysplasia. Survive as a genetic carrier for hypothyroidism. Therefore, even when cattle that do not appear abnormal are mated, even if they are mated with heterozygotes, there is a 25% probability that abnormalities will appear in the calves born. It is a problem in prevention.
[0004]
[Problems to be solved by the invention]
As a means for preventing bovine cartilage dysplasia dwarfism, a method of avoiding mating of heterozygotes of mutant genes can be considered. To that end, it is necessary to diagnose bovine cartilage dysplasia dwarfism at the gene level and find the heterozygote of the causative mutant gene. In other words, identifying the gene that causes cartilage dysplasia dwarfism and clarifying how the gene is mutated in cattle with cartilage dysplasia dwarfism makes it easier to A means that can be detected can be obtained, and a genetic diagnosis method for cartilage dysplastic dwarfism can be established.
[0005]
An object of the present invention is to provide a genetic diagnosis method for bovine cartilage dysplasia dwarfism based on such a principle.
[0006]
[Means for Solving the Problems]
As a result of researches to achieve the above object, the present inventors have succeeded in identifying a LIBIN gene that is closely related to cartilage dysplasia dwarfism. Furthermore, the present inventors detected a mutation in the translation region of the LIMBIN gene obtained from an individual with cartilage dysplasia dwarfism. And it discovered that it was the cause of this disease that the normal function of LIBIN was impaired by these mutations. Therefore, it has been clarified that when a mutation occurs in the LIMBIN gene and the normal function of LIMBIN is impaired, cartilage dysplasia hypothalasia is developed.
[0007]
Therefore, the genetic diagnosis method of the present invention comprises a step of obtaining a bovine nucleic acid sample, a base sequence represented by SEQ ID NO: 1 in the bovine LIMBIN gene, and the cause of bovine cartilage dysplasia dwarfism. The method includes a step of obtaining a nucleic acid fragment containing a certain base from the nucleic acid sample by a gene amplification reaction and a step of examining the presence or absence of a mutation in the base. This makes it possible to detect an individual having a LIBIN mutant gene mutated in the translation region of the LIMBIN gene, and to find a genetic carrier for cartilage dysplastic dwarfism.
[0008]
Further, the base may be included in the base sequence represented by SEQ ID NO: 1 in the base sequence of the LIBIN gene. Thereby, the variation | mutation in the translation area | region of a LIMBIN gene can be made into the object of gene diagnosis.
[0009]
The gene amplification reaction may include PCR (polymerase chain reaction). Thereby, a gene amplification reaction can be performed simply.
[0010]
Further, the step of examining the presence or absence of a base mutation that causes cartilage dysplasia parathyroidism may include PCR (polymerase chain reaction). This makes it possible to easily detect base mutations.
[0011]
Further, the nucleic acid sample may be a sample containing genomic DNA, cDNA, or mRNA. This makes it possible to handle molecules that actually carry genetic information.
[0012]
Furthermore, a first oligonucleotide having a continuous base sequence contained in the base sequence shown in SEQ ID NO: 1, and a continuous base contained in a base sequence complementary to the base sequence shown in SEQ ID NO: 1. A second oligonucleotide having a sequence, wherein the base sequence at the 3 ′ end of the base sequence of the first oligonucleotide is a base sequence represented by SEQ ID NO: 1. It may be a kit for detecting cartilage dysplastic dwarfism characterized by being present on the 5 ′ end side of the 5 ′ end base of the base sequence complementary to the base sequence possessed. Thereby, when detecting the individual | organism | solid with the nucleic acid which caused the variation | mutation to the LIBIN gene, the effort which adjusts each reagent separately can be saved.
[0013]
Further, the first oligonucleotide and the second oligonucleotide may be composed of 15 to 35 nucleotides. This improves the efficiency of the gene amplification reaction, particularly PCR.
[0014]
The first oligonucleotide may be any of the oligonucleotides of SEQ ID NOs: 10, 11, and 12, and the second oligonucleotide is of SEQ ID NOs: 8, 9, and 13 It may be any one of oligonucleotides. This further improves the efficiency of gene amplification reactions, particularly PCR.
[0015]
Nucleotides having the following base sequences (a) to (c) may be used.
(A) The entire base sequence represented by SEQ ID NO: 1, or a part thereof.
(B) The entire base sequence complementary to the base sequence represented by SEQ ID NO: 1, or a part thereof.
(C) In the nucleotides (a) and (b), one or several nucleotides are deleted, substituted, or added, and the entire base sequence of the LIMBIN gene or a part thereof.
Thereby, the variation | mutation in the translation area | region in a LIMBIN gene can be made into the object of gene diagnosis.
[0016]
The nucleotide having the nucleotide sequence may be any of genomic DNA, cDNA, RNA, synthetic nucleotide, or semi-synthetic nucleotide. This makes it possible to handle molecules that actually carry or transmit genetic information.
[0017]
Further, oligonucleotides having the following base sequences (a) to (c) may be used.
(A) A continuous nucleotide sequence included in the nucleotide sequence having the base sequence shown in SEQ ID NO: 1.
(B) A continuous nucleotide sequence contained in a nucleotide sequence having a base sequence complementary to the base sequence shown in SEQ ID NO: 1.
(C) A nucleotide that consists of a nucleotide sequence in which one or several nucleotides are deleted, substituted, or added in the nucleotide sequences of (a) and (b), and can be used in a gene amplification reaction of the LIBIN gene.
By using this nucleotide as a template for LIBIN gene amplification, a gene amplification reaction can be carried out specifically for the LIBBIN gene translation region.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
<Analysis of LIMBIN gene in normal cattle and cattle with dysplasia of cartilage dysplasia>
The present inventors first performed a genome linkage analysis of a family including the diseased cow, and isolated the bovine LIMBIN gene using the positional cloning method. An example in which the LIBIN gene has been isolated has not been reported so far in any species. In order to investigate the relationship between gene mutation and this disease, cDNA encoding normal bovine LIBIN was isolated and its nucleotide sequence was clarified. The entire base sequence of the cDNA encoding the obtained normal bovine LIMBIN is shown in SEQ ID NO: 1 in the sequence listing.
[0019]
Next, the mutation of the gene causing cartilage dysplasia dwarfism was clarified by comparing the nucleotide sequences of the LIBIN gene cDNAs of normal and affected cattle. That is, as in the case of normal cattle, the nucleotide sequence of the LIMBIN gene cDNA of the affected cattle was examined, and this was compared with that of the normal bovine gene to identify the mutation causing the disease. In the LIMBIN gene of a cartilage dysplastic bovine corporal bovine, a mutation from C to T at position 1356 or a mutation from CA to G at positions 2054 to 2055 was found in the base sequence of the translation region. These were mutations that resulted in a new stop codon (FIG. 1). Therefore, it was concluded that these mutations are mutations in the gene causing cartilage dysplasia dwarfism.
[0020]
Hereinafter, an experimental example will be described in more detail. In the following description, DM Glover & BD Hames (Ed.), DNA Cloning 1, Core Techniques (2nd edition), A Practical Approach, Oxford University Press, Oxford (1995); J. Sambrook, unless otherwise stated. , EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (2nd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (1989); MA Innis, DM Gelfaud, JJ Snindky & TJ White ( Ed.), PCR protocols: a guide to methods and applications, Academic Press, Inc., New York (1990); MJ McPherson, P. Quirke & GR Taylor (Ed.), PCR: a practical approach, IRL Press, Oxford (1991), or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
[0021]
[Experimental example]
(1) Determination of the base sequence of normal bovine LIMBIN gene
For the cattle dysplastic dwarfism cattle (119 heads) and their parents, the 6th region of the bovine chromosome linked to cartilage dysplastic dwarfism using polymorphic DNA markers It was determined as the telomere part of the chromosome [Literature K. Yoneda et al., Mammalian Genome, 10, 597-600 (1999)]. The region where the causative gene for cartilage dysplasia parathyroidism is present was further narrowed using a number of polymorphic DNA markers. Since the gene (EST) present in the narrowed region and actually expressed could be identified by the EST search, this gene was considered as a candidate gene for the cause of cartilage dysplasia dwarfism. In order to clone the cDNA, PCR primers of SEQ ID NOs: 2 and 3 were synthesized from the sequence AW345074.
LIBIN-1: GAGAGAGGGTGATATTCTCTGG [SEQ ID NO: 2]
LIMBIN-2: GATAAAGAGCTTTTCACCCGTG [SEQ ID NO: 3]
[0022]
By the way, since the phenotypic abnormality due to abnormality of the causative gene appears in the cartilage, it was expected that the causative gene was expressed in the cartilage. Therefore, total RNA was prepared using Trizol (trade name, manufactured by Gibco-BRL), a reagent for RNA preparation, using the epiphysis of a fresh long bone collected from a normal cow as a material. Using this total RNA as a template, cDNA was synthesized using a Superscript cDNA synthesis kit (trade name, manufactured by Gibco-BRL). PCR was performed using the primers of SEQ ID NOs: 2 and 3 and the synthesized cDNA as a template to amplify a fragment between the primers. The base sequence of the product was determined with a BigDye Terminator Cycle Sequencing reagent (trade name, manufactured by PE Biosystems) using a 3700 fluorescent DNA sequencer (PE Biosystems). Since this base sequence showed high homology with the base sequence of the above-described bovine EST and the corresponding base sequence of human EST, it was concluded that the base sequence was a part of the target bovine LIBIN cDNA.
[0023]
Therefore, using this fragment as a probe, a normal bovine kidney and long bone cDNA library was screened, and a longer bovine LIBIN cDNA clone was isolated. The bovine EST sequences are the predicted gene sequences C4000113 and C4000114 in Working Draft Genome Browser v5 published by the University of California Human Genome Project on the Internet (and the bovine RPCI-42 bacterial artificial chromosome 162M16 located in the chromosomal region). Since a high homology was observed in the base sequences of the 232M6 clone and 232M6 clone, cDNA was prepared from normal bovine total RNA as a template and amplified with PCR primers of SEQ ID NOs: 4, 5, 6, and 7 to obtain cDNA clones.
[0024]
The base sequences of these cDNAs were determined and joined together to determine the base sequence of LIBIN cDNA including the entire translation region. Among the obtained base sequences, the sequence of the translation region is shown in SEQ ID NO: 1 in the sequence listing.
LIBIN-3: GGAAGTATCTGGCCCATTTTGCC [SEQ ID NO: 4]
LIBIN-4: CTCCAGCGAGGCTGTGTAGCTGAC [SEQ ID NO: 5]
LIBIN-5: GGCTTTCAGGAAGAAGTTTCTGC [SEQ ID NO: 6]
LIBIN-6: CTCCAGGAGCTTCTGACCCTTGC [SEQ ID NO: 7]
[0025]
(2) LIMBIN gene expression
Using mRNA extraction kit, FastTrack 2.0 (trade name, manufactured by Invitrogen), mRNA was prepared from fresh tissues of normal cattle, and a superscript cDNA synthesis kit (trade name, manufactured by Gibco-BRL) was used. CDNA was synthesized and examined for expression of the LIMBIN gene. When PCR was performed using cDNA of each tissue as a template, it was expressed in all tissues examined such as epiphysis, kidney, lung, heart, spleen, brain, etc., and almost the same in the epiphysis of normal cattle and the diseased cattle It was clarified that it was expressed at the intensity of.
[0026]
(3) Determination of the nucleotide sequence of the cartilage dysplasia dwarfism bovine LIMBIN gene
As described in Experimental Example (2), since the transcription of the LIMBIN gene was observed in cattle with dysplasia of cartilage dysplasia, it was expected that the abnormality was in the base sequence itself, so the base sequence was determined. The base sequence of the normal bovine LIMBIN gene was compared. As a result, mutations were detected at two locations. Mutation 1 results in a new stop codon due to the formation of a potential splicing donor site that mutates C at position 1356 to T, and mutation 2 results in a new stop codon due to mutation of the nucleotide sequence at positions 2054-2055 from CA to G Bring. Therefore, it was considered that in cattle with dysplastic dysplasia cattle, the function of LIMBIN was impaired due to these mutations, and cartilage dysplastic dysplastic podosis developed.
[0027]
(4) Detection of mutation of LIBIN gene on bovine genomic DNA
In order to investigate the correlation between the mutation of the LIMBIN gene and the onset of cartilage dysplastic dysplasia, the bases of the mutation type 1 site and the mutation type 2 site of the LIBIN gene in normal cattle, cattle dysplastic dwarfism cattle and their parents I investigated.
[0028]
First, a primer for amplifying a DNA fragment containing a mutation type 1 site found in a cartilage dysplastic bovine scab was synthesized. LIBIN-R1 is an antisense primer that specifically binds to a normal base sequence, and LIBIN-R2 is an antisense primer that specifically binds to a mutant type 1 base sequence. Four inosines were added to the 5 ′ end of LIBIN-R2 in order to make a difference in the length of PCR products produced by these primers. In addition, for the purpose of suppressing non-specific annealing, the antisense primer has one base different from the original sequence. LIBIN-F1 is a sense primer that binds to a base sequence common to normal and mutant type 2, and a fluorescent dye FAM is added to the 5 ′ end.
[0029]
The base sequences of the primers are shown in SEQ ID NOs: 8, 9, and 10 in the sequence listing. I represents inosine.
LIBIN-R1: TTGCTGCCTCTGGCTTCAGCTGG [SEQ ID NO: 8]
LIMBIN-R2: IIIITTGCTGCCTCTGGCTTCAGGTCA [SEQ ID NO: 9]
LIBIN-F1: [FAM] -TGTGTGCTCACATGTTGCGTGC [SEQ ID NO: 10]
[0030]
Next, a primer for amplifying a DNA fragment containing a mutation type 2 site found in a cartilage dysplastic dwarf cattle was synthesized. LIBIN-F2 is a sense primer that specifically binds to a normal base sequence, and LIMBIN-F3 is a sense primer that specifically binds to a mutant type 2 base sequence. In order to make a difference in the length of the PCR product by these primers, the base at the 5 ′ end was deleted. LIBIN-R3 is an antisense primer that binds to a base sequence common to normal and variant 2 and has a fluorescent dye FAM added to the 5 'end.
[0031]
The nucleotide sequences of the primers are shown in SEQ ID NOs: 11, 12, and 13 in the sequence listing.
LIMBIN-F2: GAGCACAGCAGGGACCTGGCA [SEQ ID NO: 11]
LIMBIN-F3: AGCACAGCAGGGACCTGGG [SEQ ID NO: 12]
LIMBIN-R3: [FAM]-CGTGAAGATCAAGTGCTCCCAGTG [SEQ ID NO: 13]
[0032]
On the other hand, an automatic nucleic acid separator NA-1000 (trade name, manufactured by Kurabo Industries Co., Ltd.) is obtained from the blood of normal cattle, cattle dysplastic dysplasia cattle, and their parental cattle (including EDTA and heparin as anti-immortal agents). Genomic DNA was prepared. PCR using these genomic DNAs as a template and primers for detection of mutation type 1 (using Taq DNA polymerase, 94 ° C for 20 seconds, 67 ° C for 20 seconds, 72 ° C for 30 seconds) PCR using Taq DNA polymerase, 20 seconds at 94 ° C, 20 seconds at 65 ° C, and 30 seconds at 72 ° C for 1 cycle. And 28 cycles were performed). 0.5 microliters of these reaction solutions were electrophoresed using a 3700 fluorescent DNA sequencer (trade name, manufactured by PE Biosystems), and the size of the amplified DNA fragment was determined using GENESCAN and Genotyper software (trade name, manufactured by PE Biosystems). It measured using. As shown in FIG. 2, when PCR is carried out with primers for detecting mutation type 1, a normal type produces a 291 bp DNA fragment, whereas mutation type 1 produces a 295 bp DNA fragment. In addition, when PCR is carried out with the primer for detecting mutation type 2, a 168 bp DNA fragment is generated in the normal type, but a 166 bp DNA fragment is generated in the mutation type 2.
[0033]
Thus, as a result of investigating the correlation between the onset of cartilage dysplastic dwarfism and the mutation of the LIBIN gene, as shown in FIG. 2, the onset of cartilage dysplastic dwarfism is caused by the mutation of the LIMBIN gene. It became clear.
[0034]
<Establishment of genetic diagnosis for bovine cartilage dysplasia thymobody disease>
As described above, since a mutation in a gene causing bovine cartilage dysplasia dwarfism has been elucidated (FIG. 1), the mutation can be detected using this mutation. Hereinafter, an example of a specific genetic diagnosis method for bovine cartilage dysplasia parathyroidism will be described in detail.
[0035]
[Example]
As a step of obtaining a bovine nucleic acid sample, genomic DNA is prepared from bovine blood (including EDTA and heparin as an anti-pseudo-solid agent) using an automatic nucleic acid separation device NA-1000 (trade name, manufactured by Kurabo Industries).
[0036]
As a step of obtaining a nucleic acid fragment containing a base that can contain mutation type 1 and mutation type 2 by a gene amplification reaction, PCR (Taq DNA polymerase) is performed using the adjusted genomic DNA as a template and a primer for detection of mutation type 1. PCR using Taq DNA polymerase, a primer for detection of mutation type 2 (using 27 cycles of 94 cycles for 20 seconds at 67 ° C, 20 seconds at 67 ° C, 30 seconds at 72 ° C) , A 28-cycle reaction comprising a cycle consisting of 94 ° C. for 20 seconds, 65 ° C. for 20 seconds, and 72 ° C. for 30 seconds) to amplify a gene fragment considered to contain a mutation. PCR method: MA Innis, DM Gelfaud, JJ Snindky, & TJ White (Ed.), PCR protocols: a guide to methods and applications, Academic Press, Inc., New York (1990); MJ McPherson, P. Quirke & GR Taylor (Ed.), PCR: a practical approach, IRL Press, Oxford (1991); RK Saiki et al., Science, 239, 487-491 (1988); MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988) and the like, or modified or modified methods thereof. The PCR method can be performed using a commercially available kit suitable for the PCR method, and can also be performed according to a protocol recommended by the kit manufacturer or the kit distributor. Primers are prepared by known methods such as described in Agnew. Chem. Int. Ed. Engl., Vol. 28, p. 716-734 (1989), such as triester method, phosphite method, phosphoramidite method. And can be chemically synthesized by a method such as a phosphonate method. It is known that normal synthesis can be conveniently performed on a modified solid support. For example, the synthesis reaction can be performed using an automated synthesizer, and such an apparatus is commercially available.
[0037]
As a step to examine the presence or absence of mutation type 1 or mutation type 2, 0.5 microliters of these reaction solutions were electrophoresed using a 3700 fluorescent DNA sequencer (trade name, manufactured by PE Biosystems) to determine the size of the amplified DNA fragment. , Measured using GENESCAN and Genotyper software (trade name, manufactured by PE Biosystems).
[0038]
The results are shown in the table.
[Table 1]

[0039]
When PCR was carried out using primers for detecting mutation type 1, normal bovines were detected if only the 291 bp DNA fragment was detected, and mutation type 1 heterozygous was detected if both 291 bp and 295 bp DNA fragments were detected. If it is a zygotic cow and only a 291 bp DNA fragment is detected, it is a cartilage dysplastic dwarfism cattle due to mutation type 1.
[0040]
When PCR was performed with primers for detection of mutation type 2, normal bovines were detected if only a 168 bp DNA fragment was detected, and mutation type 2 heterozygous was detected if both 166 bp and 168 bp DNA fragments were detected. If it is a zygotic cow and only a 166 bp DNA fragment is detected, it is a cartilage dysplastic dwarf cattle due to mutation type 2.
[0041]
[Other examples]
The origin of the nucleic acid sample used in the present invention is not limited to blood, but may be derived from other appropriate cells or tissues, for example. The nucleic acid is not limited to genomic DNA but may be, for example, mRNA. In the case of mRNA, cDNA is preferably synthesized by reverse transcriptase and its base sequence is examined, but the base sequence of mRNA may be directly analyzed. Preparation of bovine nucleic acid samples is, for example, Molecular cloning, a laboratory manual (2nd edition) (J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)) It can carry out by the method as described in.
[0042]
When a region considered to contain a mutation site in the bovine LIMBIN gene is amplified, the PCR method is used as a gene amplification reaction. However, the present invention is not limited to this, for example, a nucleic acid amplification method using RNA polymerase or a strand displacement amplification method. A simple nucleic acid amplification method may be used.
[0043]
The region to be amplified is not particularly limited as long as it contains a mutation that causes bovine cartilage dysplasia dwarfism in the base sequence of the bovine LIBIN gene. The entire nucleotide sequence shown in SEQ ID NO: 1 may be used.
[0044]
As a mutation to be detected, a mutation from C to T at position 1356 or a mutation from CA to G at positions 2054 to 2055 was targeted, but it is not limited to these as long as it causes cartilage dysplasia dwarfism. .
[0045]
Primers used in the PCR method are not limited to the above primers, and (a) an oligonucleotide having a continuous base sequence of the base sequences shown in SEQ ID NO: 1 of the sequence listing and (b) the sequence listing An oligonucleotide having a continuous complementary base sequence of the base sequence shown in SEQ ID NO: 1, wherein the base sequence shown in SEQ ID NO: 1 is the 3 'end of the base sequence corresponding to the oligonucleotide a. It can be used if the base is present on the 5 ′ end side from the 5 ′ end base of the base sequence corresponding to the sequence complementary to the oligonucleotide b. (A) an oligonucleotide having a base sequence corresponding to a region close to the 5 ′ end of the base sequence shown in SEQ ID NO: 1 in the sequence listing and (2) a base shown in SEQ ID NO: 1 in the sequence listing An oligonucleotide having a complementary base sequence for the region close to the 3 ′ end of the sequence may be used. The length of the primer is preferably 10 to 50 nucleotides, more preferably 15 to 35 nucleotides. In addition, the oligonucleotide may contain one or more modified bases, for example, a non-naturally occurring base such as inosine or a tritylated base.
[0046]
PCR reaction conditions are not limited to the above-mentioned conditions, and reaction conditions can be selected with reference to the descriptions in the above-mentioned literature. 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.
[0047]
As a method for detecting the presence of a mutation causing cartilage dysplasia dwarfism, it was detected by examining the length of a DNA fragment obtained by the PCR method. However, hybridization, nucleotide sequence determination, RFLP ( restriction fragment length polymorphism). Determination of the base sequence of a DNA fragment can be performed by a chemical decomposition method (Maxam & Gilbert method), a chain terminator method (Sanger dideoxy method), or the like. Alternatively, the presence or absence of the mutation may be detected by once amplifying a gene fragment containing a base causing cartilage dysplasia dwarfism by PCR and then performing PCR using another primer.
[0048]
The label component of the primer may be detectable by spectroscopic means, optical means, biochemical means, immunological means, enzymatic chemistry means, radiochemical means and the like. For example, enzymes such as peroxidase, alkaline phosphatase, ³² Examples include radioactive labels such as P, isotopes, biotin, digoxigenin, fluorescent dyes, luminescent substances, and coloring substances.
[0049]
Objects, features, advantages and ideas of the present invention will be apparent to those skilled in the art from the description herein. It should be understood that the following description of the embodiments of the invention and descriptions of specific examples show preferred embodiments of the present invention and are presented here for illustrative purposes only. Various changes and modifications within the spirit and scope of the invention disclosed herein will be readily apparent to those skilled in the art based on the description thereof.
[0050]
【The invention's effect】
According to the present invention, it becomes possible to easily and rapidly detect and diagnose a cartilage dysplastic bovine corporal bovine and its carrier.
[0051]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1: C-to-T mutation at position 1356 of bovine LIBIN gene derived from cattle with dysplasia of cartilage dysplasia (mutation 1: C at position 1356 is surrounded by a rectangle) and nucleotide sequences from 2054 to 2055 Is a diagram showing a mutation from CA to G (mutation 2: CA at positions 2054 to 2055 is surrounded by a rectangle). → indicates the position of the PCR primer for detecting the mutation.
FIG. 2 shows a test method for detecting mutation type 1 and mutation type 2 of the LIMBIN gene. Mutant type 1 yields a 291 bp PCR product, while mutant type yields a 295 bp PCR product. In the mutant type 2, a 168 bp PCR product is obtained in the normal type, whereas a 166 bp PCR product is obtained in the mutant type.

Claims (7)

Obtaining a bovine nucleic acid sample;
SEQ ID NO: 1 includes a nucleotide corresponding to position 1356 in the base sequence shown in SEQ ID NO: 1, a nucleic acid fragment having the base sequence shown in SEQ ID NO: 1 and bases corresponding to positions 2054 and 2055, Obtaining at least one nucleic acid fragment of the nucleic acid fragments having the base sequence shown from the nucleic acid sample by a gene amplification reaction;
A step of examining whether the base corresponding to position 1356 in the obtained nucleic acid fragment is C or T and whether the base corresponding to positions 2054 and 2055 is CA or G;
A genetic diagnostic method for bovine chondrogenic dwarfism characterized by comprising   The genetic diagnosis method for bovine chondrogenic dwarfism according to claim 1, wherein the gene amplification reaction includes PCR (polymerase chain reaction).   2. The genetic diagnosis method for bovine chondrogenic dwarfism according to claim 1, wherein the step of examining the presence or absence of a mutation in the base comprises PCR (polymerase chain reaction).   The genetic diagnosis method for bovine chondrogenic dwarfism according to any one of claims 1 to 3, wherein the nucleic acid sample is a sample containing genomic DNA, cDNA, or mRNA. SEQ ID NO: 1 includes a nucleotide corresponding to position 1356 in the base sequence shown in SEQ ID NO: 1, a nucleic acid fragment having the base sequence shown in SEQ ID NO: 1 and bases corresponding to positions 2054 and 2055, Two oligonucleotides serving as primers for obtaining at least one of the nucleic acid fragments having the indicated base sequence from a bovine nucleic acid sample by a gene amplification reaction,
One of the oligonucleotides consists of a part of a complementary strand of a polynucleotide having the base sequence shown in SEQ ID NO: 1.
A kit for assaying bovine chondrogenic dwarfism using the genetic diagnostic method according to claim 1.   The kit according to claim 5, wherein the two oligonucleotides are composed of 15 to 35 nucleotides.   The kit according to claim 6, wherein the two oligonucleotides are an oligonucleotide selected from SEQ ID NOs: 10, 11, and 12 and an oligonucleotide selected from SEQ ID NOs: 8, 9, and 13.

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