JP2570928B2 - Bovine DNA, DNA probe, vector and bovine sex determination method - Google Patents
Bovine DNA, DNA probe, vector and bovine sex determination methodInfo
- Publication number
- JP2570928B2 JP2570928B2 JP3245732A JP24573291A JP2570928B2 JP 2570928 B2 JP2570928 B2 JP 2570928B2 JP 3245732 A JP3245732 A JP 3245732A JP 24573291 A JP24573291 A JP 24573291A JP 2570928 B2 JP2570928 B2 JP 2570928B2
- Authority
- JP
- Japan
- Prior art keywords
- dna
- bovine
- probe
- sequence
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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- 238000000034 method Methods 0.000 title claims description 25
- 239000003298 DNA probe Substances 0.000 title claims description 12
- 108020003215 DNA Probes Proteins 0.000 title claims description 11
- 230000020509 sex determination Effects 0.000 title description 4
- 238000002372 labelling Methods 0.000 claims description 5
- 244000309464 bull Species 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
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- 210000001182 human Y chromosome Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Description
【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION
【０００１】[0001]
【産業上の利用分野】本発明はウシの雌雄判別法に有用
なＤＮＡ、ＤＮＡプローブ、該ＤＮＡを含むベクター及
びウシの雌雄判別法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA, a DNA probe, a vector containing the DNA and a method for discriminating bovine sex, which are useful for bovine sex determination.
【０００２】[0002]
【従来の技術】家畜繁殖学の分野で胚や胎児の性別判定
が重要視され始めている。例えば、乳業分野においては
人工授精が日常的に行われているが、人工授精させた卵
の性別を母体に移植する前に知ることができれば、極め
て有利になる。つまり、乳を出す雌のみを増やすことが
できるようになるからである。2. Description of the Related Art In the field of livestock reproductive science, gender determination of embryos and fetuses is beginning to be regarded as important. For example, in the dairy industry, artificial insemination is routinely performed, but it would be extremely advantageous if the sex of the artificially inseminated egg could be known before transplantation into the mother. In other words, it is possible to increase the number of females that supply milk.
【０００３】ほ乳類の性別が性染色体によって決定され
ることは良く知られている。発生初期にＹ染色体は未分
化の生殖腺を精巣に分化させるのに重要な役目をしてい
る。従って、Ｙ染色体に特異的な遺伝子を解析すること
によって雌雄の判別ができる。It is well known that the sex of mammals is determined by sex chromosomes. Early in development, the Y chromosome plays an important role in differentiating undifferentiated gonads into testes. Therefore, by analyzing a gene specific to the Y chromosome, sex can be determined.
【０００４】最近、Ｙ染色体に特異的な遺伝子やＤＮＡ
を用いた雌雄判別法が報告されている。例えば、シンク
レア(Sinclair)らは、ヒトのＹ染色体上の未分化生殖腺
を精巣に分化させるＤＮＡの塩基配列の候補を発表し
た。さらに、彼らはその塩基配列の一部が哺乳類で良く
保存されている可能性を示唆している（ネイチャー（NA
TURE）,Vol346,19 JULY1990)。また、特開昭６３−５０
０２１４号公報には雄のウシのＤＮＡとハイブリダイズ
するウシのＤＮＡプローブが記載されている。Recently, genes and DNAs specific to the Y chromosome
A gender discriminating method has been reported. For example, Sinclair et al. Have published a candidate DNA sequence that allows undifferentiated gonads on the human Y chromosome to differentiate into testis. Furthermore, they suggest that some of their sequences may be well conserved in mammals (Nature (NA
TURE), Vol 346, 19 JULY 1990). Also, JP-A-63-50
No. 0214 discloses a bovine DNA probe that hybridizes with male bovine DNA.
【０００５】[0005]
【発明が解決しようとする課題】前記のシンクレアらの
ＤＮＡをプローブとして雌雄判別を行うこともできる
が、該ＤＮＡは人由来のものでありその特異性に問題が
残る。また、前記の特開昭６３−５００２１４号公報記
載のプローブはその配列が長すぎる点が問題である。Although gender discrimination can be performed using the DNA of Sinclair et al. As a probe, the DNA is of human origin and its specificity remains a problem. Further, the probe described in JP-A-63-500214 has a problem that the sequence is too long.
【０００６】本発明は、このような状況に鑑み、より簡
易でかつ高感度にウシの雌雄判別を行うことのできる、
雌雄判別法に有用なウシのＤＮＡ、ＤＮＡプローブ及び
該ＤＮＡを含むベクターの提供並びにウシの雌雄判別法
の提供を目的とする。The present invention has been made in view of the above circumstances, and enables sex determination of cattle to be performed more easily and with high sensitivity.
It is an object of the present invention to provide bovine DNA, a DNA probe and a vector containing the DNA, which are useful for gender discrimination, and to provide a bovine gender discrimination method.
【０００７】[0007]
【課題を解決するための手段】すなわち本発明は、雄ウ
シのＤＮＡを下記のプライマー 5'-AAGCGACCCA TGAACGCATT CATCGTGTGGT-3' および 5'-GAGGTCGATA CTTATAATTC GGGTATTTCTCTCTGTG-3' を用いてＰＣＲ法で増幅して得られることを特徴とする
ウシのＤＮＡに関する。That is, according to the present invention, bull DNA is amplified by PCR using the following primers 5'-AAGCGACCCA TGAACGCATT CATCGTGTGGT-3 'and 5'-GAGGTCGATA CTTATAATTC GGGTATTTCTCTCTGTG-3'. A bovine DNA characterized in that it is obtained.
【０００８】また本発明は、次式配列（Ｉ）で示される
塩基配列を持つウシのＤＮＡに関する。 配列（Ｉ） 5'-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3' (但し、RはAまたはGを示す)[0008] The present invention also relates to a bovine DNA having a base sequence represented by the following formula (I). Sequence (I) 5′-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3 ′ (where R represents A or G)
【０００９】さらに本発明は、上記のウシのＤＮＡを含
有し、これを標識して得られるＤＮＡプローブ、上記の
ウシのＤＮＡを含有するベクター及び上記のウシのＤＮ
Ａに相補的なＤＮＡに関する。Further, the present invention includes the above-mentioned bovine DNA.
A DNA probe obtained by labeling the same, a vector containing the above bovine DNA, and a bovine DN
For DNA complementary to A.
【００１０】さらに本発明は、前記ＤＮＡプローブを用
いることを特徴とするウシの雌雄判別法に関する。Further, the present invention relates to a method for discriminating male and female cattle comprising using the DNA probe.
【００１１】本発明における、プローブとして有用なウ
シのＤＮＡ断片は、前記プライマーを用いてＰＣＲ法
（Polymerase chain reaction）で増幅して用いること
ができるが、その工程は、例えば以下のようにして行い
得る。まず、ウシの新鮮なまたはホルマリン等で固定し
た白血球、精巣、肝臓、胸腺などの出発物質に、界面活
性剤やプロテイナーゼＫ等により酵素処理を行いタンパ
ク質を消化した後、フェノール、クロロホルム、イソプ
ロピルアルコールで抽出し、エタノール沈澱により精製
回収してＤＮＡを抽出する。In the present invention, a bovine DNA fragment useful as a probe can be used by amplifying it by the PCR method (Polymerase chain reaction) using the above primers. obtain. First, the starting material such as fresh bovine or fixed with formalin, etc., leukocytes, testis, liver, thymus, etc., is subjected to an enzyme treatment with a surfactant, proteinase K, etc. to digest the protein, followed by phenol, chloroform, isopropyl alcohol. The DNA is extracted and purified by ethanol precipitation to extract DNA.
【００１２】この抽出したＤＮＡをＰＣＲ法により増幅
する。ＰＣＲ法は公知のＤＮＡの増幅法である。このＰ
ＣＲ法に用いるプライマーは以下の通りで、ＤＮＡ合成
機（例えば、ＡＢＩ社製）で合成して用いる。 5'-AAGCGACCCA TGAACGCATT CATCGTGTGGT-3' 5'-GAGGTCGATA CTTATAATTC GGGTATTTCT CTCTGTG-3' ＰＣＲ法に用いるＤＮＡ量は、テンプレートＤＮＡは１
μｇ程度とし、プライマーＤＮＡは１００ｐｍｏｌ程度
とするのが好ましい。ＰＣＲ法の条件は常法に従うこと
ができる。[0012] The extracted DNA is amplified by the PCR method. The PCR method is a known DNA amplification method. This P
The primers used for the CR method are as follows, and are synthesized and used by a DNA synthesizer (for example, manufactured by ABI). 5'-AAGCGACCCA TGAACGCATT CATCGTGTGGT-3 '5'-GAGGTCGATA CTTATAATTC GGGTATTTCT CTCTGTG-3'
It is preferable to use about μg and the primer DNA to be about 100 pmol. The conditions for the PCR method can be in accordance with a conventional method.
【００１３】このようにして得られたＰＣＲ産物をアガ
ロースゲル電気泳動にかけ、用いたプライマーから推定
される鎖長を有するＤＮＡ断片のみを得る。そして、こ
のＤＮＡ断片を適当なプラスミド等のベクターに挿入す
る（例えば、インビトロージェン社のプラスミドｐＣＲ
１０００など）。続いて、このプラスミドを大腸菌など
の適当な宿主細胞に導入して形質転換し、該形質転換体
を適当な抗生物質が入った培地に植えて増殖させる。The PCR product thus obtained is subjected to agarose gel electrophoresis to obtain only a DNA fragment having a chain length estimated from the primers used. Then, this DNA fragment is inserted into a vector such as an appropriate plasmid (for example, plasmid pCR of Invitrogen).
1000 etc.). Subsequently, this plasmid is introduced into a suitable host cell such as Escherichia coli for transformation, and the transformant is planted and grown on a medium containing a suitable antibiotic.
【００１４】増殖後、形質転換体の有するプラスミドを
抽出し、陽性クローンを調べる。例えば、プラスミドを
適当な制限酵素で切断し、挿入ＤＮＡの鎖長を調べ、目
的の長さを持つ挿入ＤＮＡを有するクローンを陽性と判
定することができる。または、精製したＰＣＲ産物を標
識してプローブとし、プラスミドとコロニーハイブリダ
イゼイションを行い陽性を決定することもできる。After the propagation, the plasmid contained in the transformant is extracted and positive clones are examined. For example, a plasmid is cleaved with an appropriate restriction enzyme, the chain length of the inserted DNA is examined, and a clone having the inserted DNA having the desired length can be determined to be positive. Alternatively, it is also possible to label the purified PCR product as a probe and perform colony hybridization with the plasmid to determine the positive.
【００１５】得られた陽性クローンのプラスミドを適当
な制限酵素で消化して、本発明のＤＮＡを得ることがで
きる。得られたＤＮＡの塩基配列はジデオキシ法に従っ
て決定した。その結果、使用したプライマー部分を除
き、以下の配列を持っていた（配列（Ｉ））。 5'-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3'(146bp)The plasmid of the obtained positive clone is digested with an appropriate restriction enzyme to obtain the DNA of the present invention. The nucleotide sequence of the obtained DNA was determined according to the dideoxy method. As a result, it had the following sequence (sequence (I)) except for the used primer portion. 5'-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3 '(146bp)
【００１６】本発明のＤＮＡプローブは、前記のウシの
ＤＮＡを標識して得られる。標識化は、32Ｐ、3Ｈ、14
Ｃ等のような放射性同位元素による標識、ビオチン化等
の化学標識など、公知の種々の標識が適用可能であり、
標識する方法も特に制限されない。The DNA probe of the present invention comprises
Obtained by labeling DNA . Labeling was performed on 32 P, 3 H, 14
Various known labels, such as labels with radioisotopes such as C, chemical labels such as biotinylation, are applicable,
The method of labeling is not particularly limited.
【００１７】さらに、前記配列（Ｉ）のＤＮＡに相補的
なＤＮＡもまた、本発明のＤＮＡに含まれ、これらもま
たＤＮＡプローブとして使用することができる。 また、
本発明のＤＮＡプローブとして、本発明のベクターであ
るプラスミド等をニックトランスレーション等により標
識してそのまま用いることもできる。Furthermore, DNAs complementary to the DNA of the above-mentioned sequence (I) are also included in the DNA of the present invention, and these can also be used as DNA probes . Also,
As the DNA probe of the present invention, a plasmid or the like, which is the vector of the present invention, can be labeled and used by nick translation or the like.
【００１８】本発明のウシの雌雄判別法は、前述のＤＮ
Ａプローブを用いることを特徴とする。該プローブを用
いて、検体のＤＮＡとハイブリダイゼーションし、ハイ
ブリダイズするものを雄性、ハイブリダイズしないもの
を雌性と判別することができる。The bovine sex determination method of the present invention uses the aforementioned DN method.
It is characterized by using an A probe. Using the probe, it is possible to discriminate between those that hybridize with the DNA of the sample and hybridize as male and those that do not hybridize as female.
【００１９】ハイブリダイゼーションの方法としては、
上記判別が可能なものであれば特に制限はなく、例え
ば、ドットブロットハイブリダイゼーション法、サザン
ブロットハイブリダイゼーション法、in situ ハイブリ
ダイゼーション法等により行うことができる。As a hybridization method,
There is no particular limitation as long as the above-mentioned discrimination is possible, and for example, it can be performed by dot blot hybridization, Southern blot hybridization, in situ hybridization, or the like.
【００２０】[0020]
【実施例】以下、実施例により本発明を詳述するが、本
発明はこれにより何ら制限されるものではない。 実施例１ 雌雄のウシの血液より抽出したＤＮＡを試料としてドッ
トブロットハイブリダイゼーションを行い雌雄判別を行
った。 （１）プローブの製作 ホルスタイン牛（雄）の末梢血５ｍｌを遠心分離し、白
血球画分を得た。得られた白血球画分をプロテイナーゼ
Ｋ、ＲＮａｓｅ及びフェノール・クロロホルムで処理
し、エタノール沈澱法で回収、精製した。得られた牛ゲ
ノムＤＮＡを定量した後、−２０℃で保存した。EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited thereto. Example 1 A male / female bovine blood was subjected to dot blot hybridization using DNA extracted from bovine blood as a sample to determine gender. (1) Production of probe 5 ml of peripheral blood of a Holstein cow (male) was centrifuged to obtain a leukocyte fraction. The obtained leukocyte fraction was treated with proteinase K, RNase and phenol / chloroform, and recovered and purified by an ethanol precipitation method. After quantifying the obtained bovine genomic DNA, it was stored at -20 ° C.
【００２１】この抽出したＤＮＡをＰＣＲ法により増幅
した。このＰＣＲ法に用いる２種のプライマーは以下の
通りでＤＮＡ合成機（ＡＢＩ社製）で合成し用いた。 5'-AAGCGACCCA TGAACGCATT CATCGTGTGGT-3' 5'-GAGGTCGATA CTTATAATTC GGGTATTTCT CTCTGTG-3 ＰＣＲ法に用いるＤＮＡ量は、テンプレートＤＮＡは１
μｇ程度とし、プライマーＤＮＡは１００ｐｍｏｌ程度
とした。これらのＤＮＡをＰＣＲ緩衝液（インニスとゲ
ルファンド（Innis and Gelfand）の方法（Academic Pr
ess Inc.3-12,1990に記載）に従って調製）に最終容量
が１００μｌになるように加えた。２．５ユニットの耐
熱性ＤＮＡポリメラーゼ（Ｔａｑポリメラーゼ、シータ
ス社）を加えた後、プログラム式熱コントローラー（Ｍ
Ｊリサーチ社）を用いてＰＣＲ操作を行った。具体的に
は、前処理としてディネーチャー工程９５℃１０分行
い、次に１サイクルをアニーリング工程 ５４℃ １
分、イクステンション工程 ７２℃ ２．５分、ディネ
ーチャー工程 ９５℃ １分として、３０サイクル行っ
た。The extracted DNA was amplified by the PCR method. The two primers used in this PCR method were synthesized and used by a DNA synthesizer (ABI) as follows. 5'-AAGCGACCCA TGAACGCATT CATCGTGTGGT-3 '5'-GAGGTCGATA CTTATAATTC GGGTATTTCT CTCTGTG-3 The amount of DNA used for the PCR method was 1 for the template DNA.
μg and the primer DNA was about 100 pmol. These DNAs were used in PCR buffer (Innis and Gelfand) (Academic Pr
ess Inc. 3-12, 1990)) to a final volume of 100 μl. After adding 2.5 units of heat-resistant DNA polymerase (Taq polymerase, Cetus), a programmable heat controller (M
The PCR operation was performed using J Research Inc.). Specifically, a pre-treatment is performed at a denaturation step of 95 ° C. for 10 minutes, and then one cycle is performed at an annealing step of 54 ° C. 1
For 30 minutes, the extension process was performed at 72 ° C. for 2.5 minutes, and the denaturation process was performed at 95 ° C. for 1 minute.
【００２２】このようにして得られたＰＣＲ産物を４％
アガロースゲル（シーケムＭＥ、ＦＭＣ社製）上で電気
泳動にかけて分画し、臭化エチジウム染色を行った（図
１）。レーン１は分子量マーカー、レーン２はＰＣＲ産
物である。約２５０ｂｐのところにバンディングしてい
るバンドをアガロースゲルより切り出しＤＮＡ断片を抽
出、精製した。The thus obtained PCR product is 4%
Electrophoresis was performed on an agarose gel (Sechem ME, manufactured by FMC), fractionated, and stained with ethidium bromide (FIG. 1). Lane 1 is the molecular weight marker, and lane 2 is the PCR product. A band banding at about 250 bp was cut out from an agarose gel, and a DNA fragment was extracted and purified.
【００２３】次に、インビトロージェン社のＴＡクロー
ニングキットを使用して、該キットのプロトコールに従
い、このＤＮＡ断片のクローニングを行った。まず、こ
のＤＮＡ断片をプラスミドに挿入した（インビトロージ
ェン社のｐＣＲ１０００、図２）。続いて、このプラス
ミドを宿主（大腸菌）に導入した。宿主を抗生物質（カ
ナマイシン）が入った培地に植えて増殖させた。Next, this DNA fragment was cloned using a TA cloning kit of Invitrogen according to the protocol of the kit. First, this DNA fragment was inserted into a plasmid (pCR1000 of Invitrogen, FIG. 2). Subsequently, this plasmid was introduced into a host (E. coli). Hosts were seeded and grown in media containing antibiotics (kanamycin).
【００２４】次に、ＰＣＲ産物より精製したＤＮＡ断片
を、ｒ−32Ｐ（ＡＴＰ）とＴ4ポリヌクレオチドキナー
ゼを使用し、３７℃で６０分反応させ、５’末端を32Ｐ
でラベルし、精製してプローブとした。このプローブを
用いてコロニーハイブリダイゼーション（条件；プレハ
イブリダイゼーション ５０℃ １時間、ハイブリダイ
ゼーション ５０℃ １晩、洗浄 ５０℃ １時間×２
回）を行い、ＤＮＡ断片が挿入されているかを調べ、Ｄ
ＮＡ断片挿入陽性クローンを決めた。その結果を図３に
示すが、４６コロニー中１０コロニーが陽性であった。Next, the DNA fragments purified from the PCR product, using the r- 32 P (ATP) and T 4 polynucleotide kinase, reacted for 60 minutes at 37 ° C., the 5'-end 32 P
And purified to obtain a probe. Colony hybridization using this probe (conditions: prehybridization 50 ° C for 1 hour, hybridization 50 ° C overnight, washing 50 ° C for 1 hour x 2
Times) to check whether the DNA fragment has been inserted,
An NA fragment insertion positive clone was determined. The results are shown in FIG. 3, where 10 out of 46 colonies were positive.
【００２５】挿入陽性クローンの有するプラスミドをア
ルカリ分解法で抽出し、制限酵素（ＥｃｏＲｌ、Ｈｉｎ
ｄ III）で切断し、４％アガロースゲルで電気泳動（条
件；ミューピッドを使用し、５０Ｖ ９０分）した後、
臭化エチジウムで染色し、紫外線照射（３０２ｎｍ）
下、写真撮影して、切断されたＤＮＡの鎖長を調べ、目
的の長さ（約２５０ｂｐ）を持つ挿入ＤＮＡを有するク
ローンを陽性とした。図４にその結果を示すが、レーン
１は分子量マーカ、レーン２〜６はＤＮＡ断片である。The plasmid contained in the insertion-positive clone was extracted by the alkaline digestion method and digested with restriction enzymes (EcoRl, Hin).
dIII) and electrophoresed on a 4% agarose gel (conditions: using a mupid, 50 V for 90 minutes)
Stained with ethidium bromide and irradiated with ultraviolet light (302 nm)
A photograph was taken below to examine the chain length of the cleaved DNA, and a clone having the inserted DNA having the desired length (about 250 bp) was regarded as positive. FIG. 4 shows the results, wherein lane 1 is a molecular weight marker, and lanes 2 to 6 are DNA fragments.
【００２６】得られたＤＮＡの塩基配列はジデオキシ法
に従って決定した。その結果、使用したプライマー部分
及びプライマーから両末端までの部分を除き以下の配列
を持っていた。 5'-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3'(146bp) このサイズは、電気泳動度から推定される値とほぼ同じ
であった。The nucleotide sequence of the obtained DNA was determined according to the dideoxy method. As a result, it had the following sequences except for the used primer part and the part from the primer to both ends. 5'-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3 '(146 bp) This size was estimated from the electrophoresis degree.
【００２７】以上のように配列を決定されたＤＮＡ断片
を有するプラスミドｐＣＲ１０００をニックトランスレ
ーション（〔α−32Ｐ〕ｄＣＴＰ使用、１６℃ １６時
間）によって標識し（放射性同位元素：32Ｐ）プローブ
とした。The plasmid pCR1000 having the DNA fragment sequenced as described above is labeled by nick translation (using [α- 32 P] dCTP, 16 ° C. for 16 hours) (radioisotope: 32 P) and a probe. did.
【００２８】（２）ドットブロットハイブリダイゼイシ
ョン ホルスタイン牛（雌雄一頭ずつ）の末梢血５ｍｌを遠心
分離し白血球画分を得た。得られた白血球画分をプロテ
ィナーゼＫ、ＲＮａｓｅ及びフェノール・クロロホルム
で処理し、ＤＮＡを抽出した。得られた牛ゲノムＤＮＡ
を定量した後、−２０℃で保存した。(2) Dot blot hybridization 5 ml of peripheral blood of a Holstein cow (one male and one female) was centrifuged to obtain a leukocyte fraction. The obtained leukocyte fraction was treated with proteinase K, RNase and phenol / chloroform to extract DNA. Obtained bovine genomic DNA
After quantification, was stored at -20 ° C.
【００２９】ウシゲノムＤＮＡをアルカリ変性、熱変性
処理を行った後、ニトロセルロースフィルターに吸着さ
せた。同フィルターをプレハイブリダイゼーション液
（５×ＳＳＰＥ（３．６Ｍ ＮａＣｌ、２００ｍＭ Ｎ
ａＨ2ＰＯ4、２０ｍＭ ＥＤＴＡを含む、ｐＨ７．
４）、５×デンハルト溶液、０．５％ｖ／ｖＳＤＳ（ド
デシル硫酸ナトリウム））に７５℃で一時間浸漬するこ
とによって、プレハイブリダイゼーションを行った。続
いて、前述の雄のウシＤＮＡ断片を含むプラスミドを32
Ｐで標識したプローブと共に一晩ハイブリダイゼーショ
ンを行った。After bovine genomic DNA was subjected to alkali denaturation and heat denaturation treatment, it was adsorbed to a nitrocellulose filter. The filter was placed in a pre-hybridization solution (5 × SSPE (3.6 M NaCl, 200 mM N
aH 2 PO 4 , containing 20 mM EDTA, pH7.
4) Prehybridization was performed by immersing in 5 × Denhardt's solution, 0.5% v / v SDS (sodium dodecyl sulfate)) at 75 ° C. for 1 hour. Subsequently, a plasmid containing bovine DNA fragments of the aforementioned male 32
Hybridization was performed overnight with the probe labeled with P.
【００３０】次に、同フィルターを６×ＳＳＣ（Standa
rd Saline Citrate）液で一時間洗浄した後、Ｘ線フィ
ルム（コニカ社製）に−８０℃で一晩露光させた。図５
はこのようにして得られたドットブロットハイブリダイ
ゼーションの結果を示している。本発明で提供したプロ
ーブは雄のＤＮＡのみと反応し、雌のＤＮＡとは反応し
ないことが示され、ウシの雌雄判別法が可能であること
を示している。Next, the filter was replaced with a 6 × SSC (Standa
After washing with an (rd Saline Citrate) solution for 1 hour, an X-ray film (manufactured by Konica) was exposed to -80 ° C overnight. FIG.
Shows the results of the dot blot hybridization obtained in this manner. It was shown that the probe provided in the present invention reacted only with male DNA and did not react with female DNA, indicating that a method for discriminating male and female cattle was possible.
【００３１】[0031]
【発明の効果】本発明のウシのＤＮＡは、雄ウシのＤＮ
Ａとのみ反応し、雌ウシのＤＮＡとは反応しないので、
ウシの雌雄を判別するためのプローブとして有用であ
る。EFFECT OF THE INVENTION The bovine DNA of the present invention is bull DN
A only reacts with A and does not react with cow DNA.
It is useful as a probe for discriminating between male and female cattle.
【００３２】[0032]
【配列表】配列番号：１ 配列の長さ：１４６ 配列の型：核酸 鎖の数：両形態 トポロジー：直鎖状 配列の種類：Genomic DNA 起源：成熟ホルスタイン牛の末梢血の白血球 配列 CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC 50 TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC 100 TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA 146[Sequence list] SEQ ID NO: 1 Sequence length: 146 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Genomic DNA Origin: Peripheral blood leukocyte sequence of mature Holstein cow CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC 50 TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC 100 TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA 146
【００３３】配列番号：２ 配列の長さ：３１ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 起源：核酸モノマー 配列 ＡＡＧＣＧＡＣＣＣＡ ＴＧＡＡＣＧＣＡＴＴ ＣＡＴ
ＣＧＴＧＴＧＧ Ｔ ３１Sequence number: 2 Sequence length: 31 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin: Nucleic acid monomer Sequence AAGCGACCCA TGAACGCATT CAT
CGTGTGGG T31
【００３４】配列番号：３ 配列の長さ：３７ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 起源：核酸モノマー 配列 ＧＡＧＧＴＣＧＡＴＡ ＣＴＴＡＴＡＡＴＴＣ ＧＧＧ
ＴＡＴＴＴＣＴ ＣＴＣＴＧＴＧ ３７SEQ ID NO: 3 Sequence length: 37 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin: Nucleic acid monomer Sequence GAGGTCGATA CTTATAATTC GGG
TATTTCT CTCTTGTG 37
【図１】図１はＰＣＲ産物の１０％を４％アガロースゲ
ルで電気泳動した後、臭化エチジウムで染色し、紫外線
（３０２ｎｍ）照射下で撮影した結果を示す図である。
レーン１は分子量マーカーであり、レーン２はＰＣＲ産
物である。FIG. 1 is a diagram showing the results of electrophoresis of 10% of a PCR product on a 4% agarose gel, staining with ethidium bromide, and photographing under ultraviolet (302 nm) irradiation.
Lane 1 is the molecular weight marker and lane 2 is the PCR product.
【図２】図２はｐＣＲ１０００プラスミドのクローニン
グサイトを示す図である。FIG. 2 is a diagram showing a cloning site of a pCR1000 plasmid.
【図３】図３はコロニーハイブリダイゼイションの結果
を示す図である。FIG. 3 shows the results of colony hybridization.
【図４】図４は抽出したプラスミドを制限酵素で切断し
４％のアガロースゲルで電気泳動した後、臭化エチジウ
ムで染色し、紫外線（３０２ｎｍ）照射下で撮影した結
果を示す図である。レーン１は分子量マーカーであり、
レーン２〜６はＤＮＡ断片である。FIG. 4 is a diagram showing the results of digestion of the extracted plasmid with a restriction enzyme, electrophoresis on a 4% agarose gel, staining with ethidium bromide, and imaging under ultraviolet (302 nm) irradiation. Lane 1 is the molecular weight marker,
Lanes 2 to 6 are DNA fragments.
【図５】図５はドットブロットハイブリダイゼーション
分析の結果を示す図である。FIG. 5 shows the results of dot blot hybridization analysis.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭61−234798（ＪＰ，Ａ) ＮＡＴＵＲＥ，ＶＯＬ．346 ＮＯ. 19（1990）Ｐ．240−244 ＮＡＴＵＲＥ，ＶＯＬ．346 ＮＯ. 19（1990）Ｐ．245ー250 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-61-234798 (JP, A) NATURE, VOL. 346 NO. 19 (1990) p. 240-244 NATURE, VOL. 346 NO. 19 (1990) p. 245-250
Claims (6)
ウシのＤＮＡ。The present invention is characterized in that it is obtained by amplifying bull DNA by PCR using the following primers 5'-AAGCGACCCA TGAACGCATT CATCGTGTGG T-3 'and 5'-GAGGTCGATA CTTATAATTC GGGTATTTCT CTCTGTG-3'. Bovine DNA.
つウシのＤＮＡ。 配列（Ｉ） 5'-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3' (但し、RはAまたはGを示す)2. Bovine DNA having a base sequence represented by the following formula (I). Sequence (I) 5′-CTCGTGAACG AAGACGAAAG GTGGCTCTAG AGAATCCCAA AATGAAAAAC TCAGACRTCA GCAAGCAGCT GGGATATGAG TGGAAAAGGC TTACAGATGC TGAAAAGCGC CCATTCTTTG AGGAGGCACA GAGACTACTA GCCATA-3 ′ (where R represents A or G)
含有し、これを標識して得られるＤＮＡプローブ。3. The bovine DNA according to claim 1 or 2 ,
A DNA probe obtained by labeling the DNA probe.
含有するベクター。4. The bovine DNA according to claim 1 or 2 ,
The containing vector.
相補的なＤＮＡ。5. A DNA complementary to the bovine DNA according to claim 1 or 2.
ことを特徴とするウシの雌雄判別法。6. A method for discriminating male and female cattle, comprising using the DNA probe according to claim 3.
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|---|---|---|---|
| JP3245732A JP2570928B2 (en) | 1991-09-25 | 1991-09-25 | Bovine DNA, DNA probe, vector and bovine sex determination method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| Publication Number | Publication Date |
|---|---|
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| JP2570928B2 true JP2570928B2 (en) | 1997-01-16 |
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| Title |
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| ＮＡＴＵＲＥ，ＶＯＬ．３４６ ＮＯ．１９（１９９０）Ｐ．２４０−２４４ |
| ＮＡＴＵＲＥ，ＶＯＬ．３４６ ＮＯ．１９（１９９０）Ｐ．２４５ー２５０ |
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