JP2004187528A - Method for distinguishing variety of pig - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method with which distinction in variety of a pig can simply be carried out with high accuracy and to provide a kit therefor. <P>SOLUTION: The method for distinguishing the variety of a specimen pig comprises detecting one or two or more polymorphisms in a KIT gene in the specimen pig and using the resultant detected polymorphisms or a combination thereof. In the method, the polymorphisms in either one or both of positions in positions corresponding to the 1159th position and the 1209th position in a base sequence of the KIT gene based on a base sequence described in sequence number 1 are detected as the polymorphisms. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００２９】
【表２】

【００３０】
【表３】

【００３１】
本発明において、好ましくは、上記多型は、それぞれの多型をハイブリダイゼーションにより検出し得るオリゴヌクレオチドを用いて検出される。本発明においては、このようなオリゴヌクレオチドをキャプチャーオリゴヌクレオチドという。キャプチャーオリゴヌクレオチドは、多型部位を含む領域の塩基配列に相補的又は相同な塩基配列を有する。
【００３２】
キャプチャーオリゴヌクレオチドの設計は、それぞれの遺伝子に存在する各々の多型部位を含むように行う。また、１つのキャプチャーオリゴヌクレオチドのなかに２塩基以上のタイプ固有の塩基配列が存在していてもかまわない。
【００３３】
キャプチャーオリゴヌクレオチドの塩基の長さは、１０〜４０塩基が望ましい。これより短いとハイブリダイゼーションの検出が困難になり、長いとタイプ固有の配列によるハイブリダイゼーションの差が少なくなり、タイプの判定が難しくなることがある。ただし、このキャプチャーオリゴヌクレオチドの長さは、主として各塩基の含有率や同一塩基の繰り返しなどの配列の特性に依存する。また、ハイブリダイゼーションを妨げる要因である２次構造的障害がある場合は、遺伝子の多型性に関係しない任意の塩基をスペーサー化合物で置換することによりハイブリダイゼーションにおける結合親和性を緩和したオリゴヌクレオチドを用いることにより、前記障害を回避することもできる。このようなスペーサー化合物としては、いずれの種類の塩基との相補結合性を有しない核酸骨格等が挙げられる。
【００３４】
キャプチャーオリゴヌクレオチドの塩基配列の例を、配列番号３〜１２１に示す。尚、配列番号３〜１２１に示した番号のほか、各塩基配列の５’側もしくは３’側またはその両方において、各々の塩基配列を、ＭＣ１Ｒ遺伝子、ＫＩＴ遺伝子の塩基配列に対応して伸長、または短縮させてもよいが、いずれの場合もキャプチャーオリゴヌクレオチドは１０〜４０塩基の範囲とすることが好ましい。
【００３５】
キャプチャーオリゴヌクレオチドは、ＤＮＡ、ＲＮＡ、ペプチド核酸、またはロックド核酸のいずれであってもよい。キャプチャーオリゴヌクレオチドの合成方法は、通常のオリゴヌクレオチドと同様にして、例えば市販のＤＮＡ合成機を用いる方法等によって、行うことができる。
【００３６】
本発明において、設計されたキャプチャーオリゴヌクレオチドは、出願時点までの研究・調査結果を良く反映したものであるが、新しいハプロタイプの塩基配列に関して、この後、追加的な情報が得られた場合には、本出願に記載の方法に基づいて新たなキャプチャーオリゴヌクレオチドが設計されることになる。このようなキャプチャーオリゴヌクレオチドを使用する場合であっても、ＫＩＴ遺伝子の１１５９位及び１２０９位に相当する位置のいずれか一方を検出する限り、本発明に含まれる。
【００３７】
上記のようなキャプチャーオリゴヌクレオチドを用いてハイブリダイゼーションを行う際に、キャプチャーオリゴヌクレオチドと被検体豚に由来する核酸（以下、「核酸プローブ」ともいう）を液中でハイブリダイゼーションさせてもいいし、キャプチャーオリゴヌクレオチドまたは核酸プローブのどちらかを支持体上に固定化してもよい。
【００３８】
固定化を行う場合、このような支持体としては、キャプチャーオリゴヌクレオチドまたは核酸プローブを固定化でき、被検体とのハイブリダイゼーション及び、ハイブリッドの検出に必要な反応工程に耐え得るものであれば特に制限されない。このような支持体の材質として具体的にはプラスチック、無機高分子、金属、天然高分子、セラミック等が挙げられる。
【００３９】
上記プラスチックとして具体的には、ポリエチレン、ポリスチレン、ポリカーボネート、ポリプロピレン、ポリアミド、フェノール樹脂、エポキシ樹脂、ポリカルボジイミド樹脂、ポリ塩化ビニル、ポリフッ化ビリニデン、ポリフッ化エチレン、ポリイミド及びアクリル樹脂等が挙げられる。
【００４０】
また、無機高分子として具体的には、ガラス、水晶、カーボン、シリカゲル及び、グラファイト等が挙げられる。
【００４１】
また、金属として具体的には、金、白金、銀、銅、鉄、アルミニウム、磁石、パラマグネット等が挙げられる。
【００４２】
また、天然高分子としてはポリアミノ酸、セルロース、キチン、キトサン、アルギン酸及び、それらの誘導体等が挙げられる。
【００４３】
また、セラミックとして具体的にはアパタイト、アルミナ、シリカ、炭化ケイ素、窒化ケイ素及び、炭化ホウ素等が挙げられる。
【００４４】
本発明においてキャプチャーオリゴヌクレオチドまたは被検体の固定化に用いる支持体の形状はキャプチャーオリゴヌクレオチドが固定化されれば、特に制限されない。このような支持体の形状として平板、メンブレン、微粒子等が挙げられる。
【００４５】
本発明において上記支持体にキャプチャーオリゴヌクレオチドまたは核酸プローブを固定化する際、支持体にキャプチャーオリゴヌクレオチドまたは核酸プローブを直接固定化してもよく、担体を支持体に担持させて、担体を介してキャプチャーオリゴヌクレオチドまたは被検体を支持体に固定化してもよい。担体としては、担体自体がキャプチャーオリゴヌクレオチドまたは被検体に結合性を有してもよく、キャプチャーオリゴヌクレオチドまたは被検体に結合性を有するリガンドを介してキャプチャーオリゴヌクレオチドまたは被検体を固定化できるものであってもよい。ここで、「担持」とは、担体にキャプチャーオリゴヌクレオチドまたは核酸プローブを固定化する際やキャプチャーオリゴヌクレオチドまたは核酸プローブ固定化支持体を実際の検出に用いる際等に用いられる水溶性溶剤、有機溶剤等の各種溶剤中で、支持体から上記担体が実質的に脱離しないことを意味する。
【００４６】
本発明に用いられる上記担体は、上記支持体上に担持される限り、単に物理的な接着性を利用して担持されていてもよく、また、化学的に共有結合等を介して担持されていてもよい。また、上記担体は、必要に応じ、支持体上の全面において担持されていても、また、その一部において担持されていてもよい。
【００４７】
担体としては、有機低分子、プラスチック、無機高分子、金属、天然高分子、セラミック等が挙げられる。
上記有機低分子として具体的には、カルボジイミド基含有化合物、イソシアネート基含有化合物、窒素イペリット基含有化合物、アルデヒド基含有化合物、アミノ基含有化合物等が挙げられる。
【００４８】
また、プラスチック、無機高分子、金属、セラミックは、前記したものと同様のものを使用することができる。
本発明において特に好ましい担体は、カルボジイミド基含有化合物またはイソシアネート基含有化合物である。
【００４９】
各キャプチャーオリゴヌクレオチドまたは核酸プローブの支持体への供給はキャプチャーオリゴヌクレオチドまたは核酸プローブの活性が維持されるように、通常、水またはバッファー中に含まれる形で供給される。また、供給の際の温度としては固定化されるキャプチャーオリゴヌクレオチドまたは被検体の活性が失われないように０℃〜１００℃とすることが望ましい。
【００５０】
また、オリゴヌクレオチドと支持体の接触後に紫外線等の電磁波を照射することによって固定することもできる。さらに、オリゴヌクレオチドとカルボジイミド樹脂、窒素イペリット、ポリアミノ酸、ニトロセルロール等の公知の化合物を化学的に結合又は物理的に結合した状態で、これら混合物と担体を接触させ固定させても良く、また、このとき紫外線等の電磁波を照射して固定しても良い。
【００５１】
本発明においてキャプチャーオリゴヌクレオチドまたは被検体を支持体に供給する手段は、キャプチャーオリゴヌクレオチドまたは被検体の活性が維持された状態で供給されれば特に制限はされない。このような例としてディスペンサを用いる方法、ピンを用いる方法、インクジェットを用いる方法などが挙げられる。
【００５２】
キャプチャーオリゴヌクレオチドまたは核酸プローブを支持体上へ固定化する際、その形状はハイブリダイゼーションに影響を及ぼさず、また、検出が困難にならなければ特にその形状は制限されない。このような例として円形、四角形等が挙げられる。支持体上へ各キャプチャーオリゴヌクレオチドが固定化される部位のサイズは、径１０μｍ〜１ｃｍが好ましい。これより小さいと検出が困難になり、また、これより大きいと配置されたキャプチャーオリゴヌクレオチド全体の占める面積が大きくなり、操作性が悪くなるという問題が生じることがある。
【００５３】
支持体上への各キャプチャーオリゴヌクレオチドまたは核酸プローブの固定化の際、各オリゴヌクレオチドまたは被検体をそのまま固定化しても良いが、反応性を高めることによって、キャプチャーオリゴヌクレオチドまたは被検体の末端にリンカーを付加し、リンカーと支持体又は担体とを反応させてもよい。そのようなリンカーとしては、アミノ基、またはホモポリマー、例えばチミジン残基のホモポリマー等が挙げられる。
【００５４】
各キャプチャーオリゴヌクレオチドまたは核酸プローブの支持体上への配置は各多型の判定が出来れば特に制限されないが、各多型の判定を容易にするために、多型の判定に用いられるキャプチャーオリゴヌクレオチドまたは被検体を１区画にまとめるか、あるいは一列に並べるなどして配置することが好ましい。
【００５５】
支持体上へ固定されるキャプチャーオリゴヌクレオチドまたは核酸プローブは、１種であっても、２種以上であってもよい。
【００５６】
キャプチャーオリゴヌクレオチドとのハイブリダイゼーションに用いる核酸プローブは、ＭＣ１Ｒ遺伝子及びＫＩＴ遺伝子又はその一部を含む核酸である。核酸プローブは、ハイブリダイゼーションを阻害しないものであれば特に制限されないが、通常、ＤＮＡまたはＲＮＡなどが用いられる。また、核酸プローブの作製方法は、ハイブリダイゼーションを阻害しない方法であれば特に制限されない。具体的には、豚の血液または組織から核酸を抽出し、ＭＣ１Ｒ遺伝子及びＫＩＴ遺伝子の少なくとも多型部位を含む領域を、Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ（ＰＣＲ）法、ｉｎ ｖｉｒｔｏ ｔｒａｎｓｃｒｉｐｔｉｏｎ法、Ｌｏｏｐ−ｍｅｄｉａｔｅｄ Ｉｓｏｔｈｅｒｍａｌ Ａｍｐｌｉｆｉｃａｔｉｏｎ法等の方法によって増幅又は単離することによって、核酸プローブを調製することができる。核酸の調製は、通常核酸の調製に用いられている方法を採用することができる。例えばＡｎｉｍ．Ｓｃｉ．Ｊ．７１（８）：２２２−２３４，２０００で紹介されている方法が挙げられる。
【００５７】
核酸プローブは、各多型を少なくとも１箇所含むように調製する。また、核酸プローブの作製に用いるＰＣＲプライマーは、各ハプロタイプ固有の配列領域を除き、キャプチャーオリゴヌクレオチドと相補的な塩基配列になるように設計される。なお、ハイブリダイゼーションが可能である限りにおいて、核酸プローブは、キャプチャーオリゴヌクレオチドより長くても短くても良い。また、ＰＣＲ反応の特異性を高めるために、目的とする核酸プローブよりも広い領域を作製する予備的プライマーを用いて初回の作製をおこない、作製された核酸を鋳型として核酸プローブを得るための２次プライマーを用いて核酸増幅をおこなっても良い。多型が離れたところにある場合、各固有領域に応じた核酸プローブを作製することができる。
【００５８】
核酸プローブまたはキャプチャーオリゴヌクレオチドは、ハイブリダイゼーションを検出するために通常標識されている。標識された核酸プローブまたはキャプチャーオリゴヌクレオチドを得る方法は、ハイブリダイゼーションを阻害しない方法であれば特に制限されない。このような方法として、最終的な核酸プローブ作製に用いるプライマーを予め標識しておく方法、核酸プローブ作製中に標識する方法、及び、核酸プローブ作製後に標識する方法、標識したキャプチャーオリゴヌクレオチドを作製する方法等が挙げられる。
【００５９】
プライマーを予め標識しておく方法、標識したキャプチャーオリゴヌクレオチドを作製する方法としては、標識ヌクレオチドを用いてプライマー合成を行う方法などが挙げられる。核酸プローブ作製中に標識する方法として、核酸プローブ作製反応中に標識したヌクレオチドをとりこませる方法などが挙げられる。また、作製反応後に標識する方法としては、特開平１０−２８７８７０号に示される方法で標識する方法などが挙げられる。
【００６０】
核酸プローブまたはキャプチャーオリゴヌクレオチドを標識する物質は、通常のハイブリダイゼーション法に用いられる標識物質を使用することができる。このような標識物質として蛍光物質やハプテンなどが挙げられる。具体的にはフルオレセイン、ローダミン、フィコエリスリン、テキサスレッド、シアニン系蛍光色素が、ハプテンとしてはビオチン、ジゴキシゲニン、ジニトロフェニルなどが挙げられる。
【００６１】
核酸プローブを作製するためのプライマーは、キャプチャーオリゴヌクレオチドと共に、ハプロタイプタイピングキットに含めることができる。
【００６２】
ハイブリダイゼーションは、通常の核酸のハイブリダイゼーションと同様にして行うことができる。以下に具体的な方法を例示する。
ＳＳＣ（Ｓｔａｎｄａｒｄ Ｓａｌｉｎｅ Ｃｉｔｒａｔｅ）などの塩溶液、ドデシル硫酸ナトリウム（ＳＤＳ）、ウシ血清アルブミンなどのブロッキング溶液、および反応促進のための添加剤からなる混合液に核酸プローブを加える。プローブが２本鎖の場合は熱などによる変性を行う。支持体上に核酸プローブ溶液を数μＬ滴下した後、数時間加熱操作（通常３０℃〜８０℃）を行い、支持体上に固定化されているキャプチャーオリゴヌクレオチドと核酸プローブでハイブリッドを形成させる。
【００６３】
適当な濃度のＳＳＣ溶液またはテトラメチルアンモニウムクロリド溶液中に支持体を沈め、加熱（通常３７℃〜７０℃）し、特異的なハイブリッドのみを選択的に支持体上に残す。このとき、ＳＤＳなどを加えても良い。
【００６４】
ハイブリッドの検出には核酸プローブに標識されている蛍光物質またはハプテン等を使用する。蛍光物質を使用する場合は、専用の蛍光スキャナーを用いてハイブリダイズした核酸プローブに標識された蛍光を検出する。
【００６５】
ハプテンを使用する場合は、ハプテンを認識するタンパクまたはそれに結合するタンパクと、アルカリフォスファターゼまたはホースラディッシュ・パーオキシダーゼなどの結合体（酵素コンジュゲート）を含む溶液を支持体上に加え、室温で数１０分間反応させる。なお、このハプテンと酵素コンジュゲートの結合反応を行う前に、キャプチャーオリゴヌクレオチドを固定した領域以外の支持体の領域をカゼインやウシ血清アルブミンなどのタンパクを用いて被覆することによって酵素コンジュゲートと支持体の非特異的吸着を阻止することができる。この処置は、オリゴヌクレオチドを固定した後、支持体上にカゼインなどのタンパクの溶液を加え、室温で数１０分間放置することによって行うことができる。
【００６６】
酵素コンジュゲートと核酸プローブのハプテンとの結合反応後、ハプテンと結合しなかった酵素コンジュゲートを、界面活性剤を含む適当な緩衝液で洗浄することによって、支持体上にはターゲット核酸中のハプテンと結合した酵素コンジュゲートのみが残ることになる。
【００６７】
この後酵素コンジュゲートに結合されたアルカリフォスファターゼまたはホースラディッシュ・パーオキシダーゼなどの酵素によって発色し、沈着するような化合物を添加することでハイブリッドを形成している部分だけが可視化される。
【００６８】
ここで用いられる化合物としては、ニトロブルーテトラゾリウムクロライド、５−ブロモ−４−クロロ−３−インドリルリン酸−ｐ−トルイジン塩）、３，３’，５，５’−テトラメチルベンチジンなどが用いられる。
【００６９】
多型の検出は、得られたハイブリダイゼーションの結果をもとに、キャプチャーオリゴヌクレオチド毎に沈着した色素または蛍光を検出することで行う。具体的には、キャプチャーオリゴヌクレオチド毎の発色量および蛍光強度を、対応するキャプチャーオリゴヌクレオチドと比較し、その量の大きい方をハイブリダイゼーション陽性と判定する。量が同じときは共に陽性と判定する。また、その基準は特に制限されないが、用いる標識物質等によって異なるため、検出系毎に基準をきめることが望ましい。
【００７０】
上記判定を多型部位毎に行い、各々の多型またはその組み合わせがいずれかのタイプと一致したとき、その種の豚であると判定される。豚は２倍体なので交雑種の場合、２種の交雑と判定されることもある。
【００７１】
表４〜６に、配列番号３〜１２１に示す塩基配列を有するキャプチャーオリゴヌクレオチドを固定化して用いたときの、ハイブリダイゼーションシグナルと豚の種類の対応を示す。ここで表３はＭＣ１Ｒ遺伝子のものを表４はＫＩＴ遺伝子のものを示す。
【００７２】
【表４】

【００７３】
【表５】

【００７４】
【表６】

【００７５】
本発明のキットは、キャプチャーオリゴヌクレオチドを含む。また、本発明のキットは、核酸プローブを作製するためのプライマーまたは標識核酸プローブ、緩衝液、ハプテンを認識する酵素コンジュゲート等のハイブリダイゼーション用の試薬などを含めることができる。
【００７６】
【実施例】
以下、本発明を実施例によりさらに具体的に説明する。
【００７７】
＜１＞オリゴヌクレオチドの合成
定法に従い、オリゴヌクレオチド合成機（Ｐｅｒｋｉｎ−ｅｌｍｅｒ Ａｐｐｌｉｅｄ ｂｉｏｓｙｓｔｅｍｓ）を用いて、オリゴヌクレオチドを合成し、脱保護を施した後、乾燥させた。このオリゴヌクレオチド乾燥体を、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、１ｍＭ ＥＤＴＡ緩衝液を用いて溶解し、１００ｐｍｏｌ／μＬのオリゴヌクレオチド溶液を調製した。この合成法は、キャプチャーオリゴヌクレオチド又はプライマーとして使用するいずれのオリゴヌクレオチドに対しても同様である。合成したオリゴヌクレオチドの塩基配列は、配列番号３〜１２５に示す。これらのうち、配列番号３〜１２１がキャプチャーオリゴヌクレオチドであり、配列番号１２２〜１２５がプライマーである。キャプチャーオリゴヌクレオチドについては上記合成機を用いて５’末端にアミノ基を結合させ、プライマーについては５’側にローダミン標識した。
【００７８】
＜２＞支持体へのキャプチャーオリゴヌクレオチドのスポッティング
５’末端にアミノ基を有するオリゴヌクレオチド溶液１０μＬに対してマイクロスポッティング溶液（ＴｅｌｅＣｈｅｍ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃ．）を１０μＬ混合し、マイクロタイタープレート（Ｇｒｅｉｎｅｒ Ｌａｂｏｒａｔｏｒｙ Ｉｎｃ．）上に分注した。スポッティングマシンの所定の位置にシラン化スライドグラス（Ｍａｔｓｕｎａｍｉ Ｇｌａｓｓ Ｉｎｄ． Ｌｔｄ．）を配置し、スポッティングマシンを作動させた。スポッティング終了後、スライドグラスに熱水からの蒸気を数秒間あて、その後紫外線を３０ｍＪ照射した。再度蒸気に数秒間曝露した後、ホットプレート上にスライドグラスを置いて水分を除去した。０．１％ドデシル硫酸ナトリウム水溶液でスライドグラスをすすいだ後、蒸留水ですすいだ。スライドグラスを３％ ＢＳＡ（ウシ血清アルブミン）を含む１００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）， １００ｍＭ ＮａＣｌ，０．１％ Ｔｒｉｔｏｎ Ｘ−１００に室温で３０分間浸し、ブロッキングした。その後室温で乾燥させた後、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）， １ｍＭ ＥＤＴＡ緩衝液で洗浄した。スライドグラスを再度室温で乾燥させ、使用まで乾燥状態で冷暗所にて保存した。
【００７９】
＜３＞プローブ核酸の調製
豚のＤＮＡを鋳型として、ＰＣＲによりプローブ核酸を調製した。
各種の豚から得られた血液５０μＬをマイクロチューブに入れ、これに１０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ８．０， ０．１Ｍ ＮａＣｌ， １ｍＭ ＥＤＴＡ， ０．５％ ＳＤＳ， ５００μｇ／ｍＬ ｐｒｏｔｅｉｎａｓｅ Ｋを５００μｌ加え混和したのち、３７℃で一晩インキュベートした。フェノール：クロロフォルム：イソアミルアルコール（２５：２４：１）溶液を５００μＬ加え、撹拌した後遠心（１２０００ｒｐｍ、１分間）し、水相を回収した。この操作を３回繰り返した。得られた水相にクロロフォルム：イソアミルアルコール（２４：１）溶液を５００μＬ加え、撹拌した後遠心（１２０００ｒｐｍ、１分間）し、水相を回収した。この操作を２回繰り返した。得られた水相中のＤＮＡをエタノール沈殿により回収し、乾燥させ後、ＴＥバッファー（１０ｍＭ Ｔｒｉｓ−ＨＣｌ， １ｍＭ ＥＤＴＡ， ｐＨ７．５）に溶解し、ＰＣＲテンプレート溶液とした。
【００８０】
ＰＣＲ反応液の組成は、Ｔａｑポリメレースを１Ｕｎｉｔ、プライマーを各１０ｐｍｏｌ、反応用緩衝液５μｌ、ｄＮＴＰを各１０ｎｍｏｌ、テンプレートＤＮＡ溶液を０．５μｌ、および滅菌水を加えて総量５０μｌとし、１つのＰＣＲテンプレートについてＭＣ１Ｒ遺伝子を増幅するため（配列番号１２２と１２３のプライマー）とＫＩＴ遺伝子を増幅するため（配列番号１２４と１２５のプライマー）２種類用意する。チューブに入れた溶液をサーマルサイクラーにセットして、９４℃で３分間加熱した後、９４℃：４５秒間、６０℃：６０秒間、７２℃：１分間の反応を３０サイクル行い、その後７２℃：３分間反応させた。
【００８１】
本実施例では、確認試験として、次に記載のアガロースゲルを用いた電気泳動を行ったが、実際の臨床における鑑別の際にはこの操作は必須ではない。ＰＣＲ反応混合物を１μｌ取り、６×泳動色素（３０％グリセロール、０．２５％ブロモフェノールブルー、０．２５％キシレンシアノール）を１μｌ、蒸留水４μｌと混和した。２％アガロースゲル上で、１００Ｖ、９０分間の条件で泳動させた後、０．５μｇ／ｍｌエチジウムブロマイドを含む蒸留水に３０分間浸し、紫外線照射下でゲルをＣＣＤカメラで撮影した。その結果、所望の増幅断片が得られたことが確認された。
【００８２】
＜４＞ハイブリダイゼーション
＜３＞で作製した全てのプローブ核酸溶液を混ぜ、その後、４μｌ取り、ＡｒｒａｙＩｔ Ｕｎｉｈｙｂ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｓｏｌｕｔｉｏｎ（ＴｅｌｅＣｈｅｍ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃ．）１６μｌを加えて混合し、１００℃で１０分間加熱処理を行った後、氷中に５分間浸した。このプローブ核酸溶液を５μｌとり、＜２＞で作製したキャプチャーオリゴヌクレオチドを固定化したスライドグラスにのせ、その上にカバーグラスをのせた。これを保湿箱に入れ、さらに３７℃に設定した恒温器に入れて１２０分間静置した。スライドグラスを取り出し、すばやく室温下で２×ＳＳＣ， ０．１％ＳＤＳ溶液（２×ＳＳＣ：０．０３３Ｍ ＮａＣｌ， ０．０３３Ｍクエン酸ナトリウム）に浸してカバーグラスを除去した。スライドグラスを室温下で２×ＳＳＣ， ０．１％ＳＤＳ溶液に５分間浸す操作を２回行い、３７℃の０．２×ＳＳＣ， ０．１％ＳＤＳ溶液（０．２×ＳＳＣ：０．００３３Ｍ ＮａＣｌ， ０．００３３Ｍクエン酸ナトリウム）に５分間浸す操作を２回行い、最後に２×ＳＳＣに浸した。
【００８３】
＜５＞蛍光の検出
ハイブリダイゼーションを終えたスライドグラスを２×ＳＳＣから取り出し、遠心機（Ｂｅｃｋｍａｎ社製）にセットし、２０００ｒｐｍで１分間遠心した。次にＳｃａｎＡｒｒａｙ４０００（ＧＳＩ Ｌｕｍｏｎｉｃｓ社製）にスライドグラスをセットし、蛍光検出を行った。
【００８４】
以上の結果およびＤＮＡシーケンス法によるタイピングとの関係を表７に示した。本発明の方法により、豚の種別判定が可能なことが明らかである。
【００８５】
【表７】

【００８６】
【発明の効果】
本発明により、豚の種別判定を、効率よく、かつ高精度で行うことができる。
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a method and a kit for easily discriminating pig breeds.
[0002]
[Prior art]
At present, the main breeds for pork are large Yorkshire, Landrace, Duroc, Berkshire, Hampshire, Meishan, Mongkai and the like, and their crossbreeding. Among them, Berkshire varieties are called black pigs and are traded at high prices due to their high quality.
[0003]
However, some pigs are traded as black pork after taking advantage of this high price, and some pork mixed with non-Berkshire varieties or pork without any Berkshire varieties are available. Therefore, the “Meat Retail Quality Standards” were revised in June 1999 to prohibit the display of meat other than pure Berkshire breeds as black pork. However, this is not currently fully protected.
[0004]
As a method for examining pig breeds, there is a method in which after a gene is amplified, polymorphism analysis is performed by restriction fragment length polymorphisms (RFLP) (Patent Document 1). However, this method requires a large amount of DNA due to the use of RFLP, requires skill in operation because of the use of restriction enzymes, the operation becomes complicated when a plurality of restriction enzymes are used, Problems such as inability to examine polymorphic sites that are not commercially available and inability to distinguish between white varieties and colored varieties arise, and it is difficult to process multiple samples with high accuracy.
[0005]
In addition, a large number of gene polymorphisms have been reported as indicators of the classification of pigs up to the present (Patent Document 1), but it is necessary to find new polymorphisms for more accurate classification. In particular, black pigs and white pigs may not be able to be distinguished only by conventionally known polymorphisms.
[0006]
On the other hand, a microarray technology for capturing a labeled target nucleic acid on a solid phase and analyzing the presence or absence and abundance thereof has been recently developed. This is a technology that immobilizes a large number of capture nucleic acids (capture oligonucleotides) on a solid phase, hybridizes the labeled target nucleic acids to the capture nucleic acids, captures them, and comprehensively analyzes the presence or absence and abundance ratio of the target nucleic acids. (Non-Patent Document 1).
[0007]
[Patent Document 1]
Japanese Patent No. 3116049 (Japanese Patent Laid-Open No. 2000-350586)
[Non-patent document 1]
Nature genetics supplement volume 21, P10-14
[Problems to be solved by the invention]
An object of the present invention is to provide a method and a kit which are simple and suitable for multi-sample processing and can perform high-precision discrimination for one sample by a single test when discriminating the type of pig.
[0008]
[Means for Solving the Problems]
The present inventors have conducted studies to solve the above problems, and as a result, have found that a novel polymorphism suitable for discriminating the type of pig exists in the KIT gene, which is a kind of stem cell growth factor receptor gene. I found it. The present inventors have found that it is possible to determine the type of a subject pig with high accuracy by combining it with a known polymorphism of the KIT gene or a polymorphism in the melanocyte stimulating hormone receptor gene (MC1R gene). It was completed.
[0009]
That is, the present invention is as follows.
(1) The type of the subject pig is determined by a polymorphism or a combination thereof containing one or two or more capture oligonucleotides and detected by hybridization of the capture oligonucleotide with a nucleic acid derived from the subject pig. A kit for discriminating, wherein the capture oligonucleotide is located at one or both of positions corresponding to positions 1159 and 1209 in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1. A kit comprising an oligonucleotide capable of detecting a polymorphism in the above.
(2) The kit according to (1), wherein the capture oligonucleotide is immobilized on a support.
(3) In the base sequence of the KIT gene, the capture oligonucleotide is at positions 398, 549, 595, 659, 844, 884, 959, 1313, 1342, 1632, 1624, 1986, 2179, 2295, 2313, 2314, 2360, 2377, 2391, 2429, 2449, 2470, 2472, 2600, 2611, 2622, 2663, 2674 , 2680, 2717, 2834, 2840, 2899, 3020, 3049, 3207, 3221, 3258, 3296, 3317, 3323, 3341, 3373, 3387, 3420 , 3482, 3597, 3666, 3705, 3737, 773 of, 3884 positions, 3885 positions, 3886-position, and 1 or polymorphism of two or more positions further comprises an oligonucleotide capable of detecting (1) or kit according to (2) are selected from position 3887.
(4) The capture oligonucleotide includes an oligonucleotide capable of detecting a polymorphism at one or more positions in a melanocyte stimulating hormone receptor gene (hereinafter, referred to as “MC1R gene”). The kit according to any one of the above.
(5) The polymorphism in the MC1R gene corresponds to a position selected from positions 154, 176, 234, 241, 361, 367, 598, and 600 in the nucleotide sequence shown in SEQ ID NO: 2. The kit according to (4), which is a polymorphism at the position
(6) The capture oligonucleotide according to any one of (1) to (5), wherein the capture oligonucleotide has a nucleotide sequence complementary or homologous to the nucleotide sequence of the region containing the polymorphic site and has a length of 10 to 40 nucleotides. kit.
(7) An oligonucleotide capable of detecting a polymorphism at a position corresponding to position 1159 or position 1209 by hybridization in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1.
(8) The oligonucleotide according to (7), which has a nucleotide sequence complementary or homologous to the nucleotide sequence of the region containing the polymorphic site and has a length of 10 to 40 nucleotides.
(9) The nucleic acid derived from the subject pig is hybridized with the capture oligonucleotide contained in the kit according to any one of (1) to (6), and the nucleic acid derived from the subject pig is subjected to each capture oligonucleotide. A method for determining the type of a subject pig by detecting the presence or absence of hybridization with the subject pig and identifying a polymorphism in the nucleotide sequence of the subject pig.
(10) A method for detecting one or more polymorphisms in the KIT gene of a subject pig and discriminating the type of the subject pig based on the detected polymorphism or a combination thereof, wherein the polymorphism is And a polymorphism at one or both of the positions corresponding to positions 1159 and 1209 in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The method of the present invention is a method for detecting one or more polymorphisms in the KIT gene of a subject pig, and discriminating the type of the subject pig based on the detected polymorphism or a combination thereof, The polymorphism includes a polymorphism at one or both of the positions corresponding to positions 1159 and 1209 in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1. It is. Specifically, the method is performed by, for example, hybridization using immobilized nucleic acid. The kit of the present invention is a kit for performing the method of the present invention by such hybridization, and can detect a polymorphism at any one or both of the positions corresponding to the positions 1159 and 1209. Oligonucleotides are included as capture oligonucleotides. The polymorphism is a novel polymorphism found by the present inventors.
[0011]
In the present invention, the detection of the polymorphism and the classification of the pig based on the result may be performed in combination with a known polymorphism of the KIT gene in addition to the above polymorphism. Furthermore, you may combine these polymorphisms with the polymorphism in MC1R gene.
[0012]
As the polymorphism of the KIT gene, in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1, positions 398, 549, 595, 659, 844, 884, 959, 1159, 1209, 1313, 1342, 1632, 1724, 1986, 2179, 2295, 2313, 2314, 2360, 2377, 2391, 2429, 2449, 2470, 2472 2600, 2611, 2622, 2663, 2674, 2680, 2717, 2834, 2840, 2899, 3020, 3049, 3207, 3221, 3258, 3296, 3317 , 3323, 3341, 3373, 3387, 3420, 3482, 3597 , 3666 positions, 3705 positions, 3737 positions, 3773 positions, 3884 positions, 3885 positions, 3886-position, and a polymorphism at a position corresponding to position 3887.
[0013]
As the polymorphism of the MC1R gene, in the base sequence of the MC1R gene based on the base sequence shown in SEQ ID NO: 2, 154, 176, 234, 241, 241, 362, 367, 598, and A polymorphism at a position corresponding to a position selected from the 600th position may be mentioned.
[0014]
The position of a polymorphism in a gene can be changed by insertion or deletion of nucleotides in a sequence upstream of the position. In the present invention, the “corresponding position” indicates the relative position in the reference sequence shown in SEQ ID NO: 1 or 2 when the absolute position in the gene sequence is changed.
[0015]
Specifically, in the KIT gene, substitution of thymine at position 398 with cytosine, substitution of adenine at position 549 with guanine, substitution of adenine at position 595 with thymine at position 595, and position 659 of the KIT gene , A substitution of guanine with adenine at position 844, a substitution of cytosine with adenine at position 884, a substitution of adenine with guanine at position 959, a substitution of thymine with cytosine at position 1159, position 1209 , A substitution of guanine with adenine at position 1313, a substitution of guanine with adenine at position 1342, a substitution of guanine with adenine at position 1632, a substitution of cytosine with thymine at position 1724, 19 Substitution of adenine for guanine at position 6, substitution of cytosine for guanine at position 2179, substitution of guanine for adenine at position 2295, substitution of cytosine for thymine at position 2313, substitution of cytosine for adenine at position 2314, Substitution of cytosine for thymine at position 2360, substitution of cytosine for thymine at position 2377, substitution of cytosine for guanine at position 2391, deletion of cytosine at position 2429, substitution of guanine for adenine at position 2449, position 2470 Substitution of adenine for adenine at position 2, substitution of cytosine for adenine at position 2472, substitution of guanine for adenine at position 2600, substitution of adenine for guanine at position 2611, and substitution at position 2622 Tosin to thymine substitution, adenine to guanine substitution at position 2663, cytosine to guanine substitution at position 2674, thymine to cytosine substitution at position 2680, cytosine to thymine substitution at position 2717, position 2834 Adenine to guanine substitution, adenine to thymine substitution at position 2840, guanine to adenine substitution at position 2899, guanine to adenine substitution at position 3020, cytosine to adenine substitution at position 3049, position 3207 Substitution of cytosine for thymine; substitution of adenine for cytosine at position 3221; substitution of guanine for adenine at position 3258; substitution of thymine for cytosine at position 3296; Substitution of thymine at position 3323 with cytosine, substitution of guanine with adenine at position 3341, substitution of guanine with thymine at position 3373, substitution of guanine with adenine at position 3487, adenine at position 3420 To guanine, guanine to adenine at position 3482, guanine to thymine at position 3597, adenine to guanine at position 3666, guanine to adenine at position 3705, adenine at position 3737 To guanine, cytosine to thymine at position 3773, adenine deletion at position 3884, guanine deletion at position 3885, thymine deletion at position 3886, and thymine at position 3887 Like deletion of.
[0016]
In the MC1R gene, substitution of guanine for adenine at position 154, substitution of thymine for cytosine at position 176, substitution of thymine for cytosine at position 234, substitution of adenine for guanine at position 241 and cytosine for position 362 For thymine, 367 for cytosine to thymine, 598 for guanine to adenine, and 600 for guanine to adenine.
[0017]
Examples of polymorphic combinations other than positions 1159 and 1209 of the KIT gene detected in the present invention include the following combinations.
(1) C659T, C2377T
[0018]
(2) T398C, A549G, C659T, A959G, C1724T, A1986G, C2179G, C2377T, G2391C, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G3020A, C3207T, A3221C, G3A3G, 3328A, G3A3G, 3328C, G3A3G, 3962C , G3482A, A3666G, G3705A
[0019]
(3) T398C, A549G, C659T, C884A, A959G, G1342A, C1724T, A1986G, C2179G, C2377T, G2391C, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G3020, C3207, 32320, 32330C , G3341A, G3487A, G3482A, A3666G, G3705A
[0020]
(4) T398C, A549G, C659T, A959G, G1313A, C1724T, A1986G, C2179G, C2377T, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G3020A, C3207T, A3221C, G3A3G, 3328C, G3A3G, 3328C , G3482A, A3666G, G3705A
[0021]
(5) T398C, A549G, C659T, A959G, C1724T, A1986G, C2179G, C2377T, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G3020A, C3207T, A3221C, G3A3A, T3296, 3329A, T3296A, T3296A , A3666G, G3705A
[0022]
(6) T398C, A549G, C659T, A959G, C1724T, A1986G, C2179G, C2377T, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G3020A, C3207T, A3221C, G3A3A, T3296A, T3296A, T3296A , A3666G, G3705A, A3884D, G3885D, T3886D, T3887D
[0023]
(7) T398C, A549G, C659T, A959G, C1724T, A1986G, C2179G, C2377T, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G3020A, C3207T, A3221C, G3A3A, T3C3A3A, T3296A, T3296A , A3666G, G3705A
[0024]
(8) T398C, A549G, C659T, A959G, C1724T, A1986G, C2179G, C2377T, G2470A, C2472A, G2600A, A2611G, A2663G, T2680C, A2840T, G2899A, G3020A, C3207T, A3221C, A3221C, A3221A , G3482A, A3666G, G3705A, C3773T
[0025]
(9) A595T, C659T, G844A, A959G, G1632A, A1986G, G2295A, C2313T, C2314A, C2360T, C2429D, G2449A, A2611G, C2622T, A2663G, C2674G, T2680C, C2717C, A2C3A, A2734G, A2734G, A2834C , T3323C, G3373T, A3420G, G3482A, G3597T, A3666G, G3705A, A3737G
(However, the numeral indicates the position in SEQ ID NO: 1, the left and right characters sandwiching the numeral indicate substitution from the left base to the right base, and D indicates deletion.)
[0026]
In addition, examples of the combination of MC1R gene polymorphisms detected in the present invention include the following combinations.
(10) C367T
(11) A241G, C362T, G598A
(12) G154A, T176C, T234C, A241G, G600A
[0027]
In the present invention, the determination of the pig species is performed by identifying the above polymorphism or a combination thereof. Table 1 shows a list of MC1R gene polymorphisms by type of pig. Tables 2 and 3 show a list of polymorphisms of the KIT gene by pig type. For example, in Table 2, four types of "white pigs" are described, which means that when the combinations of polymorphisms are classified for the four types of white pigs, they are divided into four types. In addition, in Tables 1 to 3, “polymorphism” indicates a position based on the base sequence shown in SEQ ID NO: 2 for MC1R, and a position based on the base sequence shown in SEQ ID NO: 1 for KIT gene. In addition, since both the Landrace breed and the Great Yorkshire breed are white, the following two breeds will be referred to as white pigs.
[0028]
[Table 1]

[0029]
[Table 2]

[0030]
[Table 3]

[0031]
In the present invention, preferably, the polymorphism is detected using an oligonucleotide capable of detecting each polymorphism by hybridization. In the present invention, such an oligonucleotide is referred to as a capture oligonucleotide. The capture oligonucleotide has a nucleotide sequence complementary or homologous to the nucleotide sequence of the region containing the polymorphic site.
[0032]
The design of the capture oligonucleotide is performed so as to include each polymorphic site present in each gene. In addition, a type-specific base sequence of two or more bases may exist in one capture oligonucleotide.
[0033]
The base length of the capture oligonucleotide is preferably 10 to 40 bases. If the length is shorter than the above range, it becomes difficult to detect the hybridization. If the length is longer than the range, the difference in hybridization due to the type-specific sequence is reduced, and the type may be difficult to determine. However, the length of the capture oligonucleotide mainly depends on sequence characteristics such as the content of each base and the repetition of the same base. In addition, when there is a secondary structural disorder that is a factor that hinders hybridization, an oligonucleotide whose binding affinity in hybridization has been reduced by replacing any base not related to the polymorphism of the gene with a spacer compound is used. By using this, the above obstacle can be avoided. Examples of such a spacer compound include a nucleic acid skeleton that does not have a complementary bond with any type of base.
[0034]
Examples of the base sequence of the capture oligonucleotide are shown in SEQ ID NOs: 3 to 121. In addition, in addition to the numbers shown in SEQ ID NOs: 3 to 121, on the 5 ′ side and / or 3 ′ side of each base sequence, each base sequence is extended corresponding to the base sequence of MC1R gene and KIT gene, Alternatively, the capture oligonucleotide may be shortened, but in any case, the capture oligonucleotide is preferably in the range of 10 to 40 bases.
[0035]
The capture oligonucleotide may be any of DNA, RNA, peptide nucleic acid, or locked nucleic acid. The capture oligonucleotide can be synthesized in the same manner as a normal oligonucleotide, for example, by a method using a commercially available DNA synthesizer.
[0036]
In the present invention, the designed capture oligonucleotide is a good reflection of the research and investigation results up to the filing date, but if additional information is obtained later on the base sequence of a new haplotype, A new capture oligonucleotide will be designed based on the method described in the present application. Even when such a capture oligonucleotide is used, it is included in the present invention as long as one of the positions corresponding to positions 1159 and 1209 of the KIT gene is detected.
[0037]
When performing hybridization using a capture oligonucleotide as described above, the capture oligonucleotide and a nucleic acid derived from a subject pig (hereinafter, also referred to as “nucleic acid probe”) may be hybridized in a solution, Either the capture oligonucleotide or the nucleic acid probe may be immobilized on a support.
[0038]
In the case of performing immobilization, such a support is not particularly limited as long as it can immobilize a capture oligonucleotide or a nucleic acid probe, and can withstand the reaction steps required for hybridization with an analyte and detection of the hybrid. Not done. Specific examples of the material of such a support include plastics, inorganic polymers, metals, natural polymers, and ceramics.
[0039]
Specific examples of the plastic include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenolic resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyfluoroethylene, polyimide, and acrylic resin.
[0040]
Further, specific examples of the inorganic polymer include glass, quartz, carbon, silica gel, and graphite.
[0041]
Specific examples of the metal include gold, platinum, silver, copper, iron, aluminum, a magnet, and a paramagnet.
[0042]
Examples of natural polymers include polyamino acids, cellulose, chitin, chitosan, alginic acid, and derivatives thereof.
[0043]
Specific examples of the ceramic include apatite, alumina, silica, silicon carbide, silicon nitride, and boron carbide.
[0044]
In the present invention, the shape of the capture oligonucleotide or the support used for immobilizing the analyte is not particularly limited as long as the capture oligonucleotide is immobilized. Examples of the shape of such a support include a flat plate, a membrane, and fine particles.
[0045]
When the capture oligonucleotide or nucleic acid probe is immobilized on the support in the present invention, the capture oligonucleotide or nucleic acid probe may be directly immobilized on the support, the carrier is supported on the support, and the capture is performed via the carrier. Oligonucleotides or analytes may be immobilized on a support. As the carrier, the carrier itself may have a binding property to the capture oligonucleotide or the analyte, and the carrier can immobilize the capture oligonucleotide or the analyte via a ligand having a binding property to the analyte. There may be. Here, "supported" refers to a water-soluble solvent or an organic solvent used when immobilizing a capture oligonucleotide or a nucleic acid probe on a carrier or when using a capture oligonucleotide or a nucleic acid probe-immobilized support for actual detection. And the like, meaning that the carrier is not substantially desorbed from the support in various solvents.
[0046]
As long as the carrier used in the present invention is supported on the support, it may be supported simply by using physical adhesiveness, or may be chemically supported via a covalent bond or the like. You may. The carrier may be supported on the entire surface of the support, or may be supported on a part of the support, if necessary.
[0047]
Examples of the carrier include low organic molecules, plastics, inorganic polymers, metals, natural polymers, and ceramics.
Specific examples of the organic low-molecular compound include a carbodiimide group-containing compound, an isocyanate group-containing compound, a nitrogen-ipert group-containing compound, an aldehyde group-containing compound, and an amino group-containing compound.
[0048]
Further, the same plastics, inorganic polymers, metals and ceramics as described above can be used.
Particularly preferred carriers in the present invention are carbodiimide group-containing compounds or isocyanate group-containing compounds.
[0049]
The supply of each capture oligonucleotide or nucleic acid probe to the support is usually provided in a form contained in water or a buffer so that the activity of the capture oligonucleotide or nucleic acid probe is maintained. Further, the temperature at the time of supply is desirably 0 ° C to 100 ° C so that the activity of the capture oligonucleotide to be immobilized or the analyte is not lost.
[0050]
Alternatively, the oligonucleotide can be immobilized by irradiating the oligonucleotide with an electromagnetic wave such as ultraviolet light after contact with the support. Further, in a state in which a known compound such as an oligonucleotide and a carbodiimide resin, a nitrogen pipette, a polyamino acid, and nitrocellulose are chemically or physically bonded, the mixture may be contacted with a carrier to be fixed, and At this time, it may be fixed by irradiating electromagnetic waves such as ultraviolet rays.
[0051]
In the present invention, the means for supplying the capture oligonucleotide or the analyte to the support is not particularly limited as long as the capture oligonucleotide or the analyte is supplied while maintaining the activity of the analyte. Examples of such a method include a method using a dispenser, a method using a pin, a method using an inkjet, and the like.
[0052]
When a capture oligonucleotide or a nucleic acid probe is immobilized on a support, its shape does not affect hybridization, and its shape is not particularly limited as long as detection is not difficult. Such examples include a circle, a square, and the like. The size of the site where each capture oligonucleotide is immobilized on the support is preferably 10 μm to 1 cm in diameter. If it is smaller than this, detection becomes difficult, and if it is larger than this, the area occupied by the entire arrangement of the capture oligonucleotides becomes large, which may cause a problem that operability is deteriorated.
[0053]
When immobilizing each capture oligonucleotide or nucleic acid probe on the support, each oligonucleotide or analyte may be directly immobilized, but by increasing the reactivity, a linker is attached to the end of the capture oligonucleotide or analyte. To react the linker with the support or carrier. Such linkers include amino groups or homopolymers, such as homopolymers of thymidine residues.
[0054]
The arrangement of each capture oligonucleotide or nucleic acid probe on the support is not particularly limited as long as each polymorphism can be determined, but in order to facilitate the determination of each polymorphism, a capture oligonucleotide used for polymorphism determination Alternatively, it is preferable that the subjects are arranged in one section or arranged in a line.
[0055]
The capture oligonucleotide or nucleic acid probe immobilized on the support may be one type or two or more types.
[0056]
The nucleic acid probe used for hybridization with the capture oligonucleotide is a nucleic acid containing the MC1R gene and the KIT gene or a part thereof. The nucleic acid probe is not particularly limited as long as it does not inhibit hybridization, and usually, DNA or RNA is used. The method for producing the nucleic acid probe is not particularly limited as long as it does not inhibit hybridization. Specifically, a nucleic acid is extracted from pig blood or tissue, and a region containing at least the polymorphic sites of the MC1R gene and the KIT gene is subjected to a polymerase chain reaction (PCR) method, an in vitro transcription method, and a loop-mediated isothermal amplification method. A nucleic acid probe can be prepared by amplifying or isolating by the method described above. For the preparation of a nucleic acid, a method usually used for preparing a nucleic acid can be employed. For example, Anim. Sci. J. 71 (8): 222-234, 2000.
[0057]
The nucleic acid probe is prepared so as to include at least one polymorphism. In addition, PCR primers used for preparing a nucleic acid probe are designed so as to have a base sequence complementary to a capture oligonucleotide, excluding a sequence region unique to each haplotype. The nucleic acid probe may be longer or shorter than the capture oligonucleotide as long as hybridization is possible. In addition, in order to enhance the specificity of the PCR reaction, an initial preparation is performed using a preliminary primer that prepares a wider region than the target nucleic acid probe, and a nucleic acid probe is obtained using the prepared nucleic acid as a template. Nucleic acid amplification may be performed using the next primer. When the polymorphism is at a distant place, a nucleic acid probe corresponding to each unique region can be prepared.
[0058]
Nucleic acid probes or capture oligonucleotides are usually labeled to detect hybridization. The method for obtaining a labeled nucleic acid probe or capture oligonucleotide is not particularly limited as long as it does not inhibit hybridization. Examples of such methods include a method in which primers used for final nucleic acid probe preparation are labeled in advance, a method in which labeling is performed during nucleic acid probe preparation, a method in which labeling is performed after nucleic acid probe preparation, and a method for preparing labeled capture oligonucleotides Method and the like.
[0059]
Examples of a method of labeling a primer in advance and a method of preparing a labeled capture oligonucleotide include a method of synthesizing a primer using a labeled nucleotide. As a method of labeling during preparation of a nucleic acid probe, a method of incorporating a labeled nucleotide during a reaction of preparing a nucleic acid probe can be exemplified. Further, as a method of labeling after the production reaction, a method of labeling by a method described in JP-A-10-287870 and the like can be mentioned.
[0060]
As a substance for labeling a nucleic acid probe or a capture oligonucleotide, a labeling substance used in a usual hybridization method can be used. Examples of such a labeling substance include a fluorescent substance and a hapten. Specifically, fluorescein, rhodamine, phycoerythrin, Texas Red, cyanine fluorescent dyes, and haptens include biotin, digoxigenin, dinitrophenyl, and the like.
[0061]
Primers for preparing a nucleic acid probe can be included in a haplotype typing kit together with a capture oligonucleotide.
[0062]
Hybridization can be performed in the same manner as ordinary nucleic acid hybridization. A specific method will be exemplified below.
The nucleic acid probe is added to a mixture of a salt solution such as SSC (Standard Saline Citrate), a blocking solution such as sodium dodecyl sulfate (SDS), bovine serum albumin, and an additive for promoting the reaction. When the probe is double-stranded, denaturation by heat or the like is performed. After dropping several μL of the nucleic acid probe solution onto the support, a heating operation (usually at 30 ° C. to 80 ° C.) is performed for several hours to form a hybrid between the capture oligonucleotide immobilized on the support and the nucleic acid probe.
[0063]
The support is submerged in an appropriate concentration of SSC solution or tetramethylammonium chloride solution and heated (usually at 37 ° C. to 70 ° C.), leaving only specific hybrids selectively on the support. At this time, SDS or the like may be added.
[0064]
For detection of the hybrid, a fluorescent substance or a hapten labeled on the nucleic acid probe is used. When a fluorescent substance is used, the fluorescence labeled on the hybridized nucleic acid probe is detected using a dedicated fluorescent scanner.
[0065]
When a hapten is used, a solution containing a hapten-recognizing protein or a protein binding thereto and a conjugate (enzyme conjugate) such as alkaline phosphatase or horseradish peroxidase is added to the support, and the solution is added at room temperature for several tens of minutes. Let react for minutes. Prior to performing the binding reaction between the hapten and the enzyme conjugate, the region of the support other than the region where the capture oligonucleotide was immobilized was coated with a protein such as casein or bovine serum albumin to support the enzyme conjugate. Non-specific adsorption of the body can be prevented. This treatment can be performed by immobilizing the oligonucleotide, adding a solution of a protein such as casein onto the support, and allowing the solution to stand at room temperature for several tens of minutes.
[0066]
After the binding reaction between the enzyme conjugate and the hapten of the nucleic acid probe, the enzyme conjugate that did not bind to the hapten was washed with an appropriate buffer containing a surfactant, whereby the hapten in the target nucleic acid was placed on the support. Only the enzyme conjugate bound to will remain.
[0067]
Thereafter, by adding a compound that develops color and is deposited by an enzyme such as alkaline phosphatase or horseradish peroxidase bound to the enzyme conjugate, only the portion forming a hybrid is visualized.
[0068]
Examples of the compound used here include nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphoric acid-p-toluidine salt), 3,3 ′, 5,5′-tetramethylbenzidine and the like. Used.
[0069]
The detection of the polymorphism is performed by detecting a dye or fluorescence deposited for each capture oligonucleotide based on the obtained hybridization results. Specifically, the color development amount and fluorescence intensity of each capture oligonucleotide are compared with the corresponding capture oligonucleotide, and the larger of the amounts is determined to be hybridization positive. When the amounts are the same, both are determined to be positive. The standard is not particularly limited, but differs depending on the labeling substance used and the like. Therefore, it is desirable to determine the standard for each detection system.
[0070]
The above determination is made for each polymorphic site, and when each polymorphism or combination thereof matches any type, it is determined that the pig is of that type. Since pigs are diploid, in the case of hybrids, they may be judged as two types of crosses.
[0071]
Tables 4 to 6 show the correspondence between the hybridization signals and the types of pigs when immobilized capture oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 3 to 121 are used. Here, Table 3 shows that of the MC1R gene and Table 4 shows that of the KIT gene.
[0072]
[Table 4]

[0073]
[Table 5]

[0074]
[Table 6]

[0075]
The kit of the present invention includes a capture oligonucleotide. Further, the kit of the present invention can include primers or labeled nucleic acid probes for preparing nucleic acid probes, buffers, hybridization reagents such as enzyme conjugates that recognize haptens, and the like.
[0076]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0077]
<1> Synthesis of oligonucleotide
According to a conventional method, an oligonucleotide was synthesized using an oligonucleotide synthesizer (Perkin-Elmer Applied biosystems), deprotected, and dried. The dried oligonucleotide was dissolved using 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA buffer to prepare a 100 pmol / μL oligonucleotide solution. This method of synthesis is similar for any capture oligonucleotide or any oligonucleotide used as a primer. The nucleotide sequences of the synthesized oligonucleotides are shown in SEQ ID NOs: 3 to 125. Of these, SEQ ID NOs: 3 to 121 are capture oligonucleotides, and SEQ ID NOs: 122 to 125 are primers. An amino group was bound to the 5 'end of the capture oligonucleotide using the synthesizer described above, and a rhodamine label was placed on the 5' side of the primer.
[0078]
<2> Spotting of capture oligonucleotide to support
10 μL of an oligonucleotide solution having an amino group at the 5 ′ end was mixed with 10 μL of a microspotting solution (TeleChem International Inc.), and the mixture was dispensed on a microtiter plate (Greiner Laboratory Inc.). A silanized slide glass (Matsunami Glass Ind. Ltd.) was placed at a predetermined position of the spotting machine, and the spotting machine was operated. After the end of the spotting, steam from hot water was applied to the slide glass for several seconds, and then irradiated with 30 mJ of ultraviolet rays. After re-exposure to steam for a few seconds, the slides were placed on a hot plate to remove moisture. After rinsing the slide glass with a 0.1% aqueous solution of sodium dodecyl sulfate, the slide glass was rinsed with distilled water. The slide glass was immersed in 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100 containing 3% BSA (bovine serum albumin) for 30 minutes at room temperature for blocking. Then, after drying at room temperature, it was washed with 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA buffer. The slide glass was dried again at room temperature and stored in a dry state in a cool and dark place until use.
[0079]
<3> Preparation of probe nucleic acid
A probe nucleic acid was prepared by PCR using pig DNA as a template.
50 μL of blood obtained from various pigs was placed in a microtube, and 500 μl of 10 mM Tris-HCl pH 8.0, 0.1 M NaCl, 1 mM EDTA, 0.5% SDS, 500 μg / mL proteinase K was added thereto, and mixed. , 37 ° C overnight. 500 μL of a phenol: chloroform: isoamyl alcohol (25: 24: 1) solution was added, and the mixture was stirred and centrifuged (12,000 rpm, 1 minute) to collect an aqueous phase. This operation was repeated three times. 500 μL of a chloroform: isoamyl alcohol (24: 1) solution was added to the obtained aqueous phase, and the mixture was stirred and centrifuged (12000 rpm, 1 minute) to collect the aqueous phase. This operation was repeated twice. The DNA in the obtained aqueous phase was recovered by ethanol precipitation, dried, and then dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to obtain a PCR template solution.
[0080]
The composition of the PCR reaction solution was 1 unit of Taq polymerase, 10 pmol of each primer, 5 μl of reaction buffer, 10 nmol of dNTP, 0.5 μl of template DNA solution, and 50 μl of sterile water to make a total volume of 50 μl. 2 are prepared for amplifying the MC1R gene (primers of SEQ ID NOs: 122 and 123) and for amplifying the KIT gene (primers of SEQ ID NOs: 124 and 125). The solution placed in the tube was set in a thermal cycler and heated at 94 ° C. for 3 minutes, and then 30 cycles of 94 ° C .: 45 seconds, 60 ° C .: 60 seconds, 72 ° C .: 1 minute were performed, and then 72 ° C .: The reaction was performed for 3 minutes.
[0081]
In this example, as a confirmation test, electrophoresis using an agarose gel described below was performed, but this operation is not essential for actual clinical discrimination. 1 μl of the PCR reaction mixture was taken and mixed with 1 μl of 6 × electrophoresis dye (30% glycerol, 0.25% bromophenol blue, 0.25% xylene cyanol) and 4 μl of distilled water. After electrophoresis on a 2% agarose gel at 100 V for 90 minutes, the gel was immersed in distilled water containing 0.5 μg / ml ethidium bromide for 30 minutes, and the gel was photographed with a CCD camera under ultraviolet irradiation. As a result, it was confirmed that the desired amplified fragment was obtained.
[0082]
<4> Hybridization
Mix all the probe nucleic acid solutions prepared in <3>, then take 4 μl, add 16 μl of ArrayIt Unihyb Hybridization Solution (TeleChem International Inc.), mix and heat-treat at 100 ° C. for 10 minutes, and then in ice. For 5 minutes. 5 μl of the probe nucleic acid solution was placed on a slide glass on which the capture oligonucleotide prepared in <2> was immobilized, and a cover glass was placed thereon. This was put in a moisturizing box, further put in a thermostat set at 37 ° C., and left still for 120 minutes. The slide glass was taken out and quickly immersed in a 2 × SSC, 0.1% SDS solution (2 × SSC: 0.033 M NaCl, 0.033 M sodium citrate) at room temperature to remove the cover glass. The operation of immersing the slide glass in a 2 × SSC, 0.1% SDS solution at room temperature for 5 minutes is performed twice, and a 0.2 × SSC, 0.1% SDS solution (0.2 × SSC: 0. (0033M NaCl, 0.0033M sodium citrate) was immersed twice for 5 minutes, and finally immersed in 2 × SSC.
[0083]
<5> Detection of fluorescence
After the hybridization, the slide glass was taken out of the 2 × SSC, set in a centrifuge (Beckman), and centrifuged at 2000 rpm for 1 minute. Next, the slide glass was set on a ScanArray 4000 (manufactured by GSI Lumonics), and fluorescence was detected.
[0084]
Table 7 shows the above results and the relationship with the typing by the DNA sequencing method. It is clear that the method of the present invention enables the type of pig to be determined.
[0085]
[Table 7]

[0086]
【The invention's effect】
According to the present invention, the type of a pig can be determined efficiently and with high accuracy.
[Sequence list]

Claims (10)

In order to discriminate the type of the subject pig by a polymorphism or a combination thereof comprising one or more capture oligonucleotides and being detected by hybridization of these capture oligonucleotides with nucleic acid derived from the subject pig Wherein the capture oligonucleotide has a polymorphism at any one or both of positions corresponding to positions 1159 and 1209 in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1. A kit, comprising an oligonucleotide capable of detecting a nucleic acid. The kit according to claim 1, wherein the capture oligonucleotide is immobilized on a support. In the base sequence of the KIT gene, the capture oligonucleotide is at positions 398, 549, 595, 659, 844, 884, 959, 1313, 1342, 1632, 1724, 1986, 2179, 2295, 2313, 2314, 2360, 2377, 2391, 2429, 2449, 2470, 2472, 2600, 2611, 2622, 2663, 2674, 2680 , 2717, 2834, 2840, 2899, 3020, 3049, 3207, 3221, 3258, 3296, 3317, 3323, 3341, 3373, 3387, 3420, 3482 , 3597, 3666, 3705, 3737, 377 Position, 3884 positions, 3885 positions, 3886 positions, and kit according to claim 1 or 2 further comprising one or more polymorphism may be detected oligonucleotides positions selected from positions 3887. The capture oligonucleotide according to any one of claims 1 to 3, wherein the capture oligonucleotide includes an oligonucleotide capable of detecting a polymorphism at one or more positions in a melanocyte stimulating hormone receptor gene (hereinafter, referred to as “MC1R gene”). The kit as described. The polymorphism in the MC1R gene is at a position corresponding to a position selected from positions 154, 176, 234, 241, 241, 361, 367, 598, and 600 in the nucleotide sequence shown in SEQ ID NO: 2. The kit according to claim 4, which is polymorphic. The kit according to any one of claims 1 to 5, wherein the capture oligonucleotide has a nucleotide sequence complementary or homologous to a nucleotide sequence of a region containing a polymorphic site, and is 10 to 40 nucleotides in length. An oligonucleotide capable of detecting, by hybridization, a polymorphism at a position corresponding to position 1159 or 1209 in the base sequence of the KIT gene based on the base sequence shown in SEQ ID NO: 1. The oligonucleotide according to claim 7, which has a nucleotide sequence complementary or homologous to the nucleotide sequence of the region containing the polymorphic site, and is 10 to 40 nucleotides in length. A nucleic acid derived from a subject pig is hybridized with the capture oligonucleotide contained in the kit according to any one of claims 1 to 6, and hybridization of each capture oligonucleotide with a nucleic acid derived from the subject pig is performed. A method for determining the type of a subject pig by detecting the presence or absence and identifying a polymorphism in the nucleotide sequence of the subject pig. A method for detecting one or more polymorphisms in a KIT gene of a subject pig and discriminating the type of the subject pig based on the detected polymorphism or a combination thereof, wherein the polymorphism is a sequence number 1. A method comprising a polymorphism at one or both of the positions corresponding to positions 1159 and 1209 in the base sequence of the KIT gene based on the base sequence shown in 1.