JP3639853B2 - How to detect mutations - Google Patents

Description

同様のレポーター遺伝子に、ADE1やGFPを挙げることができる。ADE1は、ADE2と同様の原理で変異によって着色コロニーを生じる。GFPでは、正常な場合に蛍光を発し、異常によって蛍光を消失することになる。
【００４１】
あるいは、ルシフェラーゼのような酵素活性タンパク質をコードする遺伝子をレポーター遺伝子に用いることができる。このようなレポーター遺伝子を用いる場合には、形質転換体を単離した上で、その酵素活性を測定することにより、レポーター遺伝子の発現レベルを確認することもできる。
【００４２】
本発明に用いるギャップベクター、およびレポーター遺伝子を検出するための試薬を予めセットにして、変異検出用のキットとすることができる。レポーター遺伝子の検出のための試薬としては、たとえばADE2欠損酵母の培養を行うためのアデニン制限培地などが含まれる。
このキットには、ギャップベクター、ギャップベクターによる形質転換を行うための宿主細胞等を組み合わせることもできる。更に、変異を検出すべき遺伝子に合わせて設計したPCR用のプライマーを組み合わせておくこともできる。
【００４３】
【実施例】
以下、実施例に基づいて本発明を具体的に説明する。
〔実施例１〕 ストップコドンアッセイによる、BRCA1遺伝子変異の検出
（１） 使用酵母株、培地、および遺伝子導入方法
ADE2を欠損した発芽酵母S. cerevisiae株であるyPH857(ATCC# 76628)を使用した。遺伝子型は[MATa ura3-53 lys2-801 ade2-101 his3-D200 trp1-D63 leu2-D1 cyh2R] である。通常培養用の完全培地としてはYPDA++を使用した。 ウラシル非添加、アデニン制限ストップコドンアッセイ用培地としてはCAu10を使用した。
培地の組成は以下の通りである。
・YPDA++培地：
10 g/L 酵母エキストラクト
20 g/L ペプトン
2% グルコース
200 mg/L アデニン
・CAu10培地：
0.67% 酵母ニトロゲンベース
2% グルコース
1% カザミノ酸
20 mg/L L-トリプトファン
10 mg/L アデニン
2.5% 寒天
【００４４】
（２）本発明によるギャップベクターの作製
ギャップベクターは、pLS381（スイス国立癌研究所(ISREC) Richard Iggo博士より分与）を改良することによって構築した。pLS381は、URA3マーカー遺伝子とCEN/ARS配列、AmpおよびColE1 Oriを持ち、CYCプロモータで駆動されADE2を発現する酵母/大腸菌シャトルベクターである。すなわち、ギャップベクターの自己再環状化を最小限に抑えるために、CYCプロモーターとADE2遺伝子の間のBamH I切断部位をSfo I部位に変更した。Sfo Iは、次の認識配列の|で示したc|g間を切断して平滑末端を生じる制限酵素である。
....ggC|Gcc....
....ccG|Cgg....
具体的には、次の塩基配列で示される2本鎖のオリゴヌクレオチドを、BamH Iにて線形化したpLS381に組み込んだ。
5'-CACTAAATT AATAATGACC GGCGCC ATGGATTCTA GAACAGTTG-3'（配列番号：１）
3'-GTGATTTAA TTATTACTGG CCGCGG TACCTAAGAT CTTGTCAAC-5'
すなわち、BamH Iで消化したpSL381、および上記2本鎖オリゴヌクレオチドを酵母株yPH857に同時導入した。酵母コロニーから回収したプラスミドクローンのうち、Sfo Iにて切断できるものを選択し、DNA塩基配列決定により挿入塩基配列を確認して、目的とする塩基配列を持つものを選択しpMT18と命名した。pMT18をSfo Iで切断して得られるギャップベクターの末端を構成するクローニング用の塩基配列は、それぞれ次の24 bとなる。
5'-端：... CAC ACT AAA TTA ATA ATG ACC GGC（配列番号：２）
3'-端：GCC ATG GAT TCT AGA ACA GTT GGT ...（配列番号：３）
線形化ベクターは仔牛腸アルカリホスファターゼ処理を行って脱リン酸化し自己再環状化を防止した。BamH I線状化pLS381およびSfo I線状化pMT18を、単独でそれぞれ100 ngまたは50ngを酵母株yPH857に導入し、自己環状化により生じるコロニー数を比較した。
【００４５】
（３）臨床サンプルからのRNAおよびゲノムDNAの抽出
北海道大学医学部附属病院第一外科およびその関連施設で手術が行われた大腸癌患者から摘出された大腸癌および周辺正常大腸粘膜を液体窒素にて凍結保存した。-80℃で保存されたサンプルからTrizol Reagent (Gibco)を用いて全RNAおよびゲノムDNAを抽出した。
【００４６】
（４）プライマーの設計
BRCA1遺伝子を対象として、本発明によるストップコドンアッセイを評価した。
まず、BRCA1のエキソン11について表１に示すようなプライマー対を設計して臨床サンプルゲノムDNAについてPCR増幅を行った。表中、アンダーラインを付けた部分がクローニング領域に相同な塩基配列である。得られたPCR産物をインサートＤＮＡとして、本発明によるストップコドンアッセイを実施した。
【表１】

a:INに付けた−番号は、イントロンスプライス供与部位からイントロン−エキソン接合部までの塩基数
b:INに付けた＋番号は、イントロンスプライス需要部位からイントロン−エキソン接合部までの塩基数
c:ATG開始コドンとクローニング領域に相同な塩基配列までを含む断片全体の長さ
d:エキソン１１における5'末端を１としたときの位置
【００４７】
プライマーはエキソン11を全長 (set 1)、中間部1/3 (set 2)、前2/3 (set 3)、後2/3(set 4) の各部に分けて増幅するように設定した。set １による通常のPCRと、効率良く増幅生成物を得るために、nested PCR（set 2〜set 4）を利用した。すなわち、各セットについてexternalプライマー対によるPCR増幅ののち、その産物を鋳型としてinternalプライマー対でさらにPCR増幅した。Internalプライマー対には5'側(forward)プライマーには上記pMT18の5'-断端24 bと蛋白合成開始コドンATGを付加し、3'側(reverse)プライマーにはpMT18の3'-断端24 bを付加した。set 2においてはinternalプライマー対に付加するクローニング領域に相同な塩基配列の長さを変化させた。すなわち、pMT18の末端に相同な塩基配列として、18 b、30 b、および39 b付加するものを別に作成した。
【００４８】
（５）PCR増幅
PCR反応はPerkin Elmer 2400 Thermal Cyclerを用いて行った。
セット1ではinternalプライマー対(gBR1EX11F/gBR1EX11R)を直接用い、LA Taqポリメラーゼ (TAKARA)にて反応を行った。反応液は添付1x バッファー (MgCl₂ ⁺), 2.0 mM dNTP, 0.4 μM 各プライマー, 2.5 U LA Taq ポリメラーゼに鋳型 ゲノムDNA 50 ng/μlを2μl加えて、全量25μlとした。ホットスタートを行い、94℃ 40秒, 56℃ 40秒, 68℃ 5分を35サイクル行った後、68℃ 7分の後伸展を加えた。
使用したプライマーの配列は以下の通りである。
gBR1EX11F（配列番号：４）：5'-CACACTAAATTAATAATGACCGGCATGGCTGCTTGTGAATTTTCTGAGACG-3'
ｇＢＲＥＸ１１Ｒ（配列番号：５）：5'-ACCAACTGTTCTAGAATCCATGGCTCCAATACCTAAGTTTGAATCCATGC-3'
【００４９】
セット2ではinternalプライマー対(BR1EX11F2/BR1EX11R2)ではPfu TURBO ポリメラーゼ (Stratagene) 1.25 U, 添付1x buffer, 2.0 mM dNTP, 0.4 μM 各プライマー, 鋳型 100 ngを全量25μlでホットスタートにて行った。95℃ 40秒, 58℃ 40秒, 78 ℃ 2.5分を10サイクル行った後、78℃ 7分の後伸展を行った。この反応液を1/100に希釈したもの2μlを鋳型として同組成反応液にてexternalプライマー対(gBR1EX11F2/gBR1EX11R2-2)を用いてnested PCRの第2段反応を行った。PCRサイクルは95℃ 40秒, 58℃ 40秒, 78℃ 2分を35サイクル行った後、78℃ 7分の後伸展を行った。またクローニング領域部分の長さが異なる次のプライマー対を用いて同様の第２段反応を行った。
(g18BR1EX11F2/g18BR1EX11R2-2)
（ｇ３０ＢＲ１ＥＸ１１Ｆ２／ｇ３０ＢＲ１ＥＸ１１Ｒ２−２）
（ｇ３９ＢＲ１ＥＸ１１Ｆ２／ｇ３９ＢＲ１ＥＸ１１Ｒ２−２）
【００５０】
使用したプライマーの配列は以下の通りである。
BR1EX11F2（配列番号：６）：5'-AAAAGAATAGGCTGAGGAGGAAGTC -3'
ＢＲ１ＥＸ１１Ｒ２（配列番号：７）：5'-CTCATTTCCCATTTCTCTTTCAGGT -3'
ｇＢＲ１ＥＸ１１Ｆ２（配列番号：８）：5'-CACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT -3'
ｇＢＲ１ＥＸ１１Ｒ２−２（配列番号：９）：5'-ACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3'
ｇ１８ＢＲ１ＥＸ１１Ｆ２（配列番号：１０）：5'-AAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT -3'
ｇ１８ＢＲ１ＥＸ１１Ｒ２−２（配列番号：１１）：5'-TGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3'
ｇ３０ＢＲ１ＥＸ１１Ｆ２（配列番号：１２）：5'-AATACACACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT -3'
ｇ３０ＢＲ１ＥＸ１１Ｒ２−２（配列番号：１３）：5'-TAATATACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3'
ｇ３９ＢＲ１ＥＸ１１Ｆ２（配列番号：１４）：5'-GCAAACACAAATACACACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT -3'
ｇ３９ＢＲ１ＥＸ１１Ｒ２−２（配列番号：１５）：5'-TCCCCCTCCTAATATACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3'
【００５１】
セット3ではinternalプライマー対(BR1EX11F-new/BR1EX11R2)ではPfu TURBO ポリメラーゼ (Stratagene)にて95℃ 40秒, 58℃ 40秒, 78℃ 5分を10サイクル行った後、78℃ 7分の後伸展を行った。この反応液を1/100に希釈したもの2μlを鋳型としてexternalプライマー対(gBR1EX11F/gBR1EX11R2-2)を用いてnested PCRの第2段反応を行った。PCRサイクルは95℃ 40秒, 58℃ 40秒, 78℃ 5分を35サイクル行った後、78℃ 7分の後伸展を行った。
【００５２】
使用したプライマーは以下の通りである。
BR1EX11F-new（配列番号：１６）：5'-GTACCTTGTTATTTTTGTATATTTTCAG -3'
ＢＲ１ＥＸ１１Ｒ２（配列番号：１７）：5'-CTCATTTCCCATTTCTCTTTCAGGT -3'
ｇＢＲ１ＥＸ１１Ｆ（配列番号：１８）：5'-CACACTAAATTAATAATGACCGGCATGGCTGCTTGTGAATTTTCTGAGACG -3'
ｇＢＲ１ＥＸ１１Ｒ２−２（配列番号：１９）：5'-ACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3'
【００５３】
セット4ではinternalプライマー対(BR1EX11F2/BR1EX11R-new)ではPfu TURBO ポリメラーゼ (Stratagene)にて95℃ 40秒, 60℃ 40秒, 78℃ 4.5分を10サイクル行った後、78℃ 7分の後伸展を行った。この反応液を1/100に希釈したもの2μlを鋳型としてexternalプライマー対(gBR1EX11F2/gBR1EX11R)を用いてnested PCRの第2段反応を行った。PCRサイクルは95℃ 40秒, 58℃ 40秒, 78℃ 5分を35サイクル行った後、78℃ 7分の後伸展を行った。
【００５４】
使用したプライマーは以下の通りである。
BR1EX11F2（配列番号：２０）：5'-AAAAGAATAGGCTGAGGAGGAAGTC -3’
ＢＲ１ＥＸ１１Ｒ−ｎｅｗ（配列番号：２１）：5'-GGGCAAACACAAAAACCTGGTTCC -3’
ｇＢＲ１ＥＸ１１Ｆ２（配列番号：２２）：5'-CACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT -3'
ｇＢＲ１ＥＸ１１Ｒ（配列番号：２３）：5'-ACCAACTGTTCTAGAATCCATGGCTCCAATACCTAAGTTTGAATCCATGC -3'
【００５５】
（５）本発明によるストップコドンアッセイ
PCR産物を1% アガロースゲルにて電気泳動した。臭化エチジウム(ethidium bromide)染色の後にUV下で写真撮影して良好なPCR増幅を確認した。PCRバンドの濃さに基づいて測定した1〜10μlのPCR産物を、線形化pMT18とともにリチウム酢酸(Lithium acetate)法にて酵母株yPH857に導入した(Gietz, et al. Yeast 11: 355-360, 1995)。 形質転換した酵母をCAu10プレート上にて30℃、2〜3日間培養し、4℃にて発色を増強したのち、白色および赤色コロニーを計数した。
【００５６】
（６）プラスミド抽出、制限酵素切断、DNA配列決定
赤色または白色酵母コロニーをYPDA++培地にて１晩培養後、ザイモリアーゼ(Zymolyase) 100T (生化学工業)にて処理し、酵母莢膜を除去した後、アルカリSDS溶解法(QIAprep Spin Miniprep kit; Qiagen)にてプラスミドを抽出した。このプラスミドを電気穿孔法にて大腸菌株XL-1 Blueに導入、培養したものからアルカリSDS溶解法にてプラスミドを抽出した。これを制限酵素切断にてインサートの有無をチェックし、DNA配列を決定した。
【００５７】
（７）結果
（ｉ） Sfo I平滑末端作成による自己環状化防止効果
BamH I線状化pLS381とSfo I線状化pMT18を単独でそれぞれ100 ngまたは50ngを酵母株yPH857に導入し、自己環状化により生じるコロニー数を計数した。この結果BamH I線状化pLS381では115 (100ng), 77 (50 ng), 72 (50ng)個のコロニーが生じたのに対し、Sfo I線状化pMT18では67 (100ng), 42 (50 ng), 38 (50ng)個のコロニーが生じ、約45%自己環状化が防止できることが判明した。しかし白色コロニーとはほぼ同等数の赤色コロニーが生じた。これはベクター内部での偶発的相同組換えによるものと考えられた。末端平滑化のみでは高度な自己環状化防止作用を期待できないと考えられた。
【００５８】
（ｉｉ） セット1プライマー対によるアッセイ結果
nested PCRではなく、クローニング領域に相同な塩基配列を付加したプライマー対(set 1)で直接3.4 kbのフラグメントをLA Taqを用いて増幅し、６サンプルについて本発明による検出方法を試みた。その結果、６サンプルとも十分な数のコロニー(400個以上)を確認することができた。しかし、赤色コロニーが27.4%〜82.0% (平均45%)となり、バックグラウンドの赤色コロニーが多く出現した。白色コロニー６個をとってプラスミドを抽出し、制限酵素切断で調べたところ、すべてにおいて期待される3.4 kbのインサートが入っておらず、DNA配列決定ではプライマー対が二本鎖を形成したと考えられる短いフラグメントが入っていた。赤色コロニーを６個とって調べたところ、２個に3.4 kbのインサートが入っていたが、4個においては白色コロニーと同様に短いフラグメントが入っていた。
【００５９】
（ｉｉｉ）セット2, 3, 4プライマー対によるアッセイ結果
2の結果をふまえ、セット2, 3, 4では以下の改良点を加えた。
1) 増幅サイズの制限と校正機能を持ったDNAポリメラーゼ
PCRでの誤った塩基の取り込みによる赤色コロニー（バックグラウンド）を抑える目的で増幅サイズを制限し、校正機能のあるPfu TURBOポリメラーゼを使用した。
2) プライマー同士のアニールを防止
また各プライマー設計にあたっては、プライマー同士のアニーリングによる二本鎖断片増幅を避けるため、プライマーの3'末端において二本鎖が形成されないようにコンピュータで検証した。
3) nested PCRの採用
非特異的アニーリングによる非目的産物の生成をできるだけ抑えるため、nested PCRを行うようにした。
【００６０】
以上の３つの改良の結果、次のとおり赤色コロニーの著しい現象を見た。
プライマーセット2：4.0〜10.7% (n=10, 平均8.1%)
（ＢＲ１ＥＸ１１Ｆ２／ＢＲ１ＥＸ１１Ｒ２およびgBR1EX11F2/gBR1EX11R2-2)
プライマーセット3：3.8〜7.3 (n=6, 平均5.5%)
（ＢＲ１ＥＸ１１Ｆ−ｎｅｗ／ＢＲ１ＥＸ１１Ｒ２およびgBR1EX11F/ gBR1EX11R2-2)
プライマーセット4：10.3〜16.0 (n=6, 平均13.3%)
（ＢＲ１ＥＸ１１Ｆ２／ＢＲ１ＥＸ１１Ｒ−ｎｅｗおよびgBR1EX11F2/gBR1EX11R)
【００６１】
さらにクローニング領域の長さが、相同組換え率に与える影響を確認するために、様々な長さのクローニング領域について、赤色コロニー（バックグラウンド）と、白色コロニーにおけるインサートＤＮＡを比較し、適正な相同組み換えが起きている割合を調査した。
18b、24b、30b、および39bのクローニング領域を持つプライマーで細胞株MCF-7、ZR-75-1から抽出したゲノムDNAについて本発明に基づいて変異の検出方法を行った。赤色コロニーバックグラウンドはそれぞれの細胞株サンプルで以下のように生じた。
18b 4.8% 7.9%
２４ｂ ９．８％ ７．２％
３０ｂ １１．１％ １２．７％
３９ｂ ７．７％ ８．５％
30bのクローニング領域を持つプライマーを除いて、10%以下にバックグランドを抑えることができた。バックグランドの値が10%とは、実用的に許されるレベルである。また白色コロニー各20個からプラスミドを抽出して制限酵素切断にて適正インサートの有無を確認したところ、すべてに適正にインサートＤＮＡが入っていることが確認された。
【００６２】
【発明の効果】
本発明により、遺伝子中に生じるノンセンス変異、またはフレームシフト変異を、検出するための汎用性に優れた方法が提供された。本発明では、被検遺伝子に由来するインサートＤＮＡの両端に、ギャップベクターのクローニング領域と相同な配列を付加する。そのため、インサートＤＮＡの塩基配列に関わらず、共通のギャップベクターに組み込むことが可能となる。すなわち本発明による変異検出方法は、被検遺伝子ごとにベクターを構築する必要がなく、高い汎用性を有する。
本発明を利用することにより、がん遺伝子のように長い遺伝子の全領域を対象にストップコドンアッセイを行う場合であっても、単一のベクターで変異の検出が可能となった。本発明によるストップコドンアッセイの汎用性の高さは、単に多種類の遺伝子に対応できることだけでなく、がん遺伝子のような大きな遺伝子を分割して解析する場合においても有利である。単一のベクターを用いて様々な遺伝子断片の変異を検出することができることから、大きな遺伝子を細かく分割して解析することにより、変異の位置を細かく特定することも容易である。
本発明のためのインサートＤＮＡは、たとえばクローニング領域を5'側に付加したプライマーを用いたPCRによって容易に合成することができる。したがって、本発明による変異検出方法は、任意の被検遺伝子について、簡便でかつ迅速な検査を可能とする。
【００６３】
【配列表】

【図面の簡単な説明】
【図１】本発明による変異の検出方法の原理を模式的に示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting a nonsense mutation or a frameshift mutation occurring in gene DNA.
[0002]
[Prior art]
Most of the mutations that occur in genetic DNA are base substitutions, deletions, or insertions, except for special ones. Base substitution includes missense and nonsense mutations. A missense mutation is a mutation in which a mutation occurs in one base of a codon and a different amino acid is encoded. On the other hand, a nonsense mutation that changes the codon to TGA, TAA, or TAG and terminates protein synthesis (translation) at that site is also important as a mutation. If the deletion or insertion reaches a wide range of genes, the function of the protein is lost. In reality, all the amino acids after that part are replaced by a frame shift that shifts the codon reading frame. Alternatively, a mutation results in a stop codon, and translation is stopped at the mutated part, resulting in production of a disconnected protein. According to On-line Mendelian Inheritance in Man (OMIM) covering human genetic diseases, it is known that more than half are caused by non-sense mutations or frame shifts.
[0003]
For example, BRCA1 is a tumor suppressor gene that causes familial breast cancer through its inactivation. The inactivation is said to be caused by a nonsense mutation or a frameshift mutation that occurs in the genome. Techniques that can easily find mutations in such disease-causing genes are becoming increasingly important as genome analysis information accumulates.
PCR-SSCP (PCR single strand conformation polymorphism) is known as a general technique for finding slight differences in the base sequences of genes. When DNA is electrophoresed in a single-stranded state, the degree of migration is affected by the difference in the three-dimensional structure caused by self-annealing. Based on this difference, finding the slight base sequence difference is the analysis principle of PCR-SSCP. However, in principle, the PCR-SSCP method detects all mutations including silent mutations (mutations that do not involve amino acid substitution) as mutations. As described above, if a method for detecting a mutation effective for a nonsense mutation or a frameshift mutation that is important as a mutation is provided, a more rational test can be performed.
[0004]
A method for efficiently diagnosing such nonsense mutations or frameshift mutations using yeast has been reported for MSH2, one of the genes responsible for hereditary nonpolyposis colorectal cancer (Andreutti-Zaugg, et al Cancer Res. 57: 3288-3293, 1997). This method is called a stop codon assay because it detects a stop codon caused by a non-sense mutation. In principle, the stop codon assay can detect a frameshift mutation. This is because on the vector for which the stop codon assay is performed, the frame shift extends to the translation of the reporter gene.
The stop codon assay utilizes a vector containing a reporter gene. This vector first incorporates the MSH2 cDNA and the reporter gene ADE2 (phosphoribosylaminoimidazole carboxylase, EC 4.1.1.21) in a reading frame so as to form a chimeric protein. The linearized vector was cut out in the form of leaving 3 'stumps. When a test MSH2 gene, which is an insert DNA, is inserted into the excised MSH2 gene portion, a chimeric protein of the test MSH2 gene and a reporter gene is expressed.
[0005]
When the test MSH2 cDNA amplified by RT-PCR together with this linearized vector is introduced into yeast, homologous recombination to the 5 ′ and 3 ′ stumps of the MSH2 gene occurs in the yeast. As a result, the integration of the insert DNA into the vector is completed, and the chimeric protein of MSH2 and ADE2 genes is expressed in yeast. If the yeast used is an ADE2-deficient strain, the yeast will grow normally only when ADE2 activity is expressed. However, if there is a nonsense mutation or a frameshift mutation in the MSH2 cDNA, protein synthesis is terminated in the middle, or ADE2 with a reading frame shifted is generated. As a result, the intermediate metabolites of the adenine synthesis pathway accumulate and crystallize, and the yeast colony turns red (Weisman, et al. J. Cell Biol. 105: 1539-1547, 1987), and the presence of the mutation is known visually. Can do.
[0006]
This method is an extremely excellent genetic diagnosis method that can detect nonsense mutations or frameshift mutations with a simple operation. However, in order to allow homologous recombination between the vector and the insert DNA, a region homologous to the base sequence of the insert DNA must always be prepared on the vector side. For this purpose, in the stop codon assay, a linear vector in which an insert DNA derived from the MSH2 gene, which is a test gene, is once incorporated into a vector and a part thereof is excised is used for the assay. As long as it is based on this principle, vectors prepared for a gene can only be used for testing that gene. That is, it is necessary to construct a dedicated vector for each test gene. Therefore, it is not realistic to apply to many types of gene diagnosis. There is a need to provide a technique capable of knowing nonsense mutations or frameshift mutations by a simple technique targeting not only specific genes but also a wide range of genes.
[0007]
For example, in cancer that can be said to be a typical genetic disease, many cancer-related gene mutations have been found. Activation and inactivation due to mutations in cancer-related genes are considered to be closely related to the cause of cancer. Therefore, oncogenes are important for detecting mutations.
Oncogene mutations are sometimes detected as so-called hot spots concentrated at specific sites, but many of the mutations are not concentrated on specific regions but are widely found throughout the gene. Therefore, in detecting oncogene mutations, it is necessary to detect the entire region of the gene. However, it is difficult to apply the entire region of a long gene such as BRCA1 (5592 bp) and APC (8975 bp) to the stop codon assay. First, it is difficult to accurately amplify such a long region by PCR. In addition, homologous recombination for stop codon assays and expression of fusion proteins with reporter genes are difficult. Therefore, in order to analyze such a large gene by the stop codon assay, it was necessary to divide the gene into several regions. In the known stop codon assay, in such a case, a vector of a kind corresponding to the number to be divided has to be constructed. That is, even a single gene requires a plurality of vectors. Against this background, provision of a stop codon assay with excellent versatility is demanded.
[0008]
[Problems to be solved by the invention]
An object of the present invention is to provide a highly versatile method for detecting a nonsense mutation or a frameshift mutation occurring in a gene.
[0009]
[Means for Solving the Problems]
The present inventors considered that one of the reasons why versatility cannot be achieved in a known stop codon assay is the structure of the vector. As long as a base sequence derived from a test gene is used as a cloning region for homologous recombination, a dedicated vector must be prepared according to the test gene. Therefore, the present inventors thought that the versatility of the stop codon assay can be drastically improved if the test gene can be freely integrated while keeping the vector structure constant.
[0010]
In order to achieve this object, the present inventors have studied whether a base sequence matched to the cloning region of the vector can be added to the test gene side. In other words, we tried a method for inserting a test gene based on the completely opposite idea to the conventional stop codon assay. As a result, in preparing a gene fragment (insert DNA) to be incorporated into a vector, it was found that the incorporation into a vector becomes possible by linking a specific base sequence at both ends. Using this method, in principle, mutations of all genes can be tested with a single vector. That is, the present invention relates to a method for detecting a nonsense mutation or a frameshift mutation on a gene as follows.
[0011]
[1] A method for detecting a nonsense mutation or a frameshift mutation on a test gene, comprising the following steps.
1) preparing a linear vector composed of a promoter sequence, a cloning region, and a reporter gene and having a cloning region at both ends;
However, the linear vector includes the above elements in this order by incorporating the insert DNA into the cloning region, and provides a vector capable of expressing the fusion transcript of the insert DNA and the reporter gene under the control of the promoter sequence. ,
2) preparing a DNA fragment in which a base sequence homologous to the cloning region is added to the end of the insert DNA;
3) a step of incorporating the DNA fragment of 2) into the cloning region of the vector of step 1) by homologous recombination,
4) A step of detecting the mutation using the expression of the reporter gene of the vector obtained in step 3) as an index.
[2] The method according to [1], wherein the reporter gene in step 4) is a gene that generates a visually distinguishable signal between cells expressing the gene and cells not expressing the gene.
[3] The method according to [2], wherein the cell is yeast and the reporter gene is the ADE2 gene.
[4] The method according to [3], wherein the vector comprises a CEN and / or ARS sequence.
[5] A cell transformed with a vector incorporating the insert DNA fragment described in step 2) by homologous recombination is isolated, and a mutation in the test gene is detected using the expression level of the reporter gene in the cell as an index [1] The method described.
[6] The insert DNA in step 2) is obtained by PCR amplification using a test gene as a template with a primer pair in which a base sequence homologous to the cloning region is added to the 5 ′ end portion, [1] the method of.
[7] The method according to [1], wherein both ends of the linear vector are blunt ends.
[8] The method according to [1], wherein both ends of the linear vector are dephosphorylated.
[9] A kit for detecting a nonsense mutation or a frameshift mutation on an insert DNA, comprising the following elements:
a) a linear vector composed of a promoter sequence, a cloning region, and a reporter gene and having a cloning region at both ends;
However, the linear vector includes the above elements in this order by incorporating the insert DNA into the cloning region, and provides a vector capable of expressing the fusion transcript of the insert DNA and the reporter gene under the control of the promoter sequence. ,
b) an insert DNA amplification primer pair in which a base sequence homologous to the cloning region is added to the 5 ′ end, and
c) Reagent for evaluating the expression of the reporter gene
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The mutation detection method of the present invention uses a linear vector comprising the following components and having a cloning region at both ends. This vector is referred to as a gap vector in the present invention.
Promoter sequence,
A cloning region, and
・ Reporter gene
The gap vector in the present invention is a vector that includes the above elements in this order by incorporating an insert DNA into a cloning region, and can express a fusion transcription product of an insert DNA and a reporter gene under the control of a promoter sequence. is there. The insert DNA is finally placed between the cloning regions.
[0013]
The cloning region constituting the gap vector in the present invention comprises a base sequence homologous to the base sequence added to both ends of the insert DNA described below. In other words, a base sequence homologous to the cloning region used in the gap vector is added to the insert DNA.
In the present invention, the fact that the base sequences are homologous means that two double-stranded DNAs have a highly homologous base sequence. A high degree of homology does not necessarily mean that the base sequences of both are completely identical. In the present invention, any DNA can be used as long as the insert DNA can be incorporated into the linear vector by homologous recombination with a certain probability in a host cell to be used or in vitro. For example, in a 30-base homologous base sequence, homologous recombination can be achieved with a practical probability if the base sequences of at least 20 bases, preferably 25 bases or more, match.
The length of the cloning region is not limited as long as the insert DNA can be incorporated into the linear vector by homologous recombination with a certain probability. Specifically, for example, when inserting an insert DNA in a yeast cell, a homologous base sequence comprising 10 to 200 bases, preferably 10 to 50 bases, more preferably 10 to 30 bases, should be used as a cloning region. Can do. In the present invention, an unnecessarily long cloning region is disadvantageous because a base sequence homologous to the cloning region must be added to the insert DNA by some means. Therefore, a cloning region composed of 10 to 30 bases is desirable as a cloning region in the present invention.
[0014]
The most common method of gene recombination is a method using restriction enzyme cleavage and ligation. However, in the stop codon assay, homologous recombination is used because the translation frame of the insert DNA must always be linked to the reporter gene in a certain relationship. In the method using a restriction enzyme, a cloning site adapted to the insert DNA must be prepared on the vector side, and therefore versatility cannot be expected.
Alternatively, a method for obtaining a fragment for cloning by PCR using a primer added with a base sequence suitable for a cloning site is also known. However, even when such a technique is used, there are cases where the reading frame of the insert DNA cannot be matched with the reporter gene. For these reasons, homologous recombination is a preferred recombination technique for stop codon assays.
[0015]
In the present invention, the cloning region can be any nucleotide sequence. As will be described later, in consideration of the case where addition to the insert DNA is performed as a primer for PCR, it is desirable to use a base sequence that is unlikely to interfere with PCR. Specifically, a base sequence that does not self-anneal in the primer is desirable. Moreover, the possibility of non-specific annealing can be reduced by making it difficult to find a base sequence similar to the test gene. Of course, when a base sequence homologous to the cloning region is added to the insert DNA based on a technique other than PCR, a base sequence that does not hinder the technique can be selected.
[0016]
Each element constituting the gap vector of the present invention can be used as it is as long as it has been used in a known stop codon assay except for the cloning region. The remaining elements are specifically described below.
First, as the promoter constituting the vector in the present invention, any promoter effective in the host cell used in the assay can be used. The promoter in the present invention is not limited as long as it can express the fusion transcription product. In the state where the insert DNA is incorporated into the gap vector, the insert DNA and the reporter gene are arranged in this order downstream of the promoter. The distance between the promoter and the insert DNA can be appropriately adjusted so as to be advantageous for expression. In the present invention, in this way, when the insert DNA and the reporter gene are arranged and expressed so as to be translated in the same reading frame downstream of the promoter, the fusion transcription product is expressed under the control of the promoter. say. For example, in eukaryotes such as yeast, the following promoters are effective.
CYC promoter
Alcohol dehydrogenase 1 gene (ADH1) promoter
Nmt1 promoter of fission yeast (S. pombe), etc.
[0017]
On the other hand, as the reporter gene, a wide variety of reporter genes used in various assay systems can be used. That is, a gene that changes the drug resistance or auxotrophy of a host cell, or a gene that generates some signal by expression of the gene can be used. Specifically, the following genes are known.
Reporter genes that cause changes in auxotrophy
ADE1
ADE2: phosphoribosylaminoimidazole carboxylase
HIS3: imidazoleglycerolphosphate dehydratase etc.
Gene generating signal
GFP: Green Fluorescent Protein
lacZ: Beta-D-galactosidase
Luc: Firefly luciferase etc.
[0018]
The gap vector of the present invention can be added with various known functions for further increasing the efficiency of the assay system and improving operability. For example, a vector comprising a CEN gene or an ARS gene enables stable autonomous replication with a small copy number in yeast. As a result, it is possible to transform with one molecule of vector per yeast cell. By using such a vector, the expression frequency of the reporter gene can be taken as the number of yeast cells. That is, the frequency of mutation can be determined from the number of yeast cells using the reporter gene as an index. In addition, enhancers and terminators for enhancing the expression of the fusion protein can be combined.
By clarifying the mutation frequency by the stop codon assay of the present invention, specifically, the following gene mutation can be analyzed. For example, by clarifying the frequency of mutation in the genomic DNA of an individual, it can be determined whether the mutation is heterozygous or homozygous. Alternatively, if the stop codon assay of the present invention is performed on a tissue or cell gene suspected of having cancer, the frequency of mutation in the tissue or cell population can be known. This makes it possible to grasp the dynamics of the increase of cell clones having mutations in the tumor cell population, and provides an important means for analyzing carcinogenic mechanisms.
[0019]
In the present invention, since the insert DNA is incorporated by homologous recombination, the gap vector is used as a linear shape. Homologous recombination in yeast cells is virtually impossible for circular vectors. The cut end of the gap vector is preferably a blunt end. By using a blunt end, it is possible to make it difficult to cause circularization without using insert DNA. Circularization of the vector without the insert DNA may lead to expression of the reporter gene independently of the mutation of the insert DNA. Since the expression of the reporter gene by the circularized vector without insert DNA acts as noise in the assay system of the present invention, it is desirable to make it as small as possible. In addition to blunting, cyclization can be more reliably prevented by dephosphorylating the ends. The dephosphorylated blunt end does not cause ligation by ligase or the like. Dephosphorylation can be performed with alkaline phosphatase. Such a linear vector is specifically circularized only when the insert DNA is incorporated by homologous recombination.
[0020]
Expression of the reporter gene by self-circularization of the gap vector can also be controlled by the nucleotide sequence. For example, an effective start codon ATG under the control of a promoter can be provided for the first time by incorporation of an insert DNA. In other words, unless the insert DNA is incorporated, the vector is designed so that the self-circularization of the vector does not give an appropriate start codon to the reporter gene. For this purpose, for example, the following method can be used.
(1) Use a start codon derived from the test gene
(2) Select nucleotide sequence starting with atg as insert DNA
(3) Place atg across the cloning region and insert DNA
Generation of noise in the assay system can be more effectively suppressed by combining such a device on the base sequence in accordance with the structural modification of the vector described above.
[0021]
The gap vector in the present invention can be constructed by modifying an expression vector or shuttle vector suitable for the host cell used in the assay based on a known method. For example, when a yeast such as Saccharomyces cerevisiae is used as a host, a shuttle vector such as pLS381 (distributed by Dr. Richard Iggo, Swiss National Cancer Institute (ISREC)) can be used. pLS381 expresses ADE2 that functions as a reporter gene under the control of the CYC promoter, and in addition, it is a desirable vector containing an auxotrophic marker and a CEN / ARS sequence. A similar vector can be shown pLF-ADE2 (Furuuchi, Tada, Moriuchi et al. Am J Pathol printing).
[0022]
Hereinafter, representative methods for constructing the gap vector in the present invention will be described. Specifically, DNA that provides a cloning region may be used for the vector used in the known stop codon assay, instead of the structural gene of the insert DNA.
Cloning region DNA can be made into a double strand by chemically synthesizing and further annealing with a complementary strand. Alternatively, fragments excised from restriction products or the like from PCR products synthesized using other vectors or various genes as templates can also be used. The base sequence of the cloning region DNA itself can be arbitrary. At this time, if a DNA consisting of a base sequence containing a recognition sequence for a restriction enzyme that gives a blunt end is inserted, a linear vector having the blunt end described above can be easily obtained. For example, Sfo I can be shown as a restriction enzyme that gives blunt ends. Sfo I is a restriction enzyme that recognizes a base sequence consisting of ggCGcc and cuts between the CGs to make blunt ends. Also, be careful not to cause homologous recombination between the ends when linear.
Furthermore, when the ADE2 active center is covered by the three-dimensional structure of the fusion protein to be expressed and the ADE2 enzyme activity is not sufficiently expressed, a sequence corresponding to a short peptide chain may be inserted as a spacer between the test gene and ADE2. it can. If an insertion peptide chain that can be detected by an antibody such as a polyhistidine tag, a FLAG tag, a c-myc tag, or an HA tag is employed, protein expression in yeast can be confirmed immunologically.
On the other hand, the vector pSL381 is cleaved at a cloning site located upstream of the reporter gene, and a separately prepared cloning region is inserted therein. The cloning region is inserted by ligation or homologous recombination. And if the vector in which the cloning region is inserted is collected and digested with SfoI, it can be used as the linear vector in the present invention. The blunt end of the linearized vector can be dephosphorylated if necessary. An example of constructing a vector for the present invention based on this method is specifically shown in the Examples.
[0023]
What is important at this time is that when the insert DNA is incorporated into a linearized vector, a translation frame with a reporter gene arranged downstream thereof is matched. In other words, in the present invention, a fusion protein in which the N-terminus of the reporter gene is continuous on the C-terminal side of the amino acid sequence encoded by the insert DNA must be expressed. Both may be connected or may be in a state via a linker peptide. In addition, the reporter gene and the insert DNA do not necessarily have to be composed of codon units. That is, the part which connects both can also straddle a codon. In any case, when no nonsense mutation or frameshift mutation is included, the amino acid sequence encoded by the insert DNA is translated in the same frame as the protein encoded by the reporter gene. That is, it arrange | positions so that a nonsense codon may not arise between both. The translation frame can be adjusted by the positional relationship between the insert DNA and the base sequence for homologous recombination added to the end of the insert DNA. Specifically, for example, the following arrangement can be shown.
[0024]
(1) Ensure that the linker peptide is encoded by the cloning region
In this example, the linker peptide encoded by the base sequence constituting the cloning region is interposed between the amino acid sequence encoded by the insert DNA and the amino acid sequence encoded by the reporter gene. This linker peptide can be a peptide chain that functions as a spacer.
(2) Use the end of the reporter gene as a cloning region
In this example, a fusion peptide in which the amino acid sequence encoded by the insert DNA is directly linked to the reporter gene is expressed.
(3) Use a combination of (1) and (2)
In this example, a part of the reporter gene is used as a cloning region. A linker peptide is located between the amino acid sequence encoded by the insert DNA and the amino acid sequence encoded by the reporter gene, as in (1). However, the linker peptide is encoded by a part of the cloning region.
[0025]
In the present invention, an insert DNA with a cloning region added at the end is used. The insert DNA obtained by adding a cloning region to the insert DNA achieves high versatility, which is the greatest problem of the present invention. That is, by adding a homologous base sequence to the cloning region, it became possible to apply the stop codon assay to any test gene with a single vector regardless of the base sequence of the insert DNA.
[0026]
Such insert DNA can be obtained by various methods. One of the most advantageous methods is a PCR-based method. The method for obtaining the insert DNA of the present invention by PCR will be specifically described below. First, PCR primers for obtaining insert DNA are synthesized. This primer has a structure in which a base sequence homologous to the cloning region is added to the 5 ′ side of a primer capable of amplifying a region containing a mutation of a test gene. This primer first anneals to the test gene on its 3 ′ side and starts PCR. The resulting amplification products all have a base sequence homologous to the cloning region at their 5 ′ ends. That is, it can be an insert DNA to which a homologous base sequence is added to the cloning region by PCR. A similar structure can also be obtained by linking a double-stranded oligonucleotide linker consisting of a base sequence homologous to the cloning region to a product obtainable by general PCR.
In obtaining the insert DNA, the amino acid sequence may not be encoded only by the base sequence derived from the test gene. That is, when the insert DNA is finally incorporated into the gap vector, it can be used in the stop codon assay of the present invention if it is designed so that the fusion protein with the reporter gene can be expressed in the correct frame. For example, even if the base sequence derived from the test gene is interrupted at the end at the two bases of the codon, the remaining third base can be provided by the base sequence derived from the gap vector or primer.
[0027]
Since the present invention is intended to detect mutations, in PCR, it is possible to prevent erroneous incorporation of bases as much as possible. In general, a shorter base sequence to be amplified by PCR can be expected to be synthesized more accurately. In addition, by using an enzyme having a proofreading function as a DNA polymerase, complementary strand synthesis can be advanced accurately. An example of such a DNA polymerase is pfu DNA polymerase. Taq DNA polymerase generally used for PCR is not suitable for the synthesis of insert DNA in the present invention because it is said to incorporate wrong bases at a rate of about 1 in 400 bases.
As for the primers used for PCR, care should be taken in setting the base sequences on the 3 ′ side of the oligonucleotides constituting the primer set so that the primers do not anneal with each other to produce an amplification product. Annealing between primers results in insert DNA that is not derived from the target gene, causing false results. Furthermore, nested PCR can be used to obtain a large amount of PCR products in which the base sequence of the target gene serving as a template is faithfully synthesized. Nested PCR is PCR performed by two sets of primer pairs that are nested with respect to a certain template base sequence. First, PCR is performed using a primer pair located outside, and PCR is performed using the amplification product as a template and a primer set inside. Nested PCR dramatically increases amplification efficiency. At the same time, for example, products generated by annealing of primers are not used as templates in the second PCR, so accurate synthesis is possible. .
[0028]
In the present invention, “adding a homologous base sequence to the cloning region” means, in principle, an artificial base sequence capable of achieving homologous recombination with a gap vector to a base sequence derived from a test gene. Means adding. In order to achieve homologous recombination with the insert DNA efficiently with the gap vector, the base sequence homologous to the cloning region is 10 to 50b, more specifically, an artificial length of about 10 to 30b. It is desirable to add a simple sequence.
[0029]
Next, the relationship between the insert DNA and the test gene in the present invention will be described. In the present invention, the insert DNA means a DNA consisting of a base sequence derived from a test gene and actually incorporated into a gap vector. The insert DNA can include the entire length of the protein coding region of the test gene, or can be a part thereof. When a part of a gene is used as an insert DNA, it goes without saying that the original translation frame of the gene should be maintained when it is incorporated into a gap vector. The insert DNA does not necessarily have to be obtained in codon units. When the 3 ′ side of the insert DNA ends in the middle of the codon, the codon can be constructed by preparing a base capable of supplementing the codon on the gap vector side as shown below.
Insert DNA → | | ← Gap vector
.... nnn nnn nn n nnn nnn nnn ....
In the present invention, since the presence of a nonsense mutation or a frameshift mutation is detected in the insert, when a part of the test gene is used as an insert DNA, a region where the mutation to be detected is expected. Is included in the insert DNA. Alternatively, the mutation in the entire gene can be detected regardless of the site by using the entire length of the test gene as the insert DNA. The length of the insert DNA in the present invention is not particularly limited as long as it can express a fusion protein of homologous recombination and a protein encoded by a reporter gene. For example, when using homologous recombination with yeast, the efficiency of homologous recombination can be maintained high by generally setting the length to 500 to 3000 bp, desirably 500 to 2000 bp, more specifically 1000 to 1500 bp. it can. Furthermore, when an insert DNA is obtained by PCR, it is desirable that the length be such that erroneous incorporation of bases does not occur as described later.
[0030]
In the present invention, the test gene is used as a term indicating a gene whose mutation is to be detected. Therefore, the test gene can be obtained from any gene source such as cDNA, genomic DNA, or mRNA. Insertion DNA can be obtained by performing PCR using these genes as templates and using a primer to which the base sequence constituting the cloning region as described above is added. If the template is RNA, RT-PCR may be used. Alternatively, when these genes can be obtained in a sufficient amount as a double strand, after being cleaved with a restriction enzyme or the like, an oligonucleotide consisting of a base sequence corresponding to the cloning region is ligated to form an insert DNA for the present invention. You can also
[0031]
The mutation detection method of the present invention can be applied to detection of mutations in all kinds of genes. Specifically, it is possible to detect mutations in the genome, splicing abnormalities in mRNA, and the like. More specifically, the following gene mutations can be detected.
BRCA1
BRCA1 is a tumor suppressor gene that causes familial breast cancer due to its inactivation (Bishop DT: BRCA1 and BRCA2 and breast cancer incidence: a review. Ann. Oncol. 10 Suppl 6: 113-119, 1999, GenBank Accession # U14680). In addition to familial breast cancer, mutations have been observed in juvenile breast cancer, bilateral breast cancer, breast cancer combined with multiple organ cancers (ovarian cancer, etc.), male breast cancer, and familial ovarian cancer. The coding region is 5592 bp and is a long gene encoding 1864 amino acids. Because nonsense or frameshift mutations account for 80% of the mutations, the stop codon assay is useful.However, since the distribution of mutations extends to the entire region, the fragment is divided into several regions as insert DNA. Mutations can be detected by a stop codon assay based on the invention. Thus, the versatility of the present invention is particularly useful when the region where mutation detection is to be performed is wide.
[0032]
APC
APC is known to recurrent familial colon polyposis and subsequent colon cancer by inactivation due to germ cell mutation (Groden J, et al .: Identification and characterization of the familial adenomatous polyposis coli gene. 66: 589-600, 1991, GenBank Accession # M74088). In addition, somatic mutations are also known in pancreatic cancer, gastric cancer, and esophageal cancer, including arc colon cancer. More recently, the inventors have found that mutations are also observed in breast cancer (Furuuchi K, Tada M, Moriuchi T et al .: Am J Pathol in press). About 95% of the mutations are germ cells and somatic mutations. The site of mutation is a frequent site called mutation cluster region, especially in somatic mutations, but there are many reports of mutation sites. This gene is also a long gene encoding 2843 amino acids, and mutation can be detected by dividing into several regions and utilizing the versatility of the present invention.
[0033]
hMSH2 (L47581-3), hMLH1 (U07343), hMSH6 (U73732-7)
Both are mismatch repair genes identified as causative genes for hereditary non-adenomatous colorectal cancer. Abnormalities in these genes often result in replication errors (RER) or microsatellite instability (MSI) in tumors that develop as mutator phenotypes (Lynch HT, de la Chapelle A: Genetic susceptibility to non-polyposis colorectal cancer. J. Med. Genet. 36: 801-18, 1999). The frequency of hMSH2 and hMLH1 germ cell mutations is particularly high. By type of mutation, the stop codon type is about 90% for hMSH2, about 70% for hMLH1, and almost no frameshift mutation is reported for hMSH6. There are no mutation-concentrated sites in both hMSH2 and hMLH1. Since these are genes encoding 934, 756, and 1361 amino acids, respectively, the stop codon assay can be performed without division or by dividing into 2 to 3 regions. In order to detect these mutations of a plurality of genes as a set, the versatility of the present invention, in which a separate vector is not required, is important.
[0034]
Other examples of tumor suppressor genes for which a stop codon assay is useful include the following.
MEN1 (U93236〜7)
It is a causative gene for multiple endocrine neoplasia type 1. About 70% of germ cell mutations are of stop codon type. The site of mutation does not have a particularly frequent hot spot, and it exists in all exons.
[0035]
WT1 (X51630)
Identified as a gene associated with Wilms tumor development. It is a causative gene in Denys-Drash syndrome, which is associated with pseudohemiyang and glomerulonephritis. Sporadic Wilms tumors are also abnormal in about 15%. About 40% of the mutation types are stop codon type mutations. The site of mutation spans most exons.
[0036]
NF1 (M82814)
It is the causative gene of neurofibromatosis 1 (Reclinghausen disease) and is involved in the regulation of ras protein. Mutation sites are widely distributed in 60 exons and there are no hot spots. The mutation type is a mutation in which about 70% is a stop codon type.
[0037]
PTEN (U92436)
It is a causative gene of Cowden's disease and is involved in dephosphorylation of phosphatidyl / inositol triphosphate and negatively regulates PKB / Akt signal. Somatic mutations are known in brain tumors and pancreatic cancer. 70% of these are stop codon type mutations.
[0038]
The method for detecting a mutation according to the present invention can be used not only when the position of mutation can be specified in advance, but also for clarifying the mutation site of a gene whose position of mutation is unknown. That is, the following analysis can be performed based on the present invention in order to clarify the site where a mutation has occurred in a test gene whose structure of a normal gene is clear. First, a target gene is divided into arbitrary regions, and a primer capable of amplifying each region is designed. Each region may be set to be continuous or may be set to partially overlap. A base sequence homologous to the cloning region in the gap vector is linked to the 5 ′ side of each primer. By inserting an insert DNA synthesized by a series of primer sets into a gap vector and detecting a mutation, it is possible to clarify which region has the mutation. Furthermore, a vector containing a mutation can be isolated and its nucleotide sequence can be clarified.
[0039]
The stop codon assay of the present invention detects the presence or absence of mutation in the gap vector circularized by incorporating the insert DNA, using the expression state of the reporter gene as an index. That is, if there is no nonsense codon mutation or frameshift mutation in the insert DNA, a normal fusion transcript with the reporter gene should be obtained. If the reporter gene is a protein having a normal amino acid sequence, its activity can be detected. A fusion transcript can be obtained by translating a circularized gap vector in vivo or in vitro. For example, a yeast cell that has undergone homologous recombination becomes a transformant by using a circularized gap vector as it is to give the fusion transcript.
This process is basically the same as the known stop codon assay. In the known stop codon assay, the step of circularizing the gap vector by incorporating the insert DNA is expressed as repair. Actually, various confirmation methods can be performed depending on the activity of the expression product of the gene used as the reporter gene.
[0040]
A desirable activity as a reporter gene is an activity that produces coloration or fluorescence. Since such activity can be confirmed macroscopically, the assay system is simplified. Specifically, the ADE2 gene deficiency in yeast can be detected as a red colony on an adenine-restricted medium. Therefore, mutations in the insert DNA can be detected as red colonies by using ADE2 as a reporter gene and combining ADE2-deficient yeast as a host. When ADE2 is expressed by transformation of the vector, ADE2-deficient yeast can grow normally and a normal white colony is generated. As the ADE2-deficient yeast, yPH857 (ATCC # 76628), yPH500 (ATCC # 76626), yPH499 (ATCC # 76625) and the like can be used. These yeasts can be obtained from ATCC (American Type Culture Collection; http://bioscience.igh.cnrs.fr//urllists/atcc.htm).
The adenine-restricted medium is a medium that can grow and grow even in cells that do not express ADE2, but produces a red colony color. This medium contains the following composition per liter of medium. 10 g of casamino acid can also be used as a mixture of amino acids below L-tryptophane.

Similar reporter genes include ADE1 and GFP. ADE1 produces a colored colony by mutation on the same principle as ADE2. GFP emits fluorescence when normal, and disappears due to abnormality.
[0041]
Alternatively, a gene encoding an enzyme active protein such as luciferase can be used as a reporter gene. When such a reporter gene is used, the expression level of the reporter gene can also be confirmed by isolating the transformant and measuring the enzyme activity.
[0042]
A gap vector used in the present invention and a reagent for detecting a reporter gene can be set in advance to form a mutation detection kit. Examples of the reagent for detecting the reporter gene include an adenine-restricted medium for culturing ADE2-deficient yeast.
This kit can be combined with a gap vector and a host cell for transformation with the gap vector. Furthermore, PCR primers designed according to the gene whose mutation should be detected can be combined.
[0043]
【Example】
Hereinafter, the present invention will be specifically described based on examples.
[Example 1] Detection of BRCA1 gene mutation by stop codon assay
(1) Yeast strain used, medium, and gene transfer method
A budding yeast S. cerevisiae strain lacking ADE2 yPH857 (ATCC # 76628) was used. The genotype is [MATa ura3-53 lys2-801 ade2-101 his3-D200 trp1-D63 leu2-D1 cyh2R]. YPDA ++ was used as the complete medium for normal culture. CAu10 was used as a medium for addition of uracil and adenine-restricted stop codon assay.
The composition of the medium is as follows.
・ YPDA ++ medium:
10 g / L yeast extract
20 g / L peptone
2% glucose
200 mg / L adenine
・ CAu10 medium:
0.67% yeast nitrogen base
2% glucose
1% casamino acid
20 mg / L L-tryptophan
10 mg / L adenine
2.5% agar
[0044]
(2) Preparation of gap vector according to the present invention
The gap vector was constructed by modifying pLS381 (provided by Dr. Richard Iggo, Swiss National Cancer Institute (ISREC)). pLS381 is a yeast / E. coli shuttle vector that has the URA3 marker gene and CEN / ARS sequences, Amp and ColE1 Ori, and is driven by the CYC promoter to express ADE2. That is, the BamH I cleavage site between the CYC promoter and the ADE2 gene was changed to the Sfo I site in order to minimize the self-recirculation of the gap vector. Sfo I is a restriction enzyme that generates a blunt end by cleaving between c | g indicated by | in the next recognition sequence.
.... ggC | Gcc ....
.... ccG | Cgg ....
Specifically, a double-stranded oligonucleotide represented by the following base sequence was incorporated into pLS381 linearized with BamHI.
5'-CACTAAATT AATAATGACC GGCGCC ATGGATTCTA GAACAGTTG-3 '(SEQ ID NO: 1)
3'-GTGATTTAA TTATTACTGG CCGCGG TACCTAAGAT CTTGTCAAC-5 '
That is, pSL381 digested with BamHI and the above double-stranded oligonucleotide were simultaneously introduced into the yeast strain yPH857. Among the plasmid clones recovered from the yeast colony, those clones that can be cleaved with SfoI were selected, the inserted nucleotide sequence was confirmed by DNA nucleotide sequencing, and the clone having the desired nucleotide sequence was selected and named pMT18. The base sequence for cloning that constitutes the end of the gap vector obtained by cleaving pMT18 with Sfo I is the following 24b.
5'-end: ... CAC ACT AAA TTA ATA ATG ACC GGC (SEQ ID NO: 2)
3'-end: GCC ATG GAT TCT AGA ACA GTT GGT ... (SEQ ID NO: 3)
The linearized vector was treated with calf intestine alkaline phosphatase to dephosphorylate to prevent self-recirculation. 100 ng or 50 ng of BamH I linearized pLS381 and Sfo I linearized pMT18 alone were introduced into the yeast strain yPH857, respectively, and the number of colonies generated by autocyclization was compared.
[0045]
(3) Extraction of RNA and genomic DNA from clinical samples
Colon cancer and peripheral normal colon mucosa removed from patients with colorectal cancer who had undergone surgery at Hokkaido University Medical Hospital First Surgery and related facilities were frozen and stored in liquid nitrogen. Total RNA and genomic DNA were extracted from samples stored at -80 ° C using Trizol Reagent (Gibco).
[0046]
(4) Primer design
 The stop codon assay according to the present invention was evaluated for the BRCA1 gene.
First, primer pairs as shown in Table 1 were designed for exon 11 of BRCA1, and PCR amplification was performed on genomic DNA of clinical samples. In the table, the underlined portion is a base sequence homologous to the cloning region. Using the obtained PCR product as insert DNA, the stop codon assay according to the present invention was performed.
[Table 1]

a: The number attached to IN is the number of bases from the intron splice donor site to the intron-exon junction
b: The + number attached to IN is the number of bases from the intron splice demand site to the intron-exon junction
c: Length of the entire fragment including the ATG initiation codon and the base sequence homologous to the cloning region
d: Position when the 5 'end in exon 11 is 1
[0047]
The primers were set to amplify exon 11 separately for the full length (set 1), middle 1/3 (set 2), front 2/3 (set 3), and back 2/3 (set 4). In order to obtain normal PCR by set 1 and efficient amplification products, nested PCR (set 2 to set 4) was used. That is, after PCR amplification with an external primer pair for each set, PCR amplification was further performed with an internal primer pair using the product as a template. The 5'-end 24b of pMT18 and the protein synthesis initiation codon ATG are added to the 5'-forward primer for the internal primer pair, and the 3'-end of pMT18 for the 3'-reverse primer. 24 b was added. In set 2, the length of the base sequence homologous to the cloning region added to the internal primer pair was changed. In other words, as a base sequence homologous to the end of pMT18, ones to which 18 b, 30 b, and 39 b were added were separately prepared.
[0048]
(5) PCR amplification
PCR reaction was performed using Perkin Elmer 2400 Thermal Cycler.
In Set 1, an internal primer pair (gBR1EX11F / gBR1EX11R) was directly used, and the reaction was performed with LA Taq polymerase (TAKARA). The reaction solution is attached 1x buffer (MgCl₂ ⁺), 2.0 mM dNTP, 0.4 μM Each primer, 2.5 U LA Taq polymerase was added with 2 μl of template genomic DNA to a total volume of 25 μl. A hot start was performed, followed by 35 cycles of 94 ° C. for 40 seconds, 56 ° C. for 40 seconds, and 68 ° C. for 5 minutes, followed by post-extension at 68 ° C. for 7 minutes.
The primer sequences used are as follows.
gBR1EX11F (SEQ ID NO: 4): 5'-CACACTAAATTAATAATGACCGGCATGGCTGCTTGTGAATTTTCTGAGACG-3 '
gBREX11R (SEQ ID NO: 5): 5′-ACCAACTGTTCTAGAATCCATGGCTCCAATACCTAAGTTTGAATCCATGC-3 ′
[0049]
In set 2, the internal primer pair (BR1EX11F2 / BR1EX11R2) was hot-started with Pfu TURBO polymerase (Stratagene) 1.25 U, attached 1x buffer, 2.0 mM dNTP, 0.4 μM each primer, 100 ng template in a total volume of 25 μl. Ten cycles of 95 ° C. for 40 seconds, 58 ° C. for 40 seconds, and 78 ° C. for 2.5 minutes were performed, followed by post-extension at 78 ° C. for 7 minutes. A second stage reaction of nested PCR was performed using an external primer pair (gBR1EX11F2 / gBR1EX11R2-2) in the same composition reaction solution using 2 μl of the reaction solution diluted to 1/100 as a template. PCR cycles were 95 ° C 40 seconds, 58 ° C 40 seconds, 78 ° C 2 minutes 35 cycles, followed by 78 ° C 7 minutes extension. The same second stage reaction was performed using the following primer pairs with different lengths of the cloning region.
(g18BR1EX11F2 / g18BR1EX11R2-2)
(G30BR1EX11F2 / g30BR1EX11R2-2)
(G39BR1EX11F2 / g39BR1EX11R2-2)
[0050]
The primer sequences used are as follows.
BR1EX11F2 (SEQ ID NO: 6): 5'-AAAAGAATAGGCTGAGGAGGAAGTC -3 '
BR1EX11R2 (SEQ ID NO: 7): 5′-CTCATTTCCCATTTCTCTTTCAGGT -3 ′
gBR1EX11F2 (SEQ ID NO: 8): 5′-CACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT-3 ′
gBR1EX11R2-2 (SEQ ID NO: 9): 5′-ACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT-3 ′
g18BR1EX11F2 (SEQ ID NO: 10): 5′-AAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT-3 ′
g18BR1EX11R2-2 (SEQ ID NO: 11): 5'-TGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT-3 '
g30BR1EX11F2 (SEQ ID NO: 12): 5′-AATACACACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT-3 ′
g30BR1EX11R2-2 (SEQ ID NO: 13): 5′-TAATATACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3 ′
g39BR1EX11F2 (SEQ ID NO: 14): 5′-GCAAACACAAATACACACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT -3 ′
g39BR1EX11R2-2 (SEQ ID NO: 15): 5′-TCCCCCTCCTAATATACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3 ′
[0051]
In Set 3, internal primer pair (BR1EX11F-new / BR1EX11R2) was subjected to 10 cycles of 95 ° C 40 seconds, 58 ° C 40 seconds, 78 ° C 5 minutes with Pfu TURBO polymerase (Stratagene), followed by extension at 78 ° C 7 minutes Went. A second-stage reaction of nested PCR was performed using 2 μl of the reaction solution diluted to 1/100 as a template and an external primer pair (gBR1EX11F / gBR1EX11R2-2). PCR cycles were 95 ° C 40 seconds, 58 ° C 40 seconds, 78 ° C 5 minutes 35 cycles, followed by 78 ° C 7 minutes extension.
[0052]
The used primers are as follows.
BR1EX11F-new (SEQ ID NO: 16): 5'-GTACCTTGTTATTTTTGTATATTTTCAG-3 '
BR1EX11R2 (SEQ ID NO: 17): 5′-CTCATTTCCCATTTCTCTTTCAGGT -3 ′
gBR1EX11F (SEQ ID NO: 18): 5′-CACACTAAATTAATAATGACCGGCATGGCTGCTTGTGAATTTTCTGAGACG -3 ′
gBR1EX11R2-2 (SEQ ID NO: 19): 5′-ACCAACTGTTCTAGAATCCATGGCAGTAATGAGTCCAGTTTCGTT -3 ′
[0053]
In set 4, the internal primer pair (BR1EX11F2 / BR1EX11R-new) was subjected to 10 cycles of 95 ° C for 40 seconds, 60 ° C for 40 seconds, and 78 ° C for 4.5 minutes with Pfu TURBO polymerase (Stratagene), followed by extension at 78 ° C for 7 minutes Went. A second-stage reaction of nested PCR was performed using 2 μl of the reaction solution diluted to 1/100 as a template and an external primer pair (gBR1EX11F2 / gBR1EX11R). PCR cycle was 95 ° C 40 seconds, 58 ° C 40 seconds, 78 ° C 5 minutes 35 cycles, followed by 78 ° C 7 minutes extension.
[0054]
The used primers are as follows.
BR1EX11F2 (SEQ ID NO: 20): 5'-AAAAGAATAGGCTGAGGAGGAAGTC-3 '
BR1EX11R-new (SEQ ID NO: 21): 5'-GGGCAAACACAAAAACCTGGTTCC-3 '
gBR1EX11F2 (SEQ ID NO: 22): 5′-CACACTAAATTAATAATGACCGGCATGAGAAATCTAAGCCCACCTAAT-3 ′
gBR1EX11R (SEQ ID NO: 23): 5′-ACCAACTGTTCTAGAATCCATGGCTCCAATACCTAAGTTTGAATCCATGC -3 ′
[0055]
(5) Stop codon assay according to the present invention
PCR products were electrophoresed on a 1% agarose gel. A good PCR amplification was confirmed by photography under UV after ethidium bromide staining. 1-10 μl of the PCR product measured based on the intensity of the PCR band was introduced into the yeast strain yPH857 by the lithium acetate method with linearized pMT18 (Gietz, et al. Yeast 11: 355-360, 1995). The transformed yeast was cultured on a CAu10 plate at 30 ° C. for 2 to 3 days, and after enhancing color development at 4 ° C., white and red colonies were counted.
[0056]
(6) Plasmid extraction, restriction enzyme cleavage, DNA sequencing
Red or white yeast colonies are cultured overnight in YPDA ++ medium, treated with Zymolyase 100T (Seikagaku Corporation), the yeast capsule is removed, and then the alkaline SDS lysis method (QIAprep Spin Miniprep kit; Qiagen) The plasmid was extracted. This plasmid was introduced into E. coli strain XL-1 Blue by electroporation and cultured, and the plasmid was extracted by alkaline SDS lysis. The presence or absence of an insert was checked by restriction enzyme digestion, and the DNA sequence was determined.
[0057]
(7) Results
(I) Prevention of self-cyclization by creating Sfo I blunt ends
100 ng or 50 ng of BamH I linearized pLS381 and Sfo I linearized pMT18, respectively, were introduced into the yeast strain yPH857, and the number of colonies generated by autocyclization was counted. This resulted in 115 (100 ng), 77 (50 ng), 72 (50 ng) colonies with BamH I linearized pLS381, whereas 67 (100 ng), 42 (50 ng) with Sfo I linearized pMT18. ), 38 (50 ng) colonies were generated, and it was found that about 45% self-cyclization could be prevented. However, approximately the same number of red colonies as white colonies were formed. This was thought to be due to accidental homologous recombination inside the vector. It was thought that a high degree of self-cyclization prevention could not be expected only by end smoothing.
[0058]
(Ii) Result of assay with set 1 primer pair
Instead of nested PCR, a 3.4 kb fragment was directly amplified using LA Taq with a primer pair (set 1) to which a homologous base sequence was added to the cloning region, and the detection method according to the present invention was attempted on 6 samples. As a result, a sufficient number of colonies (400 or more) could be confirmed in all six samples. However, red colonies were 27.4% to 82.0% (average 45%), and many background red colonies appeared. The plasmid was extracted from 6 white colonies and examined by restriction enzyme digestion. As a result, all of the expected 3.4 kb inserts were not included, and DNA sequencing considered that the primer pair formed a double strand. It contained a short fragment that could be When six red colonies were examined, two had 3.4 kb inserts, but four had short fragments as well as white colonies.
[0059]
(Iii) Assay results with set 2, 3, 4 primer pairs
Based on the results of 2, the following improvements were added to sets 2, 3, and 4.
1) DNA polymerase with amplification size limitation and calibration function
The amplification size was limited to suppress red colonies (background) due to erroneous base incorporation by PCR, and Pfu TURBO polymerase with proofreading function was used.
2) Prevent annealing between primers
In designing each primer, in order to avoid double-stranded fragment amplification by annealing between primers, a computer was verified so that no double strand was formed at the 3 ′ end of the primer.
3) Adoption of nested PCR
Nested PCR was performed to minimize the generation of non-target products due to non-specific annealing.
[0060]
As a result of the above three improvements, a remarkable phenomenon of red colonies was observed as follows.
Primer set 2: 4.0-10. 7% (n = 10, average 8.1%)
(BR1EX11F2 / BR1EX11R2 and gBR1EX11F2 / gBR1EX11R2-2)
Primer set 3: 3.8 to 7.3 (n = 6, average 5.5%)
(BR1EX11F-new / BR1EX11R2 and gBR1EX11F / gBR1EX11R2-2)
Primer set 4: 10.3 to 16.0 (n = 6, average 13.3%)
(BR1EX11F2 / BR1EX11R-new and gBR1EX11F2 / gBR1EX11R)
[0061]
Furthermore, in order to confirm the influence of the length of the cloning region on the homologous recombination rate, the cloning DNA of various lengths was compared with the insert DNA in the red colony (background) and the white colony, and the proper homology was compared. We investigated the rate of recombination.
Based on the present invention, a mutation detection method was performed on genomic DNA extracted from cell lines MCF-7 and ZR-75-1 with primers having cloning regions of 18b, 24b, 30b, and 39b. A red colony background was generated in each cell line sample as follows.
18b 4.8% 7.9%
24b 9.8% 7.2%
30b 11.1% 12.7%
39b 7.7% 8.5%
Except for the primer with the 30b cloning region, the background could be reduced to 10% or less. A background value of 10% is a practically acceptable level. Moreover, when plasmids were extracted from 20 white colonies and the presence or absence of appropriate inserts was confirmed by restriction enzyme digestion, it was confirmed that all contained insert DNA properly.
[0062]
【The invention's effect】
The present invention provides a versatile method for detecting nonsense mutations or frameshift mutations occurring in genes. In the present invention, sequences homologous to the cloning region of the gap vector are added to both ends of the insert DNA derived from the test gene. Therefore, it can be incorporated into a common gap vector regardless of the base sequence of the insert DNA. That is, the mutation detection method according to the present invention does not need to construct a vector for each test gene and has high versatility.
By utilizing the present invention, even when a stop codon assay is performed on the entire region of a long gene such as an oncogene, mutation can be detected with a single vector. The high versatility of the stop codon assay according to the present invention is not only capable of dealing with many types of genes, but also advantageous when dividing and analyzing large genes such as cancer genes. Since it is possible to detect mutations in various gene fragments using a single vector, it is also easy to pinpoint the position of the mutation by finely dividing a large gene and analyzing it.
The insert DNA for the present invention can be easily synthesized, for example, by PCR using a primer having a cloning region added to the 5 ′ side. Therefore, the mutation detection method according to the present invention enables a simple and rapid test for any test gene.
[0063]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram schematically showing the principle of a mutation detection method according to the present invention.

Claims (11)

A method for detecting a nonsense mutation or a frameshift mutation on a test gene, comprising the following steps.
1) preparing a linear vector composed of a promoter sequence, a cloning region, and a reporter gene and having a cloning region at both ends;
However, the linear vector includes the above elements in this order by incorporating the insert DNA into the cloning region, and provides a vector capable of expressing the fusion transcript of the insert DNA and the reporter gene under the control of the promoter sequence. ,
2) a step of preparing a DNA fragment in which a base sequence having a length of 10b to 50b homologous to the cloning region is added to the end of the insert DNA;
3) a step of incorporating the DNA fragment of 2) into the cloning region of the vector of step 1) by homologous recombination,
4) A step of detecting the mutation using the expression of the reporter gene of the vector obtained in step 3) as an index. The length of the base sequence homologous to the cloning region 10b From 30b The method of claim 1, wherein The method according to claim 1 or 2 , wherein the reporter gene in step 4) is a gene that generates a visually distinguishable signal between cells that expressed the gene and cells that do not express the gene. The method according to claim 3 , wherein the cell is yeast and the reporter gene is the ADE2 gene. The method of claim 4 , wherein the vector comprises CEN and / or ARS sequences. Step 2) a cell transformed with a vector incorporating by homologous recombination the insert DNA fragment according isolated claim 1 or claim detecting mutations in a subject gene expression levels of the reporter gene in the cell as an index 2. The method according to 2 . Insert DNA in step 2) is one in which the test gene was obtained by PCR amplification of a template by a nucleotide sequence homologous with cloning region 5 'primer pair added to the terminal portion, claim 1 or claim 2 The method described. The method according to claim 1 or 2 , wherein both ends of the linear vector are blunt ends. The method according to claim 1 or 2 , wherein both ends of the linear vector are dephosphorylated. A kit for detecting a nonsense mutation or a frameshift mutation on an insert DNA, comprising the following elements:
a) a linear vector composed of a promoter sequence, a cloning region, and a reporter gene and having a cloning region at both ends;
However, the linear vector includes the above elements in this order by incorporating the insert DNA into the cloning region, and provides a vector capable of expressing the fusion transcript of the insert DNA and the reporter gene under the control of the promoter sequence. ,
b) Insert DNA amplification primer pair in which a base sequence having a length of 10b to 50b that is homologous to the cloning region is added to the 5 ′ end, and c) a reagent for evaluating the expression of the reporter gene The length of the base sequence homologous to the cloning region 10b From 30b The kit according to claim 10, wherein

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