JP2010268812A - 遺伝子発現の定量方法 - Google Patents
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Abstract
【解決手段】対象遺伝子すなわち「標的核酸配列」と比較して1塩基の差を有するように設計されている標準を、例えば、変異部位の右側における塩基伸長反応により、標準核酸と試験核酸試料の差を「強化する」方法を併用することで、標準核酸と標的核酸の同じ効率による増幅が可能となり、かつ標的核酸の定量が促進される。この後に、「強化された」標準核酸および標的核酸試料を定量する手段によって標的核酸の量を決定する。好ましい態様では、定量手段は質量分析である。
【選択図】なし
Description
さまざまな良性および悪性の腫瘍、神経疾患、心疾患、および自己免疫疾患などの、いくつかの病理学的症状で発現の異なる遺伝子の検出および定量は、これらの病理学的症状の診断、予後予測、および治療に有用な場合がある。遺伝子発現の定量は、感染症の診断、および薬物またはトキシンの分子レベルにおける作用を追跡する段階にも有用な場合がある。例えば、遺伝子発現データを用いて、薬剤またはトキシンの薬理機構を決定することができる(Libutti et al., Microarray technology and gene expression analysis for the study of angiogenesis. Expert Opin Biol Ther. 2002 Jun; 2 (5): 545-56(非特許文献1))。
本発明は、遺伝子発現または試料中の核酸の量の新しい測定法に関する。この方法は、競合PCR(ポリメラーゼ連鎖反応)と塩基伸長、そして後の測定段階を組み合わせたものである。この方法で、特定の遺伝子のコピー数を直接測定することができるほか、異なる試料に由来する特定の遺伝子の発現の相対的な増加または減少の調節を比較することができる。
質量スペクトルのピーク面積によって測定される絶対シグナルは、マスアレイ系におけるMALDI-TOF MS実験で比較的一貫している(図2)。これは、正確な定量的解析には十分とは言えない。しかし、内部対照と配列の似たオリゴを用いることで、オリゴ濃度を正確に測定することができる(図3)。
この実験では、1個のヌクレオチドのみが異なる2つのDNAを、総濃度(2*10-7 μg/μL)は一定として、さまざまな比(10:1、3:1、1:1、1:3、1:10)で混合する。ホットスタート(HotStart)DNAポリメラーゼによるPCRによる増幅の実施後に、エビアルカリホスファターゼ(SAP)処理を行って過剰なdNTPを除去した。次に塩基伸長実験を、適切なddNTP/dNTP混合物(一般に3種類の異なるddNTPと1種類のdNTP)を用いてThermoSequenaseで実施した。この伸長産物をMALDI-TOFで検出し、ピーク面積をRT(リアルタイム)ソフトウェア(Sequenom Inc.)で解析した。図4は、5つの異なる比のテンプレート混合物に関する質量スペクトルを示す。図5は、質量スペクトルのピーク面積比と、解析に先だって決定されたDNAテンプレート比との間に相関があることを示す。
培養細胞におけるGAPDH、HMBS、およびCXCR4の発現を、リアル競合PCRおよびMALDI-TOF法で解析した。各遺伝子の競合分子をcDNA試料に濃度を高めながら個別に添加する。内因性遺伝子およびこの競合分子の頻度をリアル競合PCRおよびMALDI-TOF MSで測定する。競合分子の濃度はわかっているので、対象遺伝子の発現レベルを計算することができる。
マイクロアレイは、小さな集団/条件のスケール(典型的には50を超えない)で数万の遺伝子をスクリーニングする、少なくとも現時点では理想的な方法である。また一般的には、数百の遺伝子が、対照と試料間で有意に異なるいくつかの統計標準によって選択されている。例えばGolubらは、38の骨髄試料をマイクロアレイ解析に用いて、50の遺伝子を選択し、急性リンパ芽球性白血病(ALL)と急性骨髄性白血病(AML)を集団的に区別できたと報告している。小さな試料数(38試料)および大きな遺伝子数(6817遺伝子)から統計的に大きく自由であることは、マイクロアレイ法の低い精度とあいまって、この予測因子(50の遺伝子)が、どの程度適切に大きな患者試料サイズを対象に実施できるかということに関して無視できない疑念を投げかけている。経済的には、これを数百の患者試料のサイズを対象にマイクロアレイで検討することは可能ではない。発明者らの方法では、約100個の遺伝子の発現を384チップ上で容易に測定することが可能であり、また数百の患者試料を検討することができる。マイクロアレイは遺伝子数に関してハイスループットであるが、発明者らの方法は患者数に関してハイスループットであり、これら2つの方法は高度に相補的である。
1番の問題はPCR用オリゴの設計である。リアルタイムPCRなどの他のRT-PCR法の場合、これは、増幅が対象遺伝子に関して非特異的な場合に大きな問題となる場合がある。というのは、発現レベルの有意な過小評価につながるからである。またさらに厄介な問題として、非特異的な増幅が試料に依存する場合があることが挙げられる。発明者らの場合では、対象遺伝子を含む同じ反応系中に内部基準を常に設けているので、この問題は、それほど深刻とはならないと考えられる。この件に関しては、非特異的な増幅を避けることが現在でも重要である。増幅用オリゴを設計する上での別の問題は、PCRを多重化する際に生じる。プライマーどうしの相互作用を避ける際には一層の注意を要すべきである。
Claims (9)
- 以下の段階を含む、生物試料中の標的核酸配列の量を測定する方法:
(a)少なくとも1塩基が標的核酸配列とは異なるヌクレオチド配列を有する既知量の標準核酸を標的核酸を含む生物試料に添加することで、標的核酸と標準核酸間に相違部位を生成することによって、試料を調製する段階;
(b)段階(a)の試料を増幅する段階;
(c)標準核酸と標的核酸の配列間の差を、相違部位において強化する段階;ならびに、
(d)増幅された標準核酸に対する増幅された標的核酸の比を測定して、生物試料中に存在する標的核酸配列の量を測定することによって、段階(c)で強化された産物を定量する段階。 - 標的核酸が感染性物質に由来する、請求項1記載の方法。
- 標的核酸がmRNA転写物である、請求項1記載の方法。
- 定量段階をMALDI-TOF質量分析により実施する、請求項1記載の方法。
- 段階(c)を、相違部位におけるプライマー伸長により実施する、請求項1記載の方法。
- 段階(c)を、相違部位における対立遺伝子特異的な酵素切断により実施する、請求項1記載の方法。
- 段階(c)を、相違部位における対立遺伝子特異的なハイブリダイゼーションにより実施する、請求項1記載の方法。
- 定量段階をMALDI-TOF質量分析により実施する、請求項1、5、6、および7のいずれか一項記載の方法。
- チューブ内に含まれる、少なくとも1つの対応標的核酸と異なるように設計された少なくとも1つの標準核酸、および請求項1記載の方法にしたがって核酸試料中の対応標的核酸の量を定量する段階における標準の使用法に関する指示書を含むキット。
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