JPWO2006022370A1 - 電界効果デバイスを用いたdna塩基配列解析方法及び塩基配列解析装置 - Google Patents
電界効果デバイスを用いたdna塩基配列解析方法及び塩基配列解析装置 Download PDFInfo
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Abstract
Description
(b)洗浄液を前記試料溶液ウェルに導入して、未反応の核酸を前記試料溶液ウェルから除去するステップと、
(c)酵素タックポリメラーゼ(Taq polymerase)、及び基質となるdATP,dGTP,dCTP,dTTPのうちの一つを上記試料溶液ウェルに導入し伸長反応を行わせるステップと、
(d)洗浄液を前記試料溶液ウェルに導入して、未反応の酵素、基質を試料溶液ウェルから除去するステップと、
(e)緩衝液を前記試料溶液ウェルに導入して、前記電界効果デバイスの出力値を測定するステップと
(f)(c)のステップに戻り、酵素タックポリメラーゼ(Taq polymerase)、及び上記と異なる基質(dATP,dGTP,dCTP,dTTP)の一つを上記試料溶液ウェルに導入し第2の伸長反応を行わせ、電界効果デバイスの出力値を測定するステップと
を有することを特徴とする。
上記核酸プローブの6‘末端側にはアミノ基を修飾して、ゲート絶縁膜表面に固定化する。本実施例の電界効果トランジスタのゲート絶縁膜には窒化シリコンが用いられており、その表面をγ―アミノプロピルトリエトキシシランで化学修飾して窒化シリコン表面にアミノ基を導入する。核酸プローブのアミノ基と窒化シリコンのアミノ基を例えばグルタルアルデヒドのような2官能性試薬と反応させ、シッフ塩基による結合を形成することにより、核酸プローブを窒化シリコン表面に固定化する。
VII遺伝子10を含む試料を反応させた。試料は試料溶液として容器中に保持した。
(1)洗浄液14をフローセル11中に導入
(2)緩衝液13をフローセル11中に導入(洗浄液を置換)
(3)試料をバルブ15に分注して、フローセル11に導入
(4)フローセル11中でハイブリダイゼーション
(5)緩衝液13をフローセル11に導入して、未反応の試料を除去
(6)各遺伝子検出用電界効果デバイスのしきい値電圧出力値を測定
(7)電界効果デバイスの温度をタックDNAポリメラーゼの最適温度74℃に設定
(8)酵素タックDNAポリメラーゼ(Taq polymerase)、及び基質となるdCTP,dATP,dGTP,dTTPのいずれかをフローセル11に導入し伸長反応
(9)緩衝液13をフローセル11に導入して未反応の酵素・基質を除去
(10)各遺伝子検出用電界効果デバイスのしきい値電圧出力値を測定
(11)(7)に戻る。基質を変えて順次、伸長反応・しきい値電圧測定を繰り返す
上記測定シーケンスを図6に示す。図中、矢印は出力電位を読み取るタイミングを表す。
(実施例1)
(実施例2、3)
2…Pウェル
3…n型領域
4…二酸化シリコン
5…窒化シリコン
6…核酸プローブ
7…二酸化シリコン
8…りんガラス
9…n型領域(温度センサ)
11…フローセル
12…流路
13…緩衝液
14…洗浄液
15…バルブ
16…ポンプ
17…分注器
18…廃液ボトル
19…参照電極
20…3M KCl溶液
21…液−液接合
22…信号処理回路
23…プリント基板
24…遺伝子検出用電界効果デバイス
25…ワイヤー
26…ピン
27…保護キャップ
Claims (6)
- 電界効果デバイスのゲート部において、核酸プローブと試料DNAを反応させて二本鎖DNAを形成させ、次いで、DNAポリメラーゼ及び基質(dATP,dGTP,dCTP,dTTP)を順次添加してそれぞれ伸長反応を起こさせ、基質添加のたびに電界効果デバイスの電気的特性の変化を測定することを特徴とするDNA塩基配列解析方法。
- 電界効果デバイスは、電界効果トランジスタ又は電界効果キャパシターであることを特徴とする請求項1記載のDNA塩基配列解析方法。
- 温度センサを集積化した電界効果デバイスを用いることを特徴とする請求項1又は2記載のDNA塩基配列解析方法。
- (a)電界効果デバイスのゲート部に一本鎖核酸プローブを固定化し、該ゲート部に設けた試料溶液ウェルに、少なくとも1種類の核酸を含む試料溶液を導入して、前記一本鎖核酸プローブとハイブリダイゼーションを行わせるステップと、
(b)洗浄液を前記試料溶液ウェルに導入して、未反応の核酸を前記試料溶液ウェルから除去するステップと、
(c)酵素タックポリメラーゼ(Taq polymerase)、及び基質となるdATP,dGTP,dCTP,dTTPのうちの一つを上記試料溶液ウェルに導入し伸長反応を行わせるステップと、
(d)洗浄液を前記試料溶液ウェルに導入して、未反応の酵素、基質を試料溶液ウェルから除去するステップと、
(e)緩衝液を前記試料溶液ウェルに導入して、前記電界効果デバイスのしきい値電圧変化又はフラットバンド電圧変化を測定するステップと
(f)(c)のステップに戻り、酵素タックポリメラーゼ(Taq polymerase)、及び上記と異なる基質(dATP,dGTP,dCTP,dTTP)の一つを上記試料溶液ウェルに導入し第2の伸長反応を行わせ、電界効果デバイスのしきい値電圧変化又はフラットバンド電圧変化を測定するステップと
を有することを特徴とする塩基配列解析方法。 - プリント基板に遺伝子検出用電界効果デバイスをマウントし、ワイヤーで電気的にプリント基板と接続させ、プリント基板にピンを設けて、信号処理回路と接続し、該電界効果デバイスのゲート部に設けた試料溶液ウェルに試料溶液を流路により供給し、試料溶液が信号線となるワイヤーに接触しないようにワイヤー部分を保護キャップで保護したフローセルからなることを特徴とする請求項1ないし4のいずれかの方法に用いる塩基配列解析装置。
- 電界効果デバイスに温度センサ及びヒーターを集積化したことを特徴とする請求項5記載の塩基配列解析装置。
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US7888013B2 (en) | 2011-02-15 |
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US20080286767A1 (en) | 2008-11-20 |
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