JP2006153839A - ジップコード方式を用いたpnaチップ及びその製作方法 - Google Patents
ジップコード方式を用いたpnaチップ及びその製作方法 Download PDFInfo
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Abstract
アミン基により改質された基板の上にエポキシ化合物をリンカとしてPNAプローブを結合するジップコード方式を用いたPNAジップコードチップの製作方法を提供する。
【解決手段】
(a)アミン基により改質された基板の上にジメチルスルホキシド(DMSO)、PNAジップコードプローブ及びエポキシ化合物を含む固定化試料を滴下してPNAジップコードプローブを固定する段階と、(b)前記PNAジップコードプローブが固定された基板をSDS溶液により洗浄した後に乾燥する段階と、を含むジップコード方式を用いたPNAチップの製作方法を提供する。
【選択図】
図1
Description
Gerry, N.P. et al., J. Mol. Biol.,292:251,1999 Hirschhorn,J.N., Proc. Natl. Acad. Sci.,97:12164, 2000 Nielsen,P.E.et al., Sci.,254:1497, 1991 Nielsen, P.E., Acc. Chem. Res.,32: 624, 1999 Dean,D.A., Adv.Drug Delivery Rev.,44:81, 2000
実施例1では、表面がアミンにより改質されたガラス基板をPNAチップの基板として使用した。チップに固定するPNAジップコードプローブは10本であり、12個の塩基を有し、互いに低い相同性を有する下記の10個の塩基配列よりなる(配列番号1〜配列番号10)。PNAプローブのN末端にはアミン(−NH2)基が付着しているため、下記のプローブを基板に固定するのに前記アミン基とエポキシ基との結合が使われている。
配列番号2:N−TGCGACCTATCG−C
配列番号3:N−ATCGTGCGACCT−C
配列番号4:N−ATCGGGTATGCG−C
配列番号5:N−CAGCATCGTGCG−C
配列番号6:N−CAGCACCTTGCG−C
配列番号7:N−GGTAATCGACCT−C
配列番号8:N−GACCATCGACCT−C
配列番号9:N−GACCCAGCATCG−C
配列番号10:N−ACCTGACCATCG−C
<実施例2>PNAジップコードチップ上におけるDNA混成化
<実施例3>混成化溶液の濃度によるPNAジップコードチップの感度変化
<実施例4>PNAジップコードチップとDNAジップコードチップの1塩基差の区別能に関する実験
DNA(MM)−5'−NH2−GGTAATTGACCT−3'(配列番号7の単一の変異配列を有するDNAプローブ)
PNA(PM)−N−GGTAATCGACCT−C(配列番号7の塩基配列を有するPNAプローブ)
PNA(MM)−N−GGTAATTGACCT−C(配列番号7の単一の変異配列を有するPNAプローブ)
<実施例5>PNAジップコードチップ上におけるマルチプレクスによる塩基検査(1)
SBE01−18P:5'−CGCAAGGTGCTGCACTGGCCTCAACCAGTCC−3'(配列番号14)
SBE01−31P:5'−TGCTGGGTCAGATGGTCAAGT−3'(配列番号15)
<実施例6>PNAジップコードチップ上におけるマルチプレクスによる塩基検査(2)
配列47:5'−CGATAGGTCGCATACCTGCAGCAGCACAACATC−3'(SBE02−P2)
配列48:5'−AGGTCGCACGATCCGTGGCGTGTGGCG−3'(SBE02−P3)
配列49:5'−CGCATACCCGATAGCAGCACAACATCCCACAG−3'(SBE02−P4)
配列50:5'−CGCACGATGCTGACAGCGGGAGGTGGTCG−3'(SBE02−P5)
配列51:5'−CGCAAGGTGCTGCACTGGCCTCAACCAGTCC−3'(SBE02−P6)
配列52:5'−AGGTCGATTACCGACGCAGAAGCGGGCC−3'(SBE02−P7)
配列53:5'−AGGTCGATGGTCAACCAGTCCCACCTGTCCCAAC−3'(SBE02−P8)
配列54:5'−CGATGCTGGGTCTCCCATGAAGACGCAGAAGC−3'(SBE02−P9)
配列55:5'−CGATGGTCAGGTCCTACCTGCAGCAGCACAACA−3'(SBE02−P10)
Claims (19)
- 次の段階を含むジップコード方式を用いたPNAチップの製作方法:
(a)アミン基により改質された基板の上にジメチルスルホキシド(DMSO)、PNAジップコードプローブ及びエポキシ化合物を含む固定化試料を滴下してPNAジップコードプローブを固定する段階;及び
(b)前記PNAジップコードプローブが固定された基板をSDS溶液により洗浄した後乾燥させる段階。 - 請求項1において、前記(a)段階におけるジメチルスルホキシド(DMSO)、PNAジップコードプローブ及びエポキシ化合物の体積比が、1:1:1であることを特徴とする方法。
- 請求項2において、PNAジップコードプローブの濃度は10μM〜1mMであることを特徴とする方法。
- 請求項1において、PNAジップコードプローブは複数であり、前記複数のPNAジップコードプローブは互いに30%以下の相同性を有する塩基配列を含むことを特徴とする方法。
- 請求項5において、PNAジップコードプローブは配列番号1乃至配列番号10の塩基配列のうち何れか1つを含むことを特徴とする方法。
- 請求項5において、PNAジップコードプローブは配列番号1乃至配列番号10及び配列番号16乃至配列番号45の塩基配列のうち何れか1つを含むことを特徴とする方法。
- 請求項1乃至請求項7のうち何れか一つの方法により製造され、エポキシ化合物をリンカ(linker)としてPNAジップコードプローブが結合されていることを特徴とするPNAジップコードチップ。
- 請求項8のPNAジップコードチップに目的DNAを混成化させることを特徴とする目的DNAの検出方法。
- 請求項9において、前記検出は蛍光信号を用いて行われることを特徴とする方法。
- 次の段階を含むことを特徴とする多重塩基変異の分析方法:
(a)対象遺伝子の塩基変異の部分を選定する段階;
(b)SBEプライマーをデザインする段階;
(c)前記(a)段階における対象遺伝子をPCRにより増幅する段階;
(d)前記増幅された対象遺伝子を(b)段階により得られたSBEプライマー及び蛍光物質により標識されたddATP、ddTTP、ddCTPまたはddGTPを用いてSBE反応を行う段階;
(e)前記SBE反応物を請求項8のPNAジップコードチップに混成化させる段階;及び
(f)蛍光物質を用いて信号を確認する段階。 - 請求項11において、SBEプライマーは5’末端部位に請求項8のPNAジップコードチップに固定されているPNAジップコードと相補的な配列を含み、3’末端部位に対象遺伝子と相補的な配列を含むことを特徴とする方法。
- 請求項11において、SBEプライマーは複数であることを特徴とする方法。
- 請求項11において、前記対象遺伝子は糖尿病と関わる遺伝子HNF1−aであることを特徴とする方法。
- 次の段階を含むことを特徴とする多重塩基変異の分析方法:
(a)対象遺伝子の塩基変異の部分を選定する段階;
(b)SBEプライマーをデザインする段階;
(c)前記(a)段階における対象遺伝子をPCRにより増幅する段階;
(d)前記増幅された対象遺伝子を(b)段階により得られたSBEプライマー及び特定の化合物により標識されたddNTPを用い、ddATP、ddTTP、ddCTPまたはddGTPを入れた4本のチューブにおいてそれぞれ別々にSBE反応を行う段階;
(e)前記SBE反応物を請求項8のPNAジップコードチップに混成化させる段階;及び
(f)前記化合物標識を用いて信号を確認する段階。 - 請求項15において、SBEプライマーは5’末端部位に請求項8のPNAジップコードチップに固定されているPNAジップコードと相補的な配列を含み、3’末端部位に対象遺伝子と相補的な配列を含むことを特徴とする方法。
- 請求項15において、SBEプライマーは複数であることを特徴とする方法。
- 請求項15において、前記特定の化合物はビオチン(biotin)であり、前記信号を確認する段階は、ビオチンとの親和力が高いタンパク質であるストレプタビジン(streptavidin)が結合されているフィコエリスリン(phycoerithrin)で染色することにより行われることを特徴とする方法。
- 請求項8のPNAジップコードチップを用いることを特徴とするSNPsの分析方法。
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