JP6523429B2 - Pnaプローブを用いた融解曲線分析方法、融解曲線分析による塩基多型分析方法および塩基多型分析キット - Google Patents
Pnaプローブを用いた融解曲線分析方法、融解曲線分析による塩基多型分析方法および塩基多型分析キット Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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- Life Sciences & Earth Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
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- Immunology (AREA)
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- Biotechnology (AREA)
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- Analytical Chemistry (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明における融解曲線分析のために、表1のようなPNAプローブを合成し使用した。
ガンマグルタミン酸が結合したPNAプローブと変形されていないPNAプローブとの融解曲線の差を比較するために、表1における配列番号1、2のPNAプローブを表2における配列番号10と配列番号11〜12〜13のいずれか一つを1:1の割合で混合したオリゴマーとそれぞれ前記の実施例1の方法を用いて実験を行い、融解曲線を分析した。
PNAプローブに結合した負電荷分子であるガンマグルタミン酸の位置による融解曲線の差を比較するために、表1における配列番号3、4、5、6のPNAプローブを表2における配列番号13、14のDNAオリゴマーを1:1の割合で混合した試料とそれぞれ前記の実施例1の方法を用いて融解曲線を分析した。その結果を図3に示す。
前記の実施例2を多重検出に適用することができるか否かを確認した。表1における配列番号2のPNAプローブと表2における配列番号10〜11または両方を1:1の割合で混合した標的オリゴマーとの組み合わせ、表1における配列番号7のPNAプローブと表2における配列番号15〜16または両方を1:1の割合で混合した標的オリゴマーとの組み合わせ、また、表1における配列番号8のPNAプローブと表2における配列番号17〜18または両方を1:1の割合で混合した標的オリゴマーとの組み合わせを前記の実施例1の方法を用いて実験を行い、融解曲線を分析した。
前記の実施例4で確認した多重検出能力が一塩基多型が互いに隣接して存在する場合にも可能であるかを確認した。表1における配列番号2、7、8のPNAプローブを表2における配列番号19〜20または両方を1:1の割合で混合した標的オリゴマーと前記の実施例1の方法を用いて実験を行い、融解曲線を分析した。
標的DNAの一塩基多型位置以外の他の位置に不完全相補結合をなす塩基を導入したPNAプローブの効果を確認するために、表1における配列番号9のPNAプローブを表2における配列番号21と配列番号22〜23〜24のいずれか一つと1:1の割合で混合した標的オリゴマー、また、配列番号25と配列番号26〜27〜28のいずれか一つと1:1の割合で混合した標的オリゴマーとそれぞれ前記の実施例1の方法を用いて実験を行い、融解曲線を分析した。
Claims (6)
- レポーターおよびクエンチャーが結合したPNAプローブを用いる標的遺伝子の塩基多型検出のための融解曲線分析方法であって、前記PNAプローブは、二つの負電荷を持つアミノ酸の側鎖が二つの塩基を挟んでPNAプローブと結合していることを特徴とする融解曲線分析方法。
- 前記PNAプローブは、インターカレーティング蛍光物質を含むことを特徴とする請求項1に記載の融解曲線分析方法。
- 前記負電荷を持つアミノ酸の側鎖は、PNAプローブのPNA骨格のアルファ、ベータ、若しくはガンマ位に結合することを特徴とする請求項1又は2に記載の融解曲線分析方法。
- 検体試料から標的DNAを分離するステップと、
レポーターおよびクエンチャーが結合したPNAプローブと標的DNAを混成化するステップと、
温度を変化させながら前記混成化された生成物を融解させて融解曲線を得るステップと、前記得られた融解曲線を分析するステップと、を含む標的DNAの塩基多型分析方法であって、前記PNAプローブは、二つの負電荷を持つアミノ酸の側鎖が二つの塩基を挟んでPNAプローブと結合していることを特徴とする塩基多型分析方法。 - レポーターおよびクエンチャーが結合したPNAプローブの融解曲線分析法を用いた標的遺伝子の塩基多型分析用キットであって、前記PNAプローブは、二つの負電荷を持つアミノ酸の側鎖が二つの塩基を挟んでPNAプローブと結合していることを特徴とするキット。
- 前記キットは多重標的DNAまたは単一標的DNAの塩基多型を分析するためであることを特徴とする請求項5に記載のキット。
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PCT/KR2014/002909 WO2015152446A1 (en) | 2014-04-04 | 2014-04-04 | Melting curve analysis using PNA probe, Method and Kit for analyzing Nucleotide Polymorphism using melting curve analysis |
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JP2017510301A JP2017510301A (ja) | 2017-04-13 |
JP6523429B2 true JP6523429B2 (ja) | 2019-05-29 |
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US (1) | US10227637B2 (ja) |
EP (1) | EP3126511B1 (ja) |
JP (1) | JP6523429B2 (ja) |
CN (1) | CN106414766B (ja) |
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