キャピラリーゲル電気泳動によるリガーゼ検出反応生成物の分析
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概要
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正常型DNAから微量の変異DNAを識別する手法の一つとして,ポリメラーゼ連鎖反応(PCR)/リガーゼ検出反応(LDR)が知られている.このPCR/LDRアッセイによって得られたLDR生成物は,マイクロアレイやスラブゲル電気泳動によって分析されるのが一般的である.本研究で,筆者らは,LDR生成物の分析のために,キャピラリーゲル電気泳動(CGE)-蛍光(FL)検出システムを適用した.CGE-FL分析システムにおいて,一本鎖DNAとも結合し蛍光を発するSYBR Goldを分離マトリックスであるポリエチレンオキシド(PEO)ポリマー溶液に添加し,分析を行った.本分析システムにおいて,LDR生成物をLDRプライマーやテンプレートなどの他のDNAフラグメントから分離・検出することが可能となり,K-ras遺伝子のコドン12における一塩基置換の識別が達成された.One technique that can distinguish low-abundant mutant DNA from wild-type DNA is the ligase detection reaction (LDR) coupled to a primary polymerase chain reaction (PCR). The LDR products obtained by the PCR/LDR assay can be analyzed in a variety of fashions such as microarray and slab gel electrophoresis. In the present work, we applied capillary gel electrophoresis (CGE) with fluorescence detection (FL) (CGE-FL) for analyzing the LDR products. Polyethylene oxide (PEO) polymer solution containing SYBR Gold, which could bind to a single strand DNA, was used as a separation matrix in the CGE-FL system. The LDR products could successfully be separated from other DNA fragments such as LDR primers and template, enabling the determination of the single base substitutions on codon 12 of K-ras gene.
著者
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橋本 雅彦
同志社大学理工学部化学システム創成工学科
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塚越 一彦
同志社大学理工学部化学システム創成工学科
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橋本 雅彦
Department of Chemical Engineering and Materials Science, Doshisha University
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上郡 純
Former graduate student of Doshisha University
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塚越 一彦
Department of Chemical Engineering and Materials Science, Doshisha University
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橋本 雅彦
Department Of Chemical Engineering And Materials Science Doshisha University
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塚越 一彦
Department Of Chemical Engineering And Materials Science Doshisha University
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