ストレプトアビジン修飾磁性微粒子を利用したDNA点突然変異の解析
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概要
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正常型DNAから微量の変異DNAを識別する有力な手法の一つとして,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.本研究で行ったPCR/LDRアッセイでは,3'末端においてターゲットシークエンスの一塩基の差異をアレル特異的に認識し、3'末端がビオチン化された共通プライマーとライゲートする識別プライマーの5'末端が,異なる蛍光色素(ROX, HEX, TET, TAMRA)で標識されている.PCR/LDR反応によって得られたビオチン化LDRプロダクトは,ストレプトアビジンがコートされた磁性ビーズにビオチン-ストレプトアビジン結合を介し固定化された後、ビーズを磁石で回収・洗浄することにより精製された.その後,精製されたLDRプロダクトを,10 mM EDTAを含むホルムアミド中において75℃で5分間インキュベートすることにより,ビオチン-ストレプトアビジン結合を切断し,溶媒中に解離させた.回収されたLDRプロダクト溶液を含む溶液の蛍光スペクトルを蛍光分光光度計を用いて測定した.LDRプロダクトに標識されている上記蛍光色素の蛍光スペクトルの違いにより,K-ras遺伝子のエクソン1,コドン12.2における一塩基置換を迅速かつ簡便に特定することが可能となった.One of the promising techniques 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). In the present PCR/LDR assay, we used discriminating primers that were labeled with different fluorescent dyes (ROX, HEX, TET, & TAMRA) at their 5'-ends and can discriminate a single nucleotide difference at the mutation site of the target sequence at their 3'-ends by the allele-specific ligation with the common primer which was labeled with biotin at its 3'-end. Following the PCR/LDR processing, we immobilized the resultant LDR products on streptavidin-coated magnetic beads via biotin-streptavidin bonding and purified the product by magnetically collecting and rinsing the beads. Then, the purified LDR products were released in a solvent of formamide containing 10 mM EDTA by thermally cleaving the streptavidin-biotin bonding at 75°C for 5 min. Fluorescent signatures could be acquired from the recovered LDR products by fluorescence monochrometer measurements, enabling rapid and simple determination of single nucleotide substitutions on codon 12.2 of exon 1 of K-ras oncogene based on the difference in fluorescence spectra of the labeled dyes.
著者
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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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藤田 卓
Former Undergraduate Student Of Doshisha University
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