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제106회 대한화학회 학술발표회, 총회 및 기기전시회 안내 A New Platform for Endonuclease Activity Assay Based on Graphene Oxide

2010년 8월 31일 14시 55분 28초
Ⅳ-MAT.P-157 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
금 <발표Ⅳ>
저자 및
이지언, 김미희1, 민달희1
한국과학기술원 화학과, Korea
1KAIST 화학과, Korea
Endonucleases, a family of nucleases that can hydrolyze the internal phosphodiester bonds in DNA or RNA, are one of the most important enzymes in molecular biology. Among them, restriction enzyme(type Ⅱ) used in the laboratory for DNA analysis and gene cloning cut DNA at defined positions close to or within their recognition sequences. Therefore, assay of restriction enzyme activities and evaluations of the kinetic parameters are valuable in the fields of clinical diagnostics, drug discovery, and nanoscience. Currently, the detection methods have included gel electrophoresis, high-performance liquid chromatography (HPLC), filter binding, and enzyme-linked immunosorbent assay (ELISA). Although extensively used, the above-mentioned assays suffer from serious disadvantages. They are time consuming and generally not quantitative. Moreover, many assays only allow for a discontinuous detection of DNA cleavage, because the cleavage products must be separated from the substrate. Herein, we describe a new restriction activity assay method based on the difference of binding affinity between single stranded DNA and double stranded DNA with graphene oxide(GO). The substrate DNA of the enzyme consists of single and double strand part containing recognition site. By measuring the quenching and recovery of fluorescence of dye conjugated to double strand part, restriction enzyme activity can be assayed. This GO-based method allows real-time measurement and quantitative assay of restriction enzyme in short time. We believe that this new GO based restriction enzyme assay platform will be a widely applicable tool in endonuclease related basic research and drug development.