CAJ | 학술논문

利用线性荧光探针作为核酸连接反应的模板和信号分子,通过实时监测荧光信号的降低来表征连接产物的生成过程,从而建立了一种连续、简单且特异性高的T4 DNA连接酶活性分析的新方法,检出限可达1.2 U/mL;同时,该方法还可用于快速考察金属离子和化学药物对酶促反应的影响.实验结果表明,该法不仅为灵敏、实时监测核酸连接反应提供了一种简便快捷的非同位素分析方法,也为开展核酸连接酶活性分析、反应动力学机制探讨和药物快速筛选提供了一种新技术.
이용선성형광탐침작위핵산련접반응적모판화신호분자,통과실시감측형광신호적강저래표정련접산물적생성과정,종이건립료일충련속、간단차특이성고적T4 DNA련접매활성분석적신방법,검출한가체1.2 U/mL;동시,해방법환가용우쾌속고찰금속리자화화학약물대매촉반응적영향.실험결과표명,해법불부위령민、실시감측핵산련접반응제공료일충간편쾌첩적비동위소분석방법,야위개전핵산련접매활성분석、반응동역학궤제탐토화약물쾌속사선제공료일충신기술.
A continuous, simple method with high specificity for analyzing T4 DNA ligase activity is introduced by monitoring the fluorescence change in real time. Linear DNA probes that can occur in fluorescent resonance energy transfer were used as templates and detecting probes in a homogeneous solution. The limit of detection of T4 DNA ligase was 1.2 U/mL, the K_M and K_(cat) were (1.18±0.06) μmol/L and 40 min~(-1), respectively. In addition, the method was applied to investigate the influence of chemical drugs and metal ions on T4 DNA ligase activity. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also can be applied to the activity assay, kinetics investigation and drug screening of other ligases.