实验条件对荧光共振能量转移的探针熔解曲线影响

Influence of experimental conditions on probe melting curves based on fluorescence resonance energy transfer

目的 探讨基于荧光共振能量转移的杂交探针熔解曲线的实验影响因素 ,优化此法检测基因的实验条件。方法 利用荧光共振能量转移的原理 ,设计检测人葡萄糖 6 磷酸脱氢酶 (G6PD)基因的荧光杂交探针 ,在熔解曲线模式下 ,设定不同模板量、Mg2 + 浓度 ,观察熔解曲线峰面积及其Tm值。结果 在设定的反应体系中 ,随着模板量从 1 0ng渐次上升至 1 0 0ng ,熔解曲线峰面积亦逐渐增大 ;随着Mg2 + 终浓度的增加 ,峰面积也增加 ,而且熔解曲线的Tm值也呈升高趋势。结论 探针熔解曲线法准确、迅速、操作简单 ,在应用中应考虑到模板量及Mg2 +

Objectives To investigate the experimental factors which influence the hybridization probe melting curves based on fluorescence resonance energy transfer and to optimizate the experimental conditions for detection of gene. Methods A pair of G6PD specific fluorescein labeled oligonucleotide hybridization probe were designed according to the principles of fluorescence resonance energy transfer.The melting curve peak area and melting temperature were observed at different concentrations of template and Mg 2+ for the mode of melting curves.Results Melting curve area increased gradually with increasing template from 10 ng to 100 ng into the reaction volume.A increase of melting curve area and the slightly rising trend of Tm were fonud with increasing of Mg 2+ concentrations in the reaction system.Conclusions Analytical procedures by probe melting curves based on fluorescence resonance energy transfer is accurate, simple and rapid for gene detection, but certain experimental conditions must be taken into consideration.