使用荧光相关光谱法的超灵敏杂交分析。
Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy.
作者信息
Kinjo M, Rigler R
机构信息
Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden.
出版信息
Nucleic Acids Res. 1995 May 25;23(10):1795-9. doi: 10.1093/nar/23.10.1795.
Abstract
The hybridization of fluorescently tagged 18mer deoxyribonucleotides with complementary DNA templates was analysed by fluorescence correlation spectroscopy (FCS) in a droplet under an epi-illuminated fluorescence microscope at the level of single molecules. The interaction can be monitored by the change in the translational diffusion time of the smaller (18mer) primer when binding to the bigger (7.5 kb) DNA containing the complementary sequence. The hybridization process in the presence of template M13mp18 ssDNA was monitored in a small volume (2 x 10(-16)I) at various temperatures. The Arrhenius plot of the association rate constant shows that the activation energy was 38.8 kcal/mol, but the hybridization process may involve several components. The titration experiment suggested that approximately 2 primers can be associated with one template DNA at 40 degrees C. Results of a simple homology search for the sequences complementary to the primer indicate the existence of additional sites of lower specificity.
摘要
在落射荧光显微镜下，于单分子水平通过荧光相关光谱法（FCS）分析了荧光标记的18聚体脱氧核糖核苷酸与互补DNA模板的杂交情况。当较小的（18聚体）引物与含有互补序列的较大的（7.5 kb）DNA结合时，可通过其平移扩散时间的变化来监测这种相互作用。在不同温度下，于小体积（2×10⁻¹⁶升）中监测模板M13mp18单链DNA存在时的杂交过程。缔合速率常数的阿仑尼乌斯图表明活化能为38.8千卡/摩尔，但杂交过程可能涉及多个组分。滴定实验表明，在40℃时约2个引物可与一个模板DNA缔合。对与引物互补序列的简单同源性搜索结果表明存在特异性较低的其他位点。
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