Twist Charles R, Winson Michael K, Rowland Jem J, Kell Douglas B
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth SY23 3DD, Wales, UK.
Anal Biochem. 2004 Apr 1;327(1):35-44. doi: 10.1016/j.ab.2003.12.023.
We have exploited three methods for discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation or otherwise of labeled dideoxy nucleotides at the end of a primer chain using single-molecule fluorescence detection methods. Good discrimination of incorporated vs free nucleotide may be obtained in a homogeneous assay (without washing steps) via confocal fluorescence correlation spectroscopy or by polarization anisotropy obtained from confocal fluorescence intensity distribution analysis. Moreover, the ratio of the fluorescence intensities on each polarization channel may be used directly to discriminate the nucleotides incorporated. Each measurement took just a few seconds and was done in microliter volumes with nanomolar concentrations of labeled nucleotides. Since the confocal volumes interrogated are approximately 1fL and the reaction volume could easily be lowered to nanoliters, the possibility of SNP analysis with attomoles of reagents opens up a route to very rapid and inexpensive SNP detection. The method was applied with success to the detections of SNPs that are known to occur in the BRCA1 and CFTR genes.
我们利用了三种方法，通过使用单分子荧光检测方法，检测引物链末端标记的双脱氧核苷酸的掺入情况来鉴别单核苷酸多态性（SNP）。通过共聚焦荧光相关光谱法，或通过共聚焦荧光强度分布分析获得的偏振各向异性，在均相分析（无需洗涤步骤）中可以很好地区分掺入的核苷酸与游离核苷酸。此外，每个偏振通道上的荧光强度之比可直接用于鉴别掺入的核苷酸。每次测量只需几秒钟，在纳摩尔浓度的标记核苷酸的微升体积中进行。由于所探测的共聚焦体积约为1fL，且反应体积可轻松降至纳升，使用阿托摩尔级试剂进行SNP分析的可能性为非常快速且廉价的SNP检测开辟了一条途径。该方法成功应用于检测已知存在于BRCA1和CFTR基因中的SNP。
