Chen X, Levine L, Kwok P Y
Division of Dermatology and Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Genome Res. 1999 May;9(5):492-8.
A new method for DNA diagnostics based on template-directed primer extension and detection by fluorescence polarization is described. In this method, amplified genomic DNA containing a polymorphic locus is incubated with oligonucleotide primers (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of allele-specific dye-labeled dideoxyribonucleoside triphosphates and a commercially available modified Taq DNA polymerase. The primer is extended by the dye-terminator specific for the allele present on the template, increasing approximately 10-fold the molecular weight of the fluorophore. At the end of the reaction, the fluorescence polarization of the two dye-terminators in the reaction mixture are analyzed directly without separation or purification. This homogeneous DNA diagnostic method is shown to be highly sensitive and specific and is suitable for automated genotyping of large number of samples. [The data shown in Figure 3 are available as an online supplement at.]
描述了一种基于模板导向引物延伸和荧光偏振检测的DNA诊断新方法。在该方法中，将含有多态性位点的扩增基因组DNA与寡核苷酸引物（设计用于与多态性位点相邻的DNA模板杂交）在等位基因特异性染料标记的双脱氧核糖核苷三磷酸和市售修饰的Taq DNA聚合酶存在下孵育。引物由模板上存在的等位基因特异性的染料终止剂延伸，使荧光团的分子量增加约10倍。反应结束时，直接分析反应混合物中两种染料终止剂的荧光偏振，无需分离或纯化。这种均相DNA诊断方法显示出高度灵敏和特异，适用于大量样品的自动化基因分型。[图3所示数据可作为在线补充资料获取。]
