Fluorescence polarization in nucleic acid analysis

摘要

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 fragments containing polymorphic sites are incubated with a oligonucleotide primer designed to hybridize to the DNA template adjacent to the polymorphic site in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq DNA polymerase. The primer is extended by the dye-terminator specific for the allele present on the template. 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 or samples.