Doutor

Methods

Fluorescence Polarization in Homogeneous Nucleic Acid Analysis
Xiangning Chen,1,3 Leanna Levine,2 and Pui-Yan Kwok1
1

Division of Dermatology and Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110 USA; 2Monsanto Life Sciences Company, St. Louis, Missouri 63198 USA 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 ∼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 http://www.genome.org.]

DNA analysis is becoming increasingly important in the diagnosis of hereditary diseases, detection of infectious agents, tissue typing for histocompatability, identification of individuals in forensic and paternity testing, and monitoring the genetic makeup of plants and animals in agricultural research (Alford and Caskey 1994). In addition, DNA analysis is crucial in largescale genetic studies to identify susceptibility alleles associated with common diseases involving multiples genetic and environmental factors (Risch and Merikangas 1996). Recently, attention was focused on single nucleotide polymorphisms (SNPs), the most common DNA