Fluorescence polarization in homogeneous nucleic acid analysis

Abstract

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.]

Figure 1

Fluorescence polarization.

Figure 2

FP–TDI assay.

Figure 3

Changes in fluorescence polarization for DNA samples genotyped with the FP–TDI assay. The results are plotted in mP units above the average polarization of the negative controls. A change of 40 mP for a dye-terminator is scored as positive. DNA samples from 34 individuals and 6 water blanks were used. (█) Samples positive for the G allele but negative for the A allele (homozygous G); (▴) samples positive for the A allele but negative for the G allele (homozygous A); (♦) samples positive for both alleles (heterozygotes); (●) negative controls; (○) samples with failed PCR amplification. Numerical values for the data are available as online supplementary material at http://www.genome.org.

Figure 4

FP–TDI genotyping data for the C282Y mutation in human hereditary hemochromatosis gene. DNA samples from 42 patients and 6 water blanks were tested. (▴) Samples positive for the A allele but negative for the G allele (homozygous A); (♦) samples positive for both alleles (heterozygotes); (●) negative controls; (○) samples with failed PCR amplification.