A HOMOGENEOUS METHOD FOR GENOTYPING WITH FLUORESCENCE POLARIZATION

We combined the amplification refractory mutation system (ARMS(TM)) an d fluorescence polarization (FP) to give a homogeneous genomic DNA gen otype analysis method. Oligonucleotide probes labeled with the fluores cein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl] amino)fluorescein and the rhodamine dye 6-carboxyrhodamil were include d in amplification mixes and were annealed to PCR products after ampli fication. Hybridization was accompanied by an increase in the FP of th e probe, We demonstrated homogeneous genotyping by analyzing human DNA samples for Delta F-508 mutation status of the cystic fibrosis transm embrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simulta neous detection of two PCR products in a single reaction. The assay me thod described is homogeneous and so obviates the necessity to open re action vessels after amplification. This therefore eliminates PCR carr yover contamination.