MULTIPLEX FLUORESCENCE-BASED PRIMER EXTENSION METHOD FOR QUANTITATIVEMUTATION ANALYSIS OF MITOCHONDRIAL-DNA AND ITS DIAGNOSTIC APPLICATIONFOR ALZHEIMERS-DISEASE

A sensitive and highly reproducible multiplexed primer extension assay is described for quantitative mutation analysis of heterogeneous DNA populations. Wild-type and mutant target DNA are simultaneously probed in competitive primer extension reactions using fluorophor-labeled pr imers and high fidelity thermostable DNA polymerases in the presence o f defined mixtures of deoxy- and dideoxynucleotides, Primers are diffe rentially extended and the resulting products are distinguished by siz e and dye label. Wild-type:mutant DNA ratios are determined from the f luorescence intensities associated with electrophoretically resolved r eaction products, Multiple nucleotide sites can be simultaneously inte rrogated with uniquely labeled primers of different lengths, The appli cation of this quantitative technique is shown in the analysis of hete roplasmic point mutations in mitochondrial DNA that are associated wit h Alzheimer's disease.