Assay for detection of mtDNA polymorphisms using peptide nucleic acids and capillary electrophoresis with laser-induced fluorescence

A fast, reliable assay was developed for identification of a polymorphism at position 16126 ($\rm T\to C$), in the hypervariable region 1 of the human mitochondrial genome, using peptide nucleic acids (PNAs) labeled with 6-FAM, capillary electrophoresis as a separation technique with laser induced fluorescence detection. This study involved optimization of parameters critical to DNA/PNA hybridization, including sodium ion concentration, concentration of PCR product, injection parameters and hybridization time variation. The extreme specificity of DNA/PNA hybridization was verified by experimentation. Simultaneous detection and confirmation of the nucleotide at position 16126 was achieved by multiplexing PCR products of different size and base composition. Decreasing the length of PCR amplified fragment reduced the analysis time. The application of this assay can be extended to simultaneous identification of several polymorphisms in HV1 or HV2 regions. Developed method can be improved by using dual wavelength in conjunction with ROX and FAM labeled PNA probes. (Abstract shortened by UMI.).