Separation, Quantification, and Visualization of DNA

Mitochondria are involved in numerous essential cellular pathways. Mitochondria contain their own DNA genome (mtDNA), which is distinct from nuclear DNA. A major goal of our research is to understand the mitochondrial genome as a whole as well as how mitochondrial transcription is affected through changes in the proteins involved in regulation. The direct output of mitochondrial transcription is mitochondrial RNA (mtRNA), so separation and quantification of this mtRNA would be highly beneficial to this understanding. Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) has been reported to be an effective way to analyze DNA and RNA oligonucleotides. IP RP HPLC can analyze oligonucleotide samples in less time than analysis by agarose gel electrophoresis followed by ethidium bromide detection or labelling which both take significantly more time. We report a novel IP RP HPLC method which separated ssDNA samples of 54 nt and 58 nt with good resolution. This method also quantified ssDNA samples of 54 nt and 58 nt upwards of 5000 ng. This method is further being optimized to separate similarly sized RNA samples. The ultimate goal is to separate mixtures of nucleotides generated from in vitro mitochondrial transcription reactions. We also report a method for imaging DNA molecules immobilized on mica in air using atomic force microscopy (AFM) as another method to ultimately analyze protein-mtDNA interactions