Quantification of DNA Products Using Ion-Pair Reverse Phase Liquid Chromatography

The transcription of DNA via RNA polymerases is a fundamental process in cellular systems. In eukaryotic cells, we observe transcription in the nucleus (via genomic DNA) as well as in the mitochondria (via mitochondrial DNA). There are many tools available to investigate nuclear transcription; however, few tools exist to study mitochondrial transcription even though the mitochondrial DNA encodes several essential proteins. Recently an in vitro transcription system using purified mitochondrial transcription proteins, including the mitochondrial RNA polymerase, and linear mitochondrial DNA templates has been developed. Quantitative analysis of the DNA templates can be done via ion-pair reverse-phase high performance liquid chromatography (IP-RP HPLC), a high-resolution technique in separating DNA based on size. Using IP-RP HPLC our aim is to assess the lower limits of separation, and our quantification method is based on measuring peak area and the peak height