Two-dimensional agarose gel analysis of branched double-stranded and partially single stranded synthetic DNA constructs /

Abstract

In the late 1980's, a link was established between mutations in human mitochondrial DNA (mtDNA) and certain human diseases which affect neural and muscle tissue. Individuals who have mtDNA diseases have both normal and mutant mtDNA. Increasing severity of the disease is correlated with an increase in the proportion of mutant mtDNA. To understand the abnormal mtDNA processes which cause disease, we must first be able to understand normal mtDNA processes which haven't been entirely elucidated. Today, the most widely accepted model of mammalian mtDNA replication is the D-Ioop model. The proposed asynchronous and asymmetrical D-loop model should produce regions of single stranded DNA bound to double stranded DNA. Recently, mouse and human mtDNA were analyzed by Brewer/Fangman two-dimensional agarose gel electrophoresis. Replication forms were seen which had characteristics predicted for partially single stranded D-loop DNA. However, DNA forms which are known to be partially single stranded have not been analyzed. My research involved the formation of partially single stranded DNA constructs which can be used as reference points for how D-loop replication intermediates should run on 2-D gels. Synthetic partially single stranded DNA constructs were compared with normal double stranded and single stranded total cellular mtDNA. The constructs migrated between the double stranded and single stranded arcs, as might be predicted as the constructs are neither totally single stranded nor double stranded. In the future, other forms expected by the D-loop model and alternate bi-directional synthesis may be synthesized which include double stranded Y arc constructs, whole and partially single stranded D-loop constructs, and other sizes of branched structures with one single stranded arm which may provide further insight into normal mammalian mtDNA replication processes.