Date of this Version
Jezewski, E. 2019. Detection of Heteroplasmic Single Nucleotide Polymorphisms Using Melt Curve Analysis and Dual Labeled Fluorescent Probes. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
Plant mitochondrial genomes are strange – they are unusually large, consist of huge amounts of non-coding DNA, and contain of several overlapping regions throughout the genome. The genome is made of several different sized linear and circular molecules and different mitochondria within a cell will have different pieces of the genome. Even if multiple mitochondria contain the same region of the genome, these sequences can differ by single nucleotide polymorphisms (SNPS). This is known as heteroplasmy. Heteroplasmy has been documented in Arabidopsis thaliana using whole-genome sequencing data. While heteroplasmy is well-documented, its existence in the mitochondrial genome is unexpected and it can be hard to quantify the degree to which heteroplasmy exists. This project sought to illustrate the existence of heteroplasmy in A. thaliana using melt-curve analysis and probe detection. Melt curve analysis takes advantage of the fact that DNA denatures at different temperatures depending upon the sequence; while probe detection quantifies different DNA sequences by the degree of fluorescence. The goal of this project was to show that both methods were successful in illustrating heteroplasmy in mitochondria and demonstrate heteroplasmy in DNA where one single base is different in a population. Both methods proved to be successful in illustrating heteroplasmy; however, there were drawbacks to each. Both methods required accurate knowledge of the presence of single nucleotide polymorphisms in the DNA sequences and melt curve analysis had a relatively high limit of detection.