Fluorodeoxyuridine, FUDR, is a widely used treatment for many different types of cancer. It starves cells of deoxythymidine triphosphate, dTTP, which causes an imbalance in the deoxyribonucleotide, dNTP, pool sizes in the cell during DNA replication. We are predicting that with a lack of dTTP, the DNA polymerase sometimes will substitute an incorrect nucleotide in place of dTTP causing mutations, and we believe that this will affect the number of mutations in the mitochondrial genome. To study this, we are using combinations of FUDR and O4-ethyldeoxythymidine (O4-EtdT). This second mutagen, a modification of Thymidine, is used during the salvage pathway for the synthesis of dTTP. It can compete with dTTP and causes a loss in specificity for the DNA polymerase during further rounds of replication. We are treating colon cancer cells with combinations of these mutagens and using the random mutation capture (RMC) assay to quantify the mutations in the mitochondrial genome. The mutations are enriched with a restriction enzyme that cleaves the wild type DNA leaving only mutated DNA to be amplified using qPCR. We are then using TOPO cloning to sequence the mutations. Many believe that uracil will be used as substitute when the cell is starved of thymine because of their similar structures. However, incorporation of uracil will not result in mutagenesis, while incorporation of the other three nucleotides will. With the results from TOPO cloning, we will be able to take a closer look at this hypothesis. Further on, results will be analyzed using the Student T-Test and One Way ANOVA. So far, the data shows a change in the quantity of mutations in mitochondrial DNA when either O4-EtdT or FUDR are added. Results can help us to understand and respond to post-chemotherapy health problems that may be due to mutations in the mitochondrial genome.