Date of Award
Bachelor of Science
Simple sequence repeats (SSRs) are mutation-prone DNA tracts composed of tandem repetitions of relatively short motifs (Kashi & King, 2006). SSRs are found scattered throughout the human genome in both coding and non-coding regions. Present under the category of SSRs are Trinucleotide repeats, which may vary in number and, when expanded upon, provide the basis of at least nine neurodegenerative diseases. Large degrees of structural heterogeneity are found in CAG trinucleotide repeat DNA. DNA slippage leads these hetero-structures to form, since certain repeats slip out of the origin of replication and are carried into the next generation of replication. This slippage during replication has only been seen to occur in scattered segments of the genome where repeated nucleotides reside. This can lead to diseased states involving increased amounts of repeats than in a normal-disease free individual. Current research into trinucleotide repeats involved repeat numbers ranging from 10-35 per molecule (Ning et al., 2015). This is due to the difficulty with replication of high repeat molecules. Here, an attempt to solve this problem is taken, by replication of various amplicons, ranging from 10-150, which are similar repeat numbers ranging from disease free genotypes to the most severely diseased states. This replication is to be completed by a real-time PCR assay and Evagreen binding dye as the end goal. By use of PCR with long stable primers, we are able to see amplification occur in a repeat-only manner. In order to deal with the structural heterogeneity of trinucleotide repeats alone, smaller primers were used to replicate the various lengths of DNA. Future collaboration with Nanopore technologies’ new device, MinIon, will hopefully reveal insights into the sequence of these repeats, especially at higher length, using mid ranged primers. We hope to elucidate this replication issue, with the possible application of this method to DNA repeats associated with other known diseases.
DelliCarpini, Gennaro, "Trinucleotide Heterogeneity Expansions: In vitro Molecule Amplification & Purification" (2016). Biological Sciences. 34.