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Cooperativity can be used to manipulate binding affinities of DNA biosensors – improving specificity without sacrificing sensitivity; examples include tentacle probes (TPs) and cooperative primers (CPs). This thesis body of work: (1) used TPs to develop a rapid, low-cost diagnostic for detecting the point mutation leading to Navajo Neurohepatopathy (NNH) and (2) used CPs to amplify a symmetric bowtie-barcoded origami with captured t-cell receptor (TCR) α and β mRNA of a single cell.
NNH (affecting 1-in-1600 Navajo babies) is a fatal genetic disorder often caused by 149G>A mutation and is characterized by brain damage and liver disease/failure. Phoenix Children’s Hospital currently uses gene sequencing to identify the 149G>A mutation. While this process is conclusive, there are limitations, as it requires both time (3-4 weeks) and money (>$700). Ultimately, these factors create barriers that can directly impact a patient’s quality of life. Assessment of the developed TP diagnostic, using genomic DNA derived from FFPE patient liver samples, suggests nearly 100% specificity and sensitivity while reducing cost to ~$250 (including cost of labor) and providing a diagnosis within 48 hours.
TCR specificity is dependent on V(D)J recombination as well as pairing of the αβ chains. Drs. Schoettle and Blattman have developed a solution in which a bowtie-barcoded origami strand nanostructure is transfected into individual cells of a heterogeneous cell population to capture and protect αβ mRNA. When PCR of the origami template is performed with Vα, X, Vβ, and Y primers, the α and β gene segments cannot be tied back to a barcode – and paired. Assessment of the developed CPs for PCR suggests correct individual amplification using (1) Va + Xcp and (2) Vβ + Ycp primers, whereas combination of all the primers (Va, Xcp, Vb, and Ycp) suggests hybridization of the Vα + Xcp and Vβ + Ycp products due to the origami target symmetry.
NNH (affecting 1-in-1600 Navajo babies) is a fatal genetic disorder often caused by 149G>A mutation and is characterized by brain damage and liver disease/failure. Phoenix Children’s Hospital currently uses gene sequencing to identify the 149G>A mutation. While this process is conclusive, there are limitations, as it requires both time (3-4 weeks) and money (>$700). Ultimately, these factors create barriers that can directly impact a patient’s quality of life. Assessment of the developed TP diagnostic, using genomic DNA derived from FFPE patient liver samples, suggests nearly 100% specificity and sensitivity while reducing cost to ~$250 (including cost of labor) and providing a diagnosis within 48 hours.
TCR specificity is dependent on V(D)J recombination as well as pairing of the αβ chains. Drs. Schoettle and Blattman have developed a solution in which a bowtie-barcoded origami strand nanostructure is transfected into individual cells of a heterogeneous cell population to capture and protect αβ mRNA. When PCR of the origami template is performed with Vα, X, Vβ, and Y primers, the α and β gene segments cannot be tied back to a barcode – and paired. Assessment of the developed CPs for PCR suggests correct individual amplification using (1) Va + Xcp and (2) Vβ + Ycp primers, whereas combination of all the primers (Va, Xcp, Vb, and Ycp) suggests hybridization of the Vα + Xcp and Vβ + Ycp products due to the origami target symmetry.
ContributorsDubois, Courtney Michelle (Author) / Caplan, Michael R (Thesis advisor) / Vernon, Brent (Committee member) / Carpentieri, David (Committee member) / Arizona State University (Publisher)
Created2018