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dc.contributor.advisorGu, Li-Quneng
dc.contributor.authorShi, Ruichengeng
dc.date.issued2018eng
dc.date.submitted2018 Summereng
dc.description.abstractSingle Nucleotide Polymorphisms (SNPs) are a common type of nucleotide alterations across the genome. A rapid but accurate detection of individual or SNP panels can lead to the right and in-time treatments which possibly save lives. In one of our studies, nanopore is introduced to rapidly detect BRAF 1799 T-A mutation (V600E), with the help of an Ap-dA cross-link right at the mutation site. These sequence-specific crosslinks are formed upon strong covalent interactions between probe based abasic sites (Ap) and target based deoxy-adenosine (dA) residues. Duplexes stabilized by the crosslink complexes create indefinite blocking signatures when captured in the nanopore, creating a high contrast compared to the "spike-like" translocations events produced by the un-crosslinked and wildtype duplexes. Those consistent blocking events couldn't be resolved unless an inverted voltage is applied. In a 1:1 BRAF mutant-wildtype mixture, the nanopore can successfully discriminate between the two sequences in a quantitative manner. In summary, nanopore paired with sequence-specific crosslink can detect a specific type of SNP with a high contrast manner. In another study, nanopore sensing is modified to be capable of detections with multiple SNPs in a single detection mix. To achieve this, an RNA homopolymer barcode is integrated into the probe sequence so nanopore can read out a distinctive level signature when the target-probe duplex is de-hybridizing through the pore. Since different RNA homopolymers (e.g. Poly rA and Poly rC) can generate signature levels distinctive from each other and other DNA sequences, they can be applied to generate characteristic patterns that simultaneously highlight multiple SNPs in the mixture. In this study, we assigned two different RNA barcodes (Poly rA and Poly rC) to label KRAS G12D and Tp53 R172H SNPs (both T[right arrow]A mutations) in the solution. During nanopore readout, the KRAS G12D containing duplex generates a "downward" step pattern but Tp53 R172H always has an "upward" step pattern, the high contrast between those two patterns makes recognition easy enough with naked eyes, and further statistical analysis is unnecessary. Theoretically, at least four different barcodes can be implemented at the same time, furthermore, the length of the barcode can also affect the barcode pattern. Thus, in theory, a panel of more than 10 SNPs can be identified simultaneously.eng
dc.description.bibrefIncludes bibliographical referenceseng
dc.format.extentix, 58 pages : illustrationeng
dc.identifier.urihttps://hdl.handle.net/10355/66327
dc.languageEnglisheng
dc.publisherUniversity of Missouri--Columbiaeng
dc.relation.ispartofcommunityUniversity of Missouri--Columbia. Graduate School. Theses and Dissertationseng
dc.rightsOpenAccess.eng
dc.rights.licenseThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
dc.titleSingle nucleotide polymorphism (SNP) discriminations by nanopore sensingeng
dc.typeThesiseng
thesis.degree.disciplineBiological engineering (MU)eng
thesis.degree.grantorUniversity of Missouri--Columbiaeng
thesis.degree.levelMasterseng
thesis.degree.nameM.S.eng


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