The optimization of cell-SELEX based aptamer selection through masking DNA, PCR and non-SELEX

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dc.contributor.advisorKrylov, Sergey N.
dc.contributor.advisorTsushima, Robert
dc.creatorObrecht, Natalia
dc.date.accessioned2016-09-13T13:14:18Z
dc.date.available2016-09-13T13:14:18Z
dc.date.copyright2013-09
dc.degree.disciplineBiology
dc.degree.levelMaster's
dc.degree.nameMSc - Master of Science
dc.description.abstractAptamers are short oligonucleotide sequences that are capable of binding with high affinity and specificity to a wide variety of targets. The selection of aptamers from a random oligonucleotide library, by cell-SELEX, has lead to their in vivo application in both research and clinical settings. Cell-SELEX continues to be hindered by the non-specific binding of selection sequences to the complex cell surface as well as low specificity in the PCR amplification of DNA. In this work the cell-SELEX procedure was optimized to improve the efficiency of aptamer selection using three modifications: masking DNA characterization, non-SELEX selection and touchdown PCR. An established masking DNA model was tested against the MCF-7 and 4T1 cell lines and was shown to be capable of determining the masking DNA concentration needed for aptamer selection. The traditional SELEX approach was combined with a non-SELEX aptamer selection protocol to reduce the concentration and heterogeneity of sequences collected after the first round of selection thus leading to more accurate PCR amplification and a decrease in byproduct formation. Lastly, touchdown PCR was used to successfully eliminate the amplification of genomic DNA reducing the formation of genomic DNA byproducts.
dc.identifier.urihttp://hdl.handle.net/10315/31955
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subject.keywordsAptamers
dc.subject.keywordscell-SELEX
dc.subject.keywordsDNA
dc.subject.keywordsPCR
dc.subject.keywordsnon-SELEX
dc.titleThe optimization of cell-SELEX based aptamer selection through masking DNA, PCR and non-SELEX
dc.typeElectronic Thesis or Dissertation

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