Exploring Selectivity in Nonenzymatic and Enzymatic DNA-Templated Ligation Reactions

  • Author / Creator
    Osman, Eiman A.
  • DNA-templated reactions have been utilized in many applications from the detection of a nucleic acid target to applications in nanotechnology. This thesis explores developing bioanalytical strategies based on DNA-templated ligation reactions for sensitive, rapid single nucleotide polymorphism (SNP) detection, specifically single-basepair mismatches and methylated sites. Finding a good assay that can discriminate single-base differences in a biomarker sequence is important for point-of-care diagnostics aimed at the early detection of cancer. Chemical ligation strategies have some advantages in point-of-care diagnostics compared to enzymatic ligation as they are less expensive and do not require freezer for storage. Thus, we investigated the DNA-templated Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) using 5’-azide ODN and 3’-O-propargyl ODN. Under our newly optimized conditions, the reaction is as rapid as enzymatic ligation using the highest commercially available concentration of T4 DNA ligase with kobs value of 1.1 min-1. The DNA-templated CuAAC reaction is also more selective to single nucleotide mismatches compared to the enzymatic ligation at a temperature that affords the best discrimination of T4 DNA ligase: a temperature below the thermal dissociation temperature, or melting temperature (Tm), of the matched nicked duplex and above that of the mismatched nicked duplexes. Our next aim involved improving the selectivity of T4 DNA ligase for single basepair mismatches as it is the most commonly used ligase in molecular biology and bioanalytical labs despite its low selectivity for single basepair mismatches at the site of ligation. We decided to explore the selectivity of T4 DNA ligase over a temperature range that extended far above the dissociation temperature of the nicked duplex of the perfectly matched template. Surprisingly, with the use of low concentration of T4 DNA ligase, the assay can be quite selective resulting in 100% ligation of the matched target and <2% ligation of the mismatched target when the temperature is 6-12 degrees higher than the Tm. This temperature range of high selectivity can be increased to 12-18 degrees higher than the Tm if a high concentration of enzyme is used. Finally, the assay can also work at around the Tm of the nicked duplex when we introduce an abasic group to the 5’-phosphate terminus of one of the ligating probes. Finally, I will discuss our efforts to detect methylated nucleobases as the ability to sequence such methylated groups on the KRAS gene can help in the early detection of cancer. The KRAS gene is a key controller for the expression of an epidermal growth factor receptor that is overly expressed in many types of cancer. We are able to amplify a target 18-mer sequence from the KRAS gene that contains an O6-methylated G at the codon 12 and 13 position using the isothermal amplification method that was developed in our group (lesion-induced DNA amplification, LIDA). The amplification of the methylated template resulted in a net amplification factor of 650 in a comparison of LIDA reactions initiated with 1.4 nM methylated target and 1.4 nM non-methylated target.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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