Isothermal amplification of nucleic acids and homogeneous assays for proteins

• Author / Creator

  Reid, Michael

• The detection of specific proteins and deoxyribonucleic acid (DNA) can be used for disease diagnostics, pathogen identification, and the study of biological processes. Many proteins and nucleic acids occur at low concentrations, making their detection challenging. The detection of trace-level DNA is aided by several isothermal and exponential amplification technologies that have been developed; there are no equivalent strategies for protein detection. The major goal of my research is to expand the use of isothermal and exponential amplification of DNA to facilitate the detection of proteins. Four protein detection strategies and improvements to an existing DNA amplification technique have been designed and developed in this thesis.
  I developed a homogeneous protein detection strategy with an amplified signal by incorporating isothermal amplification of DNA. I use binding induced DNA assembly (BINDA) to activate a linear amplification of DNA by enzymatic nicking and strand displacement. To increase the sensitivity of the assay, I added the exponential amplification reaction (EXPAR) to the design. The exponentially amplified signal generated by EXPAR increases the amount of DNA generated. However, the incorporation of EXPAR increased the background amplification. Therefore, I modified the design to occur at higher temperature for a reduced background amplification. The modified design had a binding phase at 37 °C to allow for antibody–target binding, followed by amplification occurring at 55 °C, which is the optimum temperature of EXPAR.
  While EXPAR is a well-used technique, it is limited by the occurrence of background amplification. Exponential amplification in EXPAR is achieved by a feedback mechanism that generates additional target sequences. Consequently, background amplification generates the same amplification products as target specific amplification products; therefore, background amplification is indistinguishable from target-specific amplification. I devised several methods to test the mechanisms that contribute to the triggering of background amplification. Blocking mechanisms that prevent nonspecific hybridization at the 3ʹ-end of EXPAR templates as well as the entire length of the template showed reduction of background amplification. By designing these blocking strategies, I was able to reduce background amplification in EXPAR, resulting in an improved detection limit by 3 orders of magnitude.
  Next, I used loop mediated isothermal amplification (LAMP) as a signal amplifier for protein detection. LAMP is a well-used DNA amplification technique capable of detecting single copies of DNA. This strategy used BINDA to trigger the ligation of DNA probes; the ligated product served as the target molecule for LAMP. Control reactions also were designed to evaluate critical reaction components. This design is isothermal, exponential, and procedurally simple, only requiring addition of the reagents in a single step.
  The techniques described in this thesis may be applied to the detection of a wide range of proteins, provided suitable affinity ligands are available. Furthermore, because of the isothermal and homogeneous nature of these techniques, they may be adapted to point-of-care diagnostics. Due to the two binding probes used in these strategies, they are well suited for the study of protein–protein interactions.

• Subjects / Keywords

  • Protein detection
  • DNA amplification
  • Bioanalytical chemistry
  • Analytical chemistry

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3BN9XJ84

• License

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