THESIS
2023
1 online resource (xvii, 147 pages) : illustrations (some color)
Abstract
Accurate, timely, and cost-efficient nucleic acid-based testing is critical to the global effort to
fight infectious and genetic diseases, which plays a vital role in selecting proper intervention
measures and preventing the deterioration of epidemics. The rationally engineered DNA probes
and primers with intended thermodynamic characteristics in nucleic acid circuitry are essential
factors to achieve the desired function of the sensing platform thus providing solutions for
bottlenecks existing in screening tests.
This dissertation focus on building a series of nucleic acid sensing platforms to attribute to the
development of large-scale screening for infectious pathogens and routine screening for genetic
diseases and leveraging on the thermodynamic prediction of Gibbs free energy and m...[
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Accurate, timely, and cost-efficient nucleic acid-based testing is critical to the global effort to
fight infectious and genetic diseases, which plays a vital role in selecting proper intervention
measures and preventing the deterioration of epidemics. The rationally engineered DNA probes
and primers with intended thermodynamic characteristics in nucleic acid circuitry are essential
factors to achieve the desired function of the sensing platform thus providing solutions for
bottlenecks existing in screening tests.
This dissertation focus on building a series of nucleic acid sensing platforms to attribute to the
development of large-scale screening for infectious pathogens and routine screening for genetic
diseases and leveraging on the thermodynamic prediction of Gibbs free energy and melting
temperature of DNA motifs based on Watson-Crick base pair nearest neighbors model. First, a sample-specific pooled testing assay using melting curve analysis was developed to solve the
issues of large-scale screening for infectious individuals, and it can analyze five individual
samples in one batch without the need for secondary testing. Subsequently, an extraction-free
mass testing platform utilizing magnetic beads was developed to detect different types of
clinical specimens in practical settings, which further improves the sensitivity and shortens the
turnaround time during the sample preparation and sample pooling. The pools with six
subsamples were identified in a single run without retesting. Third, a competitive probe-and-sink
strategy based on toehold exchange reaction at an elevated temperature was studied to
phase two single nucleotide polymorphism (SNP) sites in long DNA templates. This assay
shows the ability to differentiate four haplotypes and their respective combination of diplotypes
up to 20 nM of the target concentration. Finally, a universal reporting composition in melting
curve analysis was applied in multiplex genotyping to reduce the complexity and cost. It
achieves multiple detections of nucleic acid samples using one set of fluorophore and quencher-modified
probes.
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