UBC Theses and Dissertations
Molecular thermodynamics of the stability of natural, sugar and base-modified DNA duplexes and its application to the design of probes and primers for sensitive detection of somatic point mutations Hughesman, Curtis
Cancer is characterized as a genetic disease associated with acquired somatic mutations, a majority of which consist of only a single base change and are commonly referred to as somatic point mutations (SPM). Real-time quantitative polymerase-chain reaction (qPCR) techniques using allele specific (AS) probes or primers are widely used in genotyping assays to detect commonly known single nucleotide polymorphisms (SNP), and also have the potential to detect SPMs, provided the required analytical sensitivity and specificity can be realized. One strategy to establish the necessary performance is to introduce nucleotide analogs such as Locked Nucleic Acids (LNAs) into AS probes or primers; however the successful design requires a fundamental understanding of both the thermodynamics and kinetics of LNA-DNA heteroduplexes. Melting thermodynamic studies of DNA duplexes and LNA-DNA heteroduplexes were therefore carried out using both ultraviolet (UV) spectroscopy and differential scanning calorimetry (DSC) to quantify the thermodynamics (ΔH⁰, ΔS⁰, ΔCp and Tm) associated with the helix-to-coil transition. Data collected on DNA duplexes and DNA-LNA heteroduplexes were used to introduce improvements in the “unified” nearest-neighbor model, and for the development of a new model, referred to as the Single Base Thermodynamic (SBT) model that accurately predicts the Tm for the melting of LNA-DNA heteroduplexes. The SBT model was extended and applied to PCR conditions to design LNA-bearing AS probes for qPCR assays to detect the clinically important SPMs KIT c.1799t>a (D816V) and JAK2 c.1849g>t (V617F), and were found to significantly outperform standard AS probes containing only DNA. The interaction of Taq polymerase with heteroduplexes formed between an LNA-bearing primer and a target template were also studied and results used to generate general rules for designing LNA-bearing AS primers capable of unequivocal detection of a rare mutant allele bearing a SPM. The method was then extended to allow qPCR detection by Plexor™ technology and applied to create an AS primer directed against the JAK2 V617F SPM that can detect one mutation in a background of more than 100,000 copies of the wild-type allele and which is now used by the Cancer Genetics Laboratory of the British Columbia Cancer Agency (BCCA) to analyze patient samples.
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