Browsing by Author "Zhang, Carol"

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    Development of New Methods for Easy-to-apply, Multiplexed and Ultrasensitive Nucleic-Acid based Diagnostic Technologies
    (2021-12-09) Zhang, Carol; Zhang, David Yu; Veiseh, Omid
    The Polymerase chain reaction (PCR) has been one of the most widely used and easily accessible methods in bio-laboratories. It could be used in a fluorescence detection device together with intercalating dyes or fluorophore-labeled probes to achieve real-time quantitation of nucleic acid targets, or it could be involved in the workflow of high- throughput sequencing library preparation for target enrichment. Clinically and biologically, somatic mutations are becoming significant and informative biomarkers in many diseases, including cancer. The ability to accurately detect rare DNA variant sequences could be beneficial to early-stage cancer diagnosis, recurrence monitoring and precision treatment. However, there are some limitations for the existing common detection technologies. Some qPCR methods are restricted in the limit of detection and require multiple reactions to identify mutations; some qPCR methods are not compatible with high-fidelity DNA polymerase to achieve ultra-sensitive mutation detection and do not allow for multiplexing; some next-generation sequencing methods may require high computing resource or additional steps to reduce dimerization, improve PCR efficiency and increase sensitivity; or some high-throughput sequencing methods may be economically limited due to the expensive chemical synthesis for higher multiplex targets. During my PhD, I have developed several novel PCR-based methods to provide a solution to the unmet needs discussed above, achieving ultra-sensitive and multiplexing variant-enrichment-based detection in either the accessible quantitative PCR (qPCR) or the next-generation sequencing (NGS). This thesis is a collection of 3 manuscripts summarizing the projects during my PhD research: [1] Zhang K, Rodriguez L, Cheng LY, Zhang DY. Single Tube qPCR detection and quantification of hotspot mutations down to 0.01% VAF. Manuscript has been accepted by Analytical Chemistry. [2] Zhang K, Pinto A, Song P, Dai P, Wang MX, Cheng LY, Rodriguez L, Weller C, Zhang DY. Hairpin structure facilitates high-fidelity DNA amplification reactions in both qPCR and high-throughput sequencing. Manuscript under review. [3] Zhang K, Ping S, Zhang JX, Dai P, Wen R, Rodriguez L, Zhang DY. Non- extensible oligonucleotides in DNA amplification reactions. Manuscript under preparation.