Development of Multiplex Digital PCR and Real-time PCR Assays for Detection of DNA Mutations, RNA Fusion and miRNA in Blood and in Single Cells


Student thesis: Doctoral Thesis

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Award date1 Mar 2021


Molecular diagnostic techniques such as quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) play crucial roles in cancer diagnosis and monitoring. Advances in these technologies enable to characterize genetic alterations in limited clinical samples with low cost at desired accuracy and precision. However, the conventional assay designs currently in use are tradeoff in simultaneous detection of multiple markers in a single reaction, limiting the application of the technology in liquid biopsy analysis because this approach needs high throughput techniques to characterize the detailed genetic status of patients. To overcome this limitation, various multiplexing strategies have been reported, but most are not cost-effective. Here, we developed cost-effective multiplexed dPCR and reverse transcription PCR (RT-PCR) assays capable of detecting genetic aberrations from deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), respectively.

The first achievement of this thesis work is the development of dPCR assays that can detect 29 DNA mutations in 3 genes. These assays are multiplexed in 7 dPCR reactions to detect codon or gene-specific mutations: including 4 BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations at codon 600, 7 KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations at codon 12 or 13, and 18 EGFR (epidermal growth factor receptor) gene mutations (3 mutations at exon 18, 8 deletions at exon 19, 5 mutations at exon 20, and 2 mutations at exon 21). Unlike previous reported dPCR assays, all the multiplexed dPCR assays utilize allele-specific primer (ASP) rather than probes to detect the targeted mutation. Such design is easy, cost-effective, and enables high order multiplexing capability (8 targets the maximum we achieved) in a single reaction. Compared to the traditional probe-based duplex assays, our assays are sensitive to detect mutation abundance below 0.1% and enable high-order magnitudes quantification dynamic ranges using a DNA input ranging from 0.009 ng to 90 ng. So that, the assays we developed are named as multiplex allele-specific and sensitive dPCR (mass-dPCR). The mass-dPCR assays have consistent target quantification performance in both chip/chamber-based dPCR (cdPCR) and droplet-based dPCR (ddPCR), implying the robustness of the assay irrespective of the dPCR platform sample parting strategy. Finally, the multiplexed assays' diagnostic application was confirmed from clinical validations. It showed 100% concordance with two independent orthogonal sequencing methods: the Qiagen and the Illumine platforms. Detection of rare cancer-causing mutations from circulating tumor DNA (ctDNA) that is usually overwhelmed by background wildtype, is one of the greatest advantages of these assays because the ASPs are selective to amplify only mutant variants (MT) but not wild type (WT), which makes the assays to be more promising for the application of sensitive mutation detections in cancer management specifically for treatment follow-up and early detection of relapse. Given the involvement of various mutations in the cancer development process, these multiplexed assays are also promising for the composite characterization DNA mutations that enable patient stratifications to targeted therapies.

Another main achievement in this work was the development of methods for single-cell genetic material enrichment and subsequent analysis of multiple molecules. This work was designed mainly to analyze DNA mutations, RNA fusions and miRNAs from single cells. The enrichment method we developed is a highly specific PCR based preamplification method enabling to generate sufficient amplicon products from single cells that can be analyzed using dPCR and RT-PCR assays. The mass-dPCR assays not only compatible to analyze preamplification products but also showed consistent specificity signifying the preamplification step do not interfere with assays analytic performance. In addition, we developed three multiplexed RT-PCR assays to detect gene fusion: one for Anaplastic lymphoma kinase (ALK) fusions detection, two ROS proto-oncogene 1 (ROS1) fusions detection assays; both can detect at least 28 fusion variants. The three fusion assays are 100% specific and sensitive when applied to single cells isolated from cell lines. Simultaneously we developed an efficient RT-PCR method to detect three miRNAs: miR-16, miR-100, miR-373. All these miRNAs were successfully detected with variable amounts in single cells irrespective of cell line types. To the best of our knowledge, this is the first study to report the multiplexed analysis of multiple molecules from a single cell using dPCR and RT-PCR. Such method has a promising application in characterizations of single cells, more specifically circulating tumor cells (CTCs) for the diagnosis, prognosis, and treatment follow-up of cancer patients. In summary, we developed three multiplexed methods that provide the advantages of efficient detection of DNA mutations in extracted tumor tissue DNA/ctDNA or single cells, as well as gene fusions and miRNAs in single cells exceptionally requiring high throughput technologies. These methods have significant contributions to molecular diagnostics, particularly in prognosis and treatment follow-up of cancer patients.