Designing a Novel Primer Blocker using Bioconjugation of Single Stranded Binding Protein and Oligonucleotides for Improved KRAS Mutation Detection

Overview

The Kirsten rat sarcoma viral (KRAS) gene is the most frequently mutated oncogene, responsible for many types of highly fatal cancers — notably, pancreatic, colorectal, and non-small cell lung cancers (NSCLC). This project aims to design an improved primer-blocker mechanism to detect the KRAS G12D mutation subtype in qPCR method liquid biopsies. Early detection is paramount to increasing the chance of patient survival. In order for liquid biopsies to be a reliable method of cancer diagnosis, especially during earlier stages, there needs to be improvements in accuracy and sensitivity of detection; this is due to the lower amount of tumor genetic material (ctDNA) in the blood when tumors are localized, during early detection of cancer. This project provided a proof of concept for a novel design: replacing oligonucleotides with single stranded binding protein to act as the anchor in primer-blocker technologies. The need for a longer and more competitive primer to anneal with the template is mitigated because of the protein acting as the anchor instead, allowing a shorter, and therefore more specific, primer-blocker. The primer-blocker was constructed using copper-catalyzed azide-alkyne click chemistry, in order to connect the SSB protein anchor with KRAS G12D mutation specific primers. Multiple primers of different lengths were tested to determine the best parameters and results of qPCR trials show that the novel SSB protein primer blocker was able to selectively amplify mutant DNA when connected to the medium length (18 bp) primer, specifically in 1.0uM and 2.0uM concentrations. The RT-qPCR charts also showed that the controls (water, just oligo, or just protein) did not show any selective amplification.

Awards (1)

Category Award: 4

Competition history

CSEF 2023 Biochemistry/ Molecular Biology · Entry S0514

Resources

Related projects

ISEF · 2023