Standardizing Magnetic Bead-Based PCR for Analyzing Quantitative Gene Expression, Followed by Sequencing

Standardizing Magnetic Bead-Based PCR for Analyzing Quantitative Gene Expression, Followed by Sequencing

Presenter(s)

Malabika Bhowmik, Tracy Dentu, Jason Galaska, Tooba Momin, Sheilah Njoka, Grace Van Kerkhove

Comments

9:00-10:15, Kennedy Union Ballroom

Description

Digital PCR (dPCR) is an advanced molecular technique that enables the absolute quantification of nucleic acids with high sensitivity, precision, and reproducibility. Unlike conventional quantitative PCR (qPCR), dPCR does not rely on standard curves. Instead, it partitions the sample into thousands of individual reactions, where target DNA or cDNA (for RNA) molecules are amplified and detected in a binary (positive/negative) manner. The fraction of positive partitions can then be analyzed using Poisson statistics to determine the absolute copy number of the target sequence.This study aims to integrate magnetic bead-based PCR with dPCR. Magnetic beads provide a solid-phase support for nucleic acid binding, reducing sample loss and allowing the amplified product to be further analyzed for nucleotide sequencing. In this study, we utilized streptavidin-coated Dynabeads (1 µm diameter). A biotinylated forward primer targeting the human coronavirus OC43 (HCoV-OC43) spike protein gene was immobilized onto the Dynabeads, followed by PCR amplification.Our results showed efficient binding of the forward primer to the Dynabeads (45.22 ± 4.11% binding efficiency). The PCR product was successfully amplified on the beads, as confirmed by gel electrophoresis following the elution of DNA from the microbeads. Overall, our findings indicate that Dynabead-bound PCR products can be effectively utilized for digital PCR, followed by nucleotide sequencing using an ion-sensitive photonic field-effect waveguide (ISPFEW) chip.

Publication Date

4-23-2025

Project Designation

Independent Research

Primary Advisor

Swapnajit Chakravarty, Mrigendra Rajput

Primary Advisor's Department

Biology

Keywords

Stander Symposium, College of Arts and Sciences

Standardizing Magnetic Bead-Based PCR for Analyzing Quantitative Gene Expression, Followed by Sequencing

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