Poster Session 3, 1:15 PM - 2:00 PM: Room 165 [D3]

Genome-Scale Modeling of Restriction Enzyme Performance for Efficient iPCR Transgene Mapping in Brachypodium distachyon

Presenter: Milo Georgiev

Faculty Sponsor: Scott M. Auerbach

School: UMass Amherst

Research Area: Biochemistry and Molecular Biology

ABSTRACT

Transgene insertion site mapping is essential for accurate genetic characterization in transgenic plants, yet because mapping techniques are costly or difficult mapping is seldom done.  Inverse PCR (iPCR) is a technique used to recover DNA flanking a transgene insertion, but its success depends heavily on selecting restriction enzymes that generate fragments of appropriate size for circularization and PCR amplification, making enzyme choice a major bottleneck. To address this limitation, I developed a reproducible in silico workflow to select appropriate restriction enzymes for transgene insertion mapping in Brachypodium distachyon, a model grass species. Using the Bd21 v3.2 reference genome, I performed a genome-wide scanning of candidate restriction enzyme recognition sites, with a python-based pipeline and calculated cutting frequency, normalized site density, and fragment size distributions for ten commonly used restriction enzymes. Fragments were evaluated for suitability within a size range of 500bp and 5kb for circularization and PCR amplification, and simulated random transgene insertions were used to estimate the likelihood that each enzyme would generate recoverable insertion-associated fragments. This framework enables enzyme selection based on predicted recovery efficiency rather than trial-and-error experimentation, improving the reliability of iPCR-based insertion mapping and establishing a quantitative, species-tailored strategy to accelerate molecular genetic studies in Brachypodium distachyon.

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