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

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

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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