Fast and accurate quantification of insertion-site specific transgene levels from raw seed samples using solid-state...

Area of Science:

• Agricultural Biotechnology
• Molecular Biology
• Genetics

Background:

• Modern crops increasingly feature patented biotechnology traits, often stacked.
• Accurate transgene quantification is crucial for food, feed, and fuel industries.
• Existing quantification methods require optimization for new transgenic traits.

Purpose of the Study:

• To develop a rapid, simple, and accurate method for quantifying transgene levels in seeds.
• To integrate polymerase chain reaction (PCR) with advanced sensing technologies for seed-to-trait analysis.
• To facilitate the addition of new traits into existing quantification assays.

Main Methods:

• A novel, end-point polymerase chain reaction (PCR) assay designed for whole seeds, completed in under 5 minutes.
• Relative quantification using capillary electrophoresis across the 0-100% range.
• Integration of the PCR assay with non-optical solid-state nanopore sensors for real-time quantification.

Main Results:

• The PCR and capillary electrophoresis method achieved a mean absolute error of 1.9% for transgene quantification.
• Coupling PCR with nanopore sensors yielded seed-to-trait quantification with a mean absolute error of 2.3%.
• The integrated system provides accurate seed-to-trait quantification in under 10 minutes across the full dynamic range.

Conclusions:

• A fast and robust PCR-based method enables efficient transgene level determination in seeds.
• The integration of PCR with nanopore sensing offers a rapid, accurate, and versatile solution for seed-to-trait quantification.
• This technology supports the streamlined management of genetically modified crops throughout the value chain.