Fast and accurate quantification of insertion-site specific transgene levels from raw seed samples using solid-state nanopore technology
Autoři:
Michael D. Pearson aff001; Leslee Nguyen aff001; Yanan Zhao aff001; William L. McKenna aff001; Trevor J. Morin aff001; William B. Dunbar aff001
Působiště autorů:
Ontera, Inc., Santa Cruz, California, United States of America
aff001
Vyšlo v časopise:
PLoS ONE 14(12)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0226719
Souhrn
Many modern crop varieties contain patented biotechnology traits, and an increasing number of these crops have multiple (stacked) traits. Fast and accurate determination of transgene levels is advantageous for a variety of use cases across the food, feed and fuel value chain. With the growing number of new transgenic crops, any technology used to quantify them should have robust assays that are simple to design and optimize, thereby facilitating the addition of new traits to an assay. Here we describe a PCR-based method that is simple to design, starts from whole seeds, and can be run to end-point in less than 5 minutes. Subsequent relative quantification (trait vs. non-trait) using capillary electrophoresis performed in 5% increments across the 0–100% range showed a mean absolute error of 1.9% (s.d. = 1.1%). We also show that the PCR assay can be coupled to non-optical solid-state nanopore sensors to give seed-to-trait quantification results with a mean absolute error of 2.3% (s.d. = 1.6%). In concert, the fast PCR and nanopore sensing stages demonstrated here can be fully integrated to produce seed-to-trait quantification results in less than 10 minutes, with high accuracy across the full dynamic range.
Klíčová slova:
Crops – DNA extraction – Gel electrophoresis – Nanotechnology – Polymerase chain reaction – Soybean – Support vector machines – Capillary electrophoresis
Zdroje
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