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


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


1. Holst-Jensen A. Testing for genetically modified organisms (GMOs): Past, present and future perspectives. Biotechnology Advances. Elsevier Inc; 2009 Nov 12;27(6):1071–82. doi: 10.1016/j.biotechadv.2009.05.025 19477261

2. Nguyen HT, Jehle JA. Quantitative analysis of the seasonal and tissue-specific expression of Cry1Ab in transgenic maize Mon810. J Plant Dis Prot. Second edition. Springer Berlin Heidelberg; 2016 Mar 31;114(2):82–7. doi: 10.1007/BF03356208

3. Shrestha HK, Hwu K-K, Chang M-C. Advances in detection of genetically engineered crops by multiplex polymerase chain reaction methods. Trends in Food Science & Technology. 2010 Sep;21(9):442–54. doi: 10.1016/j.tifs.2010.06.004

4. Chaouachi M, Bérard A, Saïd K. Relative quantification in seed GMO analysis: state of art and bottlenecks. Transgenic Res. 2013 Feb 12;22(3):461–76. doi: 10.1007/s11248-012-9684-1 23400878

5. Huang C-C, Pan T-M. Event-Specific Real-Time Detection and Quantification of Genetically Modified Roundup Ready Soybean. J Agric Food Chem. 2005 May;53(10):3833–9. doi: 10.1021/jf048580x 15884804

6. Gerdes L, Busch U, Pecoraro S. A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions. BMC Bioinformatics. BioMed Central; 2014 Dec 14;15(1):407. doi: 10.1016/j.bdq.2015.12.003

7. Kiddle G, Hardinge P, Buttigieg N, Gandelman O, Pereira C, McElgunn CJ, et al. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol. BioMed Central; 2012 Dec 1;12(1):1–13. doi: 10.1186/1472-6750-12-15 22546148

8. Auer CA. Tracking genes from seed to supermarket: techniques and trends. Trends Plant Sci. 2003 Dec;8(12):591–7. doi: 10.1016/j.tplants.2003.10.010 14659708

9. Morin TJ, McKenna WL, Shropshire TD, Wride DA, Deschamps JD, Liu X, et al. A handheld platform for target protein detection and quantification using disposable nanopore strips. Sci Rep. 2018 Oct;8(1):14834. doi: 10.1038/s41598-018-33086-7 30287843

10. Li J, Stein D, McMullan C, Branton D, Aziz MJ, Golovchenko JA. Ion-beam sculpting at nanometre length scales. Nature. 2001 Jul 12;412(6843):166–9. doi: 10.1038/35084037 11449268

11. Storm AJ, Chen JH, Zandbergen HW, Dekker C. Translocation of double-strand DNA through a silicon oxide nanopore. Phys Rev E. 2005 May;71(5 Pt 1):051903. doi: 10.1103/PhysRevE.71.051903 16089567

12. Wanunu M, Sutin J, McNally B, Chow A, Meller A. DNA translocation governed by interactions with solid-state nanopores. Biophys J. 2008 Nov 15;95(10):4716–25. doi: 10.1529/biophysj.108.140475 18708467

13. Fologea D, Brandin E, Uplinger J, Branton D, Li J. DNA conformation and base number simultaneously determined in a nanopore. ELECTROPHORESIS. 2007 Sep;28(18):3186–92. doi: 10.1002/elps.200700047 17854121

14. Squires AH, Atas E, Meller A. Genomic Pathogen Typing Using Solid-State Nanopores. Ahmed N, editor. PLoS ONE. 2015 Nov 12;10(11):e0142944–16. doi: 10.1371/journal.pone.0142944 26562833

15. Zhao Y, Mckenna W, Dunbar WB. Fractional abundance of polynucleotide sequences in a sample, International patent no. WO2018081178A1. 2018.

16. Singer A, Wanunu M, Morrison W, Kuhn H, Frank-Kamenetskii M, Meller A. Nanopore based sequence specific detection of duplex DNA for genomic profiling. Nano Lett. 2010 Feb 10;10(2):738–42. doi: 10.1021/nl100058y 20088590

17. Morin TJ, Shropshire T, Liu X, Briggs K, Huynh C, Tabard-Cossa V, et al. Nanopore-based target sequence detection. Wanunu M, editor. PLoS ONE. 2016 May 5;11(5):e0154426–21. doi: 10.1371/journal.pone.0154426 27149679

18. Windels P, Taverniers I, Depicker A, Van Bockstaele E, De Loose M. Characterisation of the Roundup Ready soybean insert. Eur Food Res Technol. Springer-Verlag; 2001 Aug 1;213(2):107–12. doi: 10.1007/s002170100336

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden