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Molecular Analysis for Characterizing Transgenic Events

  • Wei Chen
  • PoHao Wang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1864)

Abstract

To develop a commercial trait product, a large number of transgenic events are often produced to obtain the event with desired level of expression. It is crucial to develop efficient and sensitive molecular characterization methods to advance events with stable transgene expression, free of vector backbone sequences and without major changes to the native genome caused by transgene insertion. Here, we discuss a variety of analytical tools, including quantitative PCR (qPCR), Southern blot analysis, and various sequencing technologies, which have been widely used to determine the insert copy number, presence/absence of vector backbone sequences, integrity of the T-DNA, and genomic location of the T-DNA insertion. Moreover, since the discovery of RNA interference in 1998 (Fire et al., Nature 391:806-811, 1998), RNAi has emerged as another powerful tool in in the development of a new transgenic trait for insect control. RNAi creates a double-stranded RNA duplex as the active molecule which forms a strong secondary structure, resulting in challenges for detection. In addition to molecular analysis at the DNA level, this chapter describes detection methods of the active molecules (i.e., double-stranded RNA) for RNAi-based traits.

Key words

Transgenic events Molecular characterization qPCR NGS RNAi 

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Corteva Agriscience™Agriculture Division of DowDuPont™JohnstonUSA

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