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Investigating Transgene Integration and Organization in Cotton (Gossypium hirsutum L.) Genome

 Last Update: 2021-03-13
 Source: Internet
 Author: User

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In this chapter, we present detailed experimental procedures for investigating integration patterns of transgenes in cotton genome. We use conventional
PCR
and genomic Southern blot hybridization to characterize integration of T-
DNA
components and vector backbone fragments. For multiple copy insertions into the same site (complex loci), transgene/transgene junctions (including canonical and truncated T-DNA and transgene involved vector backbone sequences) are characterized by PCR and sequencing. Inverse PCR (see Note 1) and sequencing is used to characterize transgene/cotton genome junctions. Distribution of T-DNA insertion in cotton genome is evaluated by analysis of transgene flanking sequences. The pre-insertion sites can also be cloned and sequenced (based on the flanking sequences) for survey of genomic structure changes brought by transgene integration by comparing a pre-insertion site with corresponding transgene/plant junctions.

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