title: Investigating transgene integration and organization in cotton (Gossypium hirsutum L.) genome.
abstract: 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.
authors.affiliation: Cotton Research Center, Shandong Academy of Agricultural Sciences, Jinan, Shandong, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Hong
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D015139:Blotting, Southern; D018744:DNA, Plant / Q000235:genetics; D018014:Gene Transfer Techniques; D018745:Genome, Plant / Q000235:genetics*; D003368:Gossypium / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: D018744:DNA, Plant