title: Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.
abstract: One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct.
authors.affiliation: Dipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente (DiSPAA), Università degli Studi di Firenze, Viale delle Idee 30, 50019, Sesto Fiorentino, FI, Italy. stefano.biricolti@unifi.it.
authors.forename: Biricolti
authors.initials: B
authors.lastname: Stefano
authors.affiliation: Dipartimento di Biologia (BIO), Università degli Studi di Firenze, Via Madonna del Piano 6, 50019, Sesto Fiorentino, FI, Italy.
authors.forename: Bogani
authors.initials: B
authors.lastname: Patrizia
authors.affiliation: Dipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente (DiSPAA), Università degli Studi di Firenze, Viale delle Idee 30, 50019, Sesto Fiorentino, FI, Italy.
authors.forename: Cerboneschi
authors.initials: C
authors.lastname: Matteo
authors.affiliation: Dipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente (DiSPAA), Università degli Studi di Firenze, Viale delle Idee 30, 50019, Sesto Fiorentino, FI, Italy.
authors.forename: Gori
authors.initials: G
authors.lastname: Massimo
mesh_terms: D015139:Blotting, Southern / Q000379:methods*; D018628:Gene Dosage; D018745:Genome, Plant; D027845:Malus / Q000235:genetics*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D019076:Transgenes
keywords: Inverse PCR; Quantitative PCR; Southern blot; Transgene copy number; Transgenic fruit trees