Development a cloning strategy based on Gateway Technology

BERTELLI, A; SETTEN, L; NELSON, G; ALVAREZ, MA; MARCONI, PL

Guadalajara, México

Simposio; VII Encuentro Latinoamericano y del Caribe sobre Biotecnología Agropecuaria. REDBIO 2010; 2010

RedBio México

In order to obtain an efficient DNA fragment single step transference to the Gateway destination vector (pK7WG2D) we have developed a method with direct recombination from PCR products. This strategy was performed because both entry and destination vectors carried the same resistance (Kanamicin). We therefore cloned the gene of interest into the pCR8(GW)TOPO vector (Invitrogen). The process was evaluated using different enzymes: PMT (putrescine N-methyltransferase from Nicotiana tabacum L.) and H6H (Hyoscyamine 6b-hydroxylase from Brugmansia x candida Pers.) encoding regions