Digestion of I-PpoI recognition sites in unfavorable sequence contexts can be achieved by changing the reaction conditions

CHRISTINA B. MCCARTHY; VICTOR ROMANOWSKI

Villa Carlos Paz, Córdoba, Argentina

Congreso; XXXVIII Reunión Anual de la de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

I-PpoI is an intron-encoded endonuclease that recognizes a 15-bp target and, therefore, is very useful for a variety of applications. However, its cutting efficiency has been reported to depend strongly upon nucleotide sequences flanking this target, which limits its performance only to favorable sequence contexts. In addition to this, the protocol provided by the manufacturer establishes very stringent conditions for its adequate performance. In our study we explored two different approaches to improve I-PpoI´s deficient performance due to adverse flanking regions. In the first case, we used a plasmid (pAgPHZ3-IPpoI) in which I-PpoI´s restriction site had been inserted in an unfavorable context, to find an experimental condition in which the enzyme worked satisfactorily. In the second case, we used a different plasmid (pZIPpoI) in which I-PpoI´s restriction site had also been incorporated, but in this case the unfavorable flanking sequences were changed via mutation to optimal flanking regions. We found that the requisite of optimal flanking sequences for I-PpoI to digest adequately is not as indispensable as previously claimed, and that I-PpoI´s cleavage reaction conditions are much more lax than those formerly reported. We also found that, by simply changing the composition of the cleavage reaction buffer, the detrimental effect some flanking sequences have on this enzyme´s cleavage efficiency, is circumvented.