Polypeptide mutagenesis method

C - Chemistry – Metallurgy – 12 – N

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C12N 15/10 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2601324

There is provided a method for altering the amino acid sequence of a target polypeptide by altering a target DNA sequence which encodes that polypeptide, the method comprising the step of introducing a transposon into the target DNA sequence, in which the transposon comprises a first restriction enzyme recognition sequence towards each of its termini, the recognition sequence not being present in the remainder of the transposon, or in the target DNA sequence, or in a construct comprising the target DNA sequence, the first restriction enzyme recognition sequence being recognised by a first restriction enzyme which is an outside cutter and being positioned such that the first restriction enzyme has a DNA cleavage site positioned beyond the end of the terminus of the transposon.

L'invention concerne un procédé d'altération de séquence d'acide aminé d'un polypeptide cible par modification d'une séquence d'ADN cible qui code ce polypeptide. Ce procédé consiste à introduire un transposon dans la séquence d'ADN cible, ce transposon comprenant une première séquence de reconnaissance d'enzyme de restriction au niveau de chacune des ses extrémités, la séquence de reconnaissance n'étant pas présente dans le reste du transposon, ou dans la séquence ADN cible, ou dans une construction comprenant la séquence d'ADN cible, la première séquence de reconnaissance d'enzyme de restriction reconnue par une première enzyme de restriction qui est une coupure externe et est positionnée de manière que la première enzyme de restriction possède u site de clivage d'ADN disposé au-delà de l'extrémité du transposon.

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