Patent ID: 6586184
Filing Date: 2003-07-01
Classification: C07H,C12N

Abstract:
A method of introducing a change in a target DNA sequence to form a different, desired sequence in a recombination/repair competent prokaryotic cell comprising:(A) transforming the prokaryotic cell with a duplex mutational vector comprising: (i) a first oligonucleobase strand of at least 12 linked nucleobases and not more than 75 linked nucleobases, that has a first and a second terminal nucleobase; (ii) a second oligonucleobase strand having a 3â€² most and a 5â€² most nucleobase and having a number of nucleobases equal to the first oligonucleobase strand, which second oligonucleobase strand is optionally divided into a first chain and a second chain; and (iii) a single 3â€² end nucleobase and a single 5â€² end nucleobase; in which (a) the 3â€² most and 5â€² most nucleobases of the second oligonucleobase strand are Watson-Crick base paired to the first terminal and the second terminal nucleobase of the first oligonucleobase strand, respectively, (b) said 3â€² most nucleobase and said second terminal nucleobase are protected from 3â€² exonuclease attack; and (c) the second oligonucleobase strand contains at least two non-overlapping regions of at least 5 contiguous nucleobases that are Watson-Crick base paired to nucleobases of the first oligonucleobase strand, provided that for not more than two contiguous nucleobases of a strand is a deoxy-type nucleobase Watson-Crick paired with a ribo-type nucleobase or a ribo-type nucleobase Watson-Crick paired with a deoxy-type nucleobase; and wherein the sequence of at least one oligonucleobase strand comprises the different, desired sequence; and (B) detecting the presence of the different desired sequence from or in the prokaryotic cell or progeny thereof.