Targeted alteration of dna with oligonucleotides

Abstract

The current invention relates to a method for targeted alteration of acceptor DNA, for example duplex acceptor DNA. The method comprises use of at least two oligonucleotides, each oligonucleotide having at least one mismatch relative to the targeted (duplex) acceptor DNA. The mismatch of the first oligonucleotide is directed to a nucleotide at a position in the first strand of the duplex and the mismatch of the second oligonucleotide is directed to the nucleotide in the second strand that occupies the complementary position in the duplex acceptor DNA (e.g. forms a base-pair with the nucleotide in the first strand). These mismatches are located at specific positions within said oligonucleotides. Also provided is a kit that comprises instructions for performing the method according to the inventions, and in a preferred embodiment, comprises oligonucleotides suitable for use in the method.

1 . A method for targeted alteration of a duplex acceptor DNA sequence comprising a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence, the method comprising
 combining the duplex acceptor DNA sequence with at least a first oligonucleotide and a second oligonucleotide,   wherein the first oligonucleotide comprises at least one domain that is capable of hybridizing to the first DNA sequence and wherein the first oligonucleotide further comprises at least one mismatch with respect to the first DNA sequence and wherein the at least one mismatch is positioned at most 2 nucleotides from the 3′ end of said first oligonucleotide;   and wherein the second oligonucleotide comprises at least one domain that is capable of hybridizing to the second DNA sequence and wherein the second oligonucleotide further comprises at least one mismatch with respect to the second DNA sequence and wherein the at least one mismatch is positioned at most 2 nucleotides from the 3′ end of said second oligonucleotide;   and wherein the at least one mismatch in the first oligonucleotide is relative to a nucleotide in the first DNA sequence of the duplex acceptor DNA sequence and wherein the at least one mismatch in the second oligonucleotide is relative to a nucleotide in the second DNA sequence of the duplex acceptor DNA, and wherein said nucleotides occupy complementary positions in the duplex acceptor DNA.   
     
     
         2 . The method according to  claim 1  wherein the mismatch in the first oligonucleotide or the mismatch in the second oligonucleotide is, independently, positioned at most 1 nucleotide from the 3′ end of said oligonucleotide, more preferably said at least one mismatch is at the 3′ end of the oligonucleotide, preferably the mismatch in both oligonucleotides is at the 3′ end of the oligonucleotides. 
     
     
         3 . The method according to  claim 1  wherein the domain in the first oligonucleotide and/or in the second oligonucleotide comprises or is directly adjacent to the at least one mismatch. 
     
     
         4 . The method according to  claim 1  wherein the first oligonucleotide is complementary to the first DNA sequence except for the mismatch and/or wherein the second oligonucleotide is complementary to the second DNA sequence except for the mismatch. 
     
     
         5 . The method according to  claim 4  wherein the mismatch in the first oligonucleotide is at the 3′ end and wherein the mismatch in the second oligonucleotide is at the 3′ end. 
     
     
         6 . The method according to  claim 1  wherein the first oligonucleotide and/or the second oligonucleotide comprises at least one section that contains at least one modified nucleotide, wherein the modification is selected from the group consisting of a base modification, a 3′ and/or 5′ end base modification, a backbone modification or a sugar modification. 
     
     
         7 . The method according to  claim 6  wherein the modified nucleotide is selected from the group consisting of LNA or phosphorothioate bonds. 
     
     
         8 . The method according to  claim 6  wherein the oligonucleotide comprises at least two, three, four, or five modified nucleotides, preferably the oligonucleotide comprises two, three, four or five modified nucleotides. 
     
     
         9 . The method according to  claim 1  wherein the mismatch is not a modified nucleotide. 
     
     
         10 . The method according to  claim 6  wherein the modified nucleotide is at least one nucleotide from the at least one mismatch located at most 2, preferably at most 1 nucleotide from the 3′ end of said oligonucleotide, most preferably said at least one mismatch is at the 3′ end of the oligonucleotide. 
     
     
         11 . The method according to  claim 1 , wherein the alteration of the duplex acceptor DNA is within a cell preferably selected from the group consisting of prokaryotic cell, a bacterial cell, a eukaryotic cell, a plant cell, an animal cell, a yeast cell, a fungal cell, a rodent cell, a human cell, a non-human cell, and/or an embryonic cell. 
     
     
         12 . The method according to  claim 1  wherein the duplex acceptor DNA is obtained from a prokaryotic organism, a bacteria, an eukaryotic organism, a plant, an animal, a yeast, a fungus, a rodent, or a human. 
     
     
         13 . The method according to  claim 1 , wherein the alteration is a deletion, a substitution and/or an insertion of at least one nucleotide. 
     
     
         14 . The method according to  claim 1 , wherein the duplex acceptor DNA is from genomic DNA, linear DNA, artificial chromosomes, mammalian artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, plant artificial chromosomes, nuclear chromosomal DNA, organellar DNA, and/or episomal DNA including plasmids. 
     
     
         15 . The method according to  claim 1 , for altering a cell, correcting a mutation by restoration to wild type, inducing a mutation, inactivating an enzyme by disruption of coding region, modifying bioactivity of an enzyme by altering coding region, modifying a protein by disrupting the coding region. 
     
     
         16 . (canceled) 
     
     
         17 . A kit comprising instructions for performing a method for targeted alteration of a duplex acceptor DNA according to  claim 1 . 
     
     
         18 . A kit according to  claim 17  further comprising at least two oligonucleotides for use in the method according to  claim 1 , preferably comprising the at least two oligonucleotides as described in  claim 1 . 
     
     
         19 . A kit according to  claim 17  wherein, when combined with a duplex acceptor DNA sequence containing a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence, the first oligonucleotide comprises at least one domain that is capable of hybridizing to the first DNA sequence and wherein the first oligonucleotide further comprises at least one mismatch with respect to the first DNA sequence and wherein the at least one mismatch is positioned at most 2 nucleotides from the 3′ end of said first oligonucleotide; and wherein the second oligonucleotide comprises at least one domain that is capable of hybridizing to the second DNA sequence and wherein the second oligonucleotide further comprises at least one mismatch with respect to the second DNA sequence and wherein the at least one mismatch is positioned at most 2 nucleotides from the 3′ end of said second oligonucleotide; and wherein the at least one mismatch in the first oligonucleotide is relative to a nucleotide in the first DNA sequence of the duplex acceptor DNA sequence and wherein the at least one mismatch in the second oligonucleotide is relative to a nucleotide in the second DNA sequence of the duplex acceptor DNA, and wherein said nucleotides occupy complementary positions in the duplex acceptor DNA.