Patent ID: 7226785

Claim:
A method of optimizing an oligonucleotide for in vitro targeted chromosomal sequence alteration, comprising: (a) Providing a first oligonucleotide and a second oligonucleotide for alteration of a targeted nucleic acid sequence, i) wherein said first oligonucleotide: (1) is a single-stranded nonhairpin oligonucleotide 17-121 nucleotides in length, (2) has an unmodified DNA domain of at least 8 contiguous deoxyribonucleotides, (3) is fully complementary in sequence to the sequence of a first strand of the nucleic acid target but for one or more mismatches as between the sequences of said deoxyribonucleotides domain and its complement on the target nucleic acid first strand, each of said mismatches positioned at least 8 nucleotides from said first oligonucleotide's 5′ and 3′ termini, and (4) has at least one terminal modification selected from the group consisting of: at least one terminal locked nucleic acid (LNA), at least one terminal 2′-O-Me base analog, and at least three terminal phophorothioate linkages, and ii) wherein said second oligonucleotide is selected from the group consisting of (1) an oligonucleotide that is fully complementary to the target and lacks the mismatch, (2) a fully modified phosphothiolated oligonucleotide, (3) a fully modified 2′-O-methylated oligonucleotide, and (4) a chimeric double-stranded double hairpin containing RNA and DNA nucleotides, and iii) wherein the targeted nucleic acid sequence alteration is not in human embryonic stem cells; (b) combining the targeted nucleic acid with said first or said second oligonucleotide in the presence of cellular repair proteins; (c) combining the targeted nucleic acid with the other of said first or second oligonucleotide in the presence of cellular repair proteins; (d) quantifying the percentage of target molecules that undergo a sequence alteration event; (e) comparing the efficiency of alteration of said targeted nucleic acid sequence by said first oligonucleotide with the efficiency of alteration of the same targeted nucleic acid sequence by said second oligonucleotide; and (f) repeating steps (a) through (e) two or more times to optimize said first oligonucleotide for in vitro targeted chromosomal sequence alteration.