US 9,518,258 B2Granted

Targeted alteration of DNA with oligonucleotides

AssigneeKeygene N.V.

InventorsMichiel Theodoor Jan De Both, Tomoyuki Furukawa

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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.

Claims25

Dependent claims

A method for targeted alteration of a duplex acceptor DNA sequence comprising the step of

combining the duplex acceptor DNA sequence with at least a first oligonucleotide and a second oligonucleotide, wherein the duplex acceptor DNA sequence comprises a first DNA sequence and a second DNA sequence, which is complementary to the first DNA sequence;
and
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 this 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 this 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 sequence, and wherein said nucleotide in the first DNA sequence and said nucleotide in the second DNA sequence occupy complementary positions in the duplex acceptor DNA sequence;
and wherein the alteration of the duplex acceptor DNA sequence is within a cell.

3 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/991,065Filed 25 Nov 2011 · us provisional application
Priority: 2 Dec 2010
Published: US 2013/0330774 A112 Dec 2013
Granted: 13 Dec 2016
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12N 15/1024
Mutagenizing nucleic acids: in vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an…
C12N 15/102
Processes for the isolation, preparation or purification of DNA or RNA: mutagenizing nucleic acids
C12N 15/1082
Isolating an individual clone by screening libraries: preparation or screening gene libraries by chromosomal integration of polynucleotide…
C12N
Microorganisms or enzymes

Family

Application61/419,183Us provisional application · filed 2 Dec 2010
US 2013/0330774 A1Pre-grant publication · published 12 Dec 2013
US 9,518,258 B2This patent

From the US filing history. Foreign family members aren’t listed.

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