High fidelity restriction endonucleases

High fidelity restriction endonucleases

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer. The restriction endonuclease enzyme is NruI, and the mutations are selected from G75, Q99, G155, or P22/R90. The mutation may be G75A, Q99A, G155A; or P22A and R90A. Also claimed are methods of identifying the FI of the aforementioned NruI restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for NruI, permitting serially diluted NruI variants to cleave the DNA substrate, and determining the FI for the NruI variant.

High fidelity restriction endonucleases

Compositions are disclosed comprising a high fidelity HindIII restriction endonuclease enzyme having an artificially introduced amino acid mutation corresponding to position K198 or Q148 in HindIII (SEQ ID No. 2). The mutation may be K198A, Q148A, or Q148I. Also claimed are methods of identifying a fidelity index (FI) of the aforementioned HindIII restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for HindIII, permitting serially diluted HindIII variants to cleave the DNA substrate, and determining the FI for the HindIII variant.

Compositions,methods and related uses for cleaving modified DNA

Compositions, methods and a kit are described relating to a novel family of enzymes which preferentially bind to a hydroxymethylated cytosine or a glucosylated hydroxymethylated cytosine and cleave double- stranded DNA at a defined distance 3' of the recognition site to produce a cleavage fragment with a 1-3 base overhang.

High fidelity restriction endonucleases

High fidelity restriction endonucleases

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer. The restriction endonuclease enzyme is NsiI, and the mutation is at F376. The mutation may be F376A. Also claimed are methods of identifying the FI of the aforementioned NsiI restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for NsiI, permitting serially diluted NsiI variants to cleave the DNA substrate, and determining the FI for the NsiI variant.

High fidelity restriction endonucleases

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer. The restriction endonuclease enzyme is MluI, and the mutations are E112 and R132. The mutations may be E112A and R132A. Also claimed are methods of identifying the FI of the aforementioned MluI restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for MluI, permitting serially diluted MluI variants to cleave the DNA substrate, and determining the FI for the MluI variant.

High fidelity restriction endonucleases

High fidelity restriction endonucleases

High fidelity restriction endonucleases

Compositions are disclosed comprising a high fidelity SpeI restriction endonuclease enzyme having an artificially introduced amino acid mutation corresponding to position E59, P65, S108, N172, K174, Q179, G182, or Y55 in SpeI (SEQ ID No. 17). The SpeI mutation may be E59A, P65A, S108A, N172A, K174A, Q179A, G182A, or Y55F. Also claimed are methods of identifying a fidelity index (FI) of the aforementioned SpeI restriction enzyme variants, wherein the methods comprise selecting a reaction buffer and DNA substrate with a binding site for SpeI, permitting serially diluted SpeI variants to cleave the DNA substrate, and determining the FI for the SpeI variant.

High fidelity restriction endonucleases

High fidelity restriction endonucleases

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.