# US 2014/0154676 A1 · High Fidelity Restriction Endonucleases

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- Number: US 2014/0154676 A1
- Document ID: US20140154676A1
- Status: Published application
- Kind: A1
- Application number: 14/137,583
- Filed: 2013-12-20
- Published: 2014-06-05
- Earliest claimed: 2007-07-12
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: Zhenyu Zhu; Aine Quimby; Shengxi Guan; Dapeng Sun; Yishu Huang; Xuhui Lai; Siu-hong Chan; Xianghui Li; Shuang-Yong Xu; Chunhua Zhang
- Web page: https://patentgrep.com/patent/US20140154676A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/16: acting on ester bonds (3.1)
- C12Q 1/34: involving hydrolase
- C12Q 1/44: involving esterase
- C12Q 1/68: involving nucleic acids
- C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4)
- G01N 2333/916: acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N 2333/922: Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim at position 1

1-74. (canceled)

### Claim 75

A method of identifying a fidelity index (FI) of a restriction endonuclease and one or more variants thereof;
comprising:
selecting a reaction buffer and a DNA substrate containing the binding and cleavage site of the restriction endonuclease;
permitting serially dilutions of the restriction endonuclease and the one or more variants thereof to cleave the DNA substrate; and
determining an FI for each of the restriction endonuclease and the one or more variants thereof.

### Claim 76 (depends on 75)

A method according to claim 75, further comprising: comparing the FI for the restriction endonuclease to the F1 for a variant thereof to obtain an improvement factor for the variant.

### Claim 77 (depends on 76)

A method according to claim 76, wherein the improvement factor is greater than 2.

### Claim 78 (depends on 75)

A method according to claim 75, wherein the reaction buffer comprises potassium acetate, Tris acetate and magnesium acetate; or magnesium chloride.