Mutant endonuclease V enzymes and applications thereof
View Patent ↗Provided herein are mutant endonuclease V enzymes that are capable of nicking an inosine-containing DNA sequence. Nucleic acid assays and agents that employ such mutant endonuclease V enzymes to introduce a nick into a target DNA including one or more inosine, and uses a DNA polymerase to generate amplicons of a target DNA are also described.
1. A mutant endonuclease V comprising amino acid sequence of SEQ ID NO: 1, wherein its Tyrosine 75 residue is replaced with Alanine.
2. The mutant endonuclease of claim 1 , wherein the mutant endonuclease is a thermo labile endonuclease.
3. The mutant endonuclease of claim 1 , wherein the mutant endonuclease nicks an inosine-containing strand of a double stranded DNA that contains an inosine residue base-paired with a cytosine residue at a position 3′ to the inosine residue.
4. The mutant endonuclease of claim 3 , wherein the mutant endonuclease has a higher nicking efficiency than a wild type endonuclease V of SEQ ID NO: 1.
5. A mutant Escherichia coli endonuclease V comprising amino acid sequence of SEQ ID NO: 2.
6. The mutant endonuclease of claim 5 , wherein the mutant endonuclease preferentially nicks an inosine-containing strand of a double stranded DNA that contains an inosine residue base-paired with a cytosine residue at a position 3′ to the inosine residue.
7. The mutant endonuclease of claim 5 , wherein the mutant endonuclease nicks a double stranded DNA at the location of a base pair mismatch or at one or more bases 3′ to the base pair mismatch.
8. A mutant Archaeoglobus fulgidus endonuclease V comprising amino acid sequence of SEQ ID NO: 3.