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 nucleic acid assay, comprising:
providing a double stranded DNA that contains an inosine residue base-paired with a cytosine residue;
nicking the inosine-containing strand of the double stranded DNA at a residue 3′ to the inosine residue using one or more of a mutant endonuclease V comprising an amino acid sequence of SEQ ID NO: 1, wherein its Tyrosine 75 residue is replaced with Alanine, a mutant endonuclease V comprising an amino acid sequence of SEQ ID NO: 2, or a mutant Archaeoglobus fulgidus endonuclease V comprising an amino acid sequence of SEQ ID NO: 3; and
performing the nucleic acid assay.
2. The nucleic acid assay of claim 1 , wherein the double stranded DNA is generated in situ by a primer-mediated replication of a target DNA.
3. The nucleic acid assay of claim 2 , comprising extending the nicked inosine-containing strand via a nucleic acid amplification reaction using a strand displacement DNA polymerase.
4. The nucleic acid assay of claim 3 , wherein the nucleic acid amplification is an isothermal amplification.
5. A nucleic acid assay comprising:
providing a target DNA;
providing a DNA polymerase;
providing a mutant endonuclease V comprising an amino acid sequence of SEQ ID NO: 2;
generating a double stranded DNA from the target DNA, wherein the double stranded DNA comprises an inosine residue base-paired with a cytosine residue;
nicking the inosine-containing strand of the double stranded employing the mutant endonuclease V to generate a nicked DNA; and
performing a DNA polymerase reaction on the nicked DNA employing the DNA polymerase.
6. A nucleic acid assay comprising:
providing a target DNA;
providing a DNA polymerase;
providing a mutant endonuclease V comprising an amino acid sequence of SEQ ID NO: 1, wherein its Tyrosine 75 residue is replaced with Alanine;
generating a double stranded DNA from the target DNA, wherein the double stranded DNA comprises an inosine residue base-paired with a cytosine residue;
nicking the inosine-containing strand of the double stranded employing the mutant endonuclease V to generate a nicked DNA; and
performing a DNA polymerase reaction on the nicked DNA employing the DNA polymerase.
7. A nucleic acid assay comprising:
providing a target DNA;
providing a DNA polymerase;
providing a mutant Archaeoglobus fulgidus endonuclease V comprising an amino acid sequence of SEQ ID NO: 3;
generating a double stranded DNA from the target DNA, wherein the double stranded DNA comprises an inosine residue base-paired with a cytosine residue;
nicking the inosine-containing strand of the double stranded employing the mutant endonuclease V to generate a nicked DNA; and
performing a DNA polymerase reaction on the nicked DNA employing the DNA polymerase.