Flap Endonuclease Mutants

• US 20080118922A1
• Filed: 01/21/2005
• Published: 05/22/2008
• Est. Priority Date: 07/23/2004
• Status: Active Grant

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Original Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Inventors: Matsui, Eriko, Matsui, Ikuo
• Granted Patent: US 7,846,704 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

A mutation is introduced into the substrate-binding site of flap endonuclease to prepare a mutant with modified substrate specificity. Using the mutant as a reagent for the analysis of genetic polymorphism, the analysis of genetic polymorphism can be performed more accurately, easily and sensitively as compared with conventional methods.

Claims

1. A flap endonuclease mutant wherein an amino acid in the amino acid sequence of the wild type flap endonuclease is substituted by other amino acid, wherein said mutant acts, among substrates for the wild type flap endonuclease, on the DNA substrates where one strand of the double-stranded DNA has been cleaved, resulting in a 3′

projection structure, and where each of the cleaved DNA strand has a nucleotide sequence portion that is complementary to the other DNA strand, and the complementary nucleotide sequence portions are linked to form base pairs devoid of deletion sites in relation to the other DNA strand, and wherein said mutant does not act or has reduced activity on other substrates.

6. A reagent for the analysis of genetic polymorphism consisting of the flap endonuclease mutant according to claim 1.

2. A mutant of the flap endonuclease depicted in the amino acid sequence of SEQ ID No. 2 of the Sequence Listing, having a mutation selected from (A) the amino acid at position 33 is alanine or leucine, (B) the amino acid at position 35 is tyrosine, (C) the amino acid at position 79 is alanine or histidine, (D) the amino acids at positions 33 and 79 are both alanine, (E) the amino acids at positions 33 and 35 are both alanine, and (F) the amino acids at positions 278 and 279 are both alanine.

3. DNA encoding the mutant of flap endonuclease according to claim 2.

4. A recombinant vector comprising the DNA encoding the mutant of flap endonuclease according to claim 3.

5. A transformant transformed by the recombinant vector according to claim 4.

7. A reagent for the analysis of genetic polymorphism consisting of the flap endonuclease mutant according to claim 2.