Cleavage of nucleic acids
View Patent ↗The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Enzymes, including 5′ nucleases and 3′ exonucleases, are used to detect and identify nucleic acids derived from microorganisms. Methods are provided which allow for the detection and identification of bacterial and viral pathogens in a sample.
1. A composition comprising an isolated modified thermostable eubacterial DNA polymerase enzyme having 5′ nuclease activity but lacking polymerase activity, and a nucleic acid molecule.
2. The composition of claim 1 , further comprising a target nucleic acid, wherein at least a portion of said nucleic acid molecule is complementary to at least a portion of said target nucleic acid.
3. The composition of claim 2 , wherein said modified thermostable eubacterial DNA polymerase enzyme and said nucleic acid molecule and said target nucleic acid are contained in a reaction mixture.
4. The composition of claim 1 , further comprising a buffer comprising magnesium.
5. The composition of claim 1 , wherein said nucleic acid molecule comprises a label.
6. The composition of claim 5 , wherein said label comprises a fluorescent label.
7. A composition comprising an isolated modified thermostable eubacterial DNA polymerase enzyme having 5′ nuclease activity but lacking polymerase activity, a first nucleic acid molecule, a second nucleic acid molecule, and a target nucleic acid, wherein said target nucleic acid comprises a first portion downstream of a second portion, wherein said first nucleic acid molecule comprises a region that is complementary to said second portion of said target nucleic acid, and wherein said second nucleic acid molecule comprises a region that is complementary said first portion of said target nucleic acid.
8. The composition of claim 7 , wherein said first and said second portions of said target nucleic acid are adjacent to one another.
9. The composition of claim 8 , wherein said first nucleic acid molecule comprises a label.
10. The composition of claim 9 , wherein said label comprises a fluorescent label.
11. The composition of claim 1 or claim 7 , wherein said DNA polymerase is from a eubacterium of genus Thermus.
12. The composition of claim 11 , wherein said eubacterium is selected from the group consisting of Thermus aquaticus, Thermus flavus , and Thermus thermophilus.