Cleavage of nucleic acid acid using thermostable methoanococcus jannaschii FEN-1 endonucleases
First Claim
1. A mixture comprising a first structure-specific nuclease, said first nuclease consisting of the Mja FEN-1 endonuclease and a second structure-specific nuclease, wherein said second nuclease is a 5'"'"' nuclease derived from a thermostable DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same 5'"'"' nuclease activity of the wild-type DNA polymerase.
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Abstract
The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Structure-specific nucleases, including 5'"'"' nucleases, thermostable FEN-1 endonucleases and 3'"'"' exonucleases, are used to detect and identify target nucleic acids. Methods are provided which allow for the detection specific nucleic acid sequences; these methods permit the detection and identification of mutant and wild-type forms of genes (e.g., human genes) as well as permit the detection and identification of bacterial and viral pathogens in a sample.
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Citations
26 Claims
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1. A mixture comprising a first structure-specific nuclease, said first nuclease consisting of the Mja FEN-1 endonuclease and a second structure-specific nuclease, wherein said second nuclease is a 5'"'"' nuclease derived from a thermostable DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same 5'"'"' nuclease activity of the wild-type DNA polymerase.
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2. A mixture comprising a first structure-specific nuclease, said first nuclease consisting of the Mja FEN-1 endonuclease and a second structure-specific nuclease, wherein said second nuclease is selected from the group consisting of the Cleavase®
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3. A method for treating nucleic acid, comprising:
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a) providing; i) the Mja FEN-1 endonuclease; and ii) a nucleic acid substrate; b) treating said nucleic acid substrate under conditions such that said substrate forms one or more cleavage structures; and c) reacting said endonuclease with said cleavage structures so that one or more cleavage products are produced. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for treating nucleic acid, comprising:
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a) providing; i) a first structure-specific nuclease consisting of the Mja FEN-1 endonuclease in a solution containing manganese; and ii) a nucleic acid substrate; b) treating said nucleic acid substrate with increased temperature such that said substrate is substantially single-stranded; c) reducing said temperature under conditions such that said single-stranded substrate forms one or more cleavage structures; d) reacting said cleavage means with said cleavage structures so that one or more cleavage products are produced; and e) detecting said one or more cleavage products. - View Dependent Claims (16, 17, 18, 19, 20)
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21. A nucleic acid treatment kit, comprising:
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a) a composition comprising the Mja FEN-1 endonuclease; and b) a solution containing manganese. - View Dependent Claims (22, 23, 24, 25, 26)
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Specification