Rapid detection of mutations in the p53 gene
First Claim
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1. A method for treating nucleic acid comprising an oligonucleotide containing human p53 gene sequences, comprising:
- a) providing;
i) an enzymatic cleavage means; and
ii) a nucleic acid substrate containing human p53 gene sequences;
b) treating said nucleic acid substrate under conditions such that said substrate forms one or more cleavage structures, said cleavage structures formed by intra-strand hydrogen bonding in the absence of a primer oligonucleotide; and
c) reacting said cleavage means with said cleavage structures so that a plurality of cleavage products are produced.
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Abstract
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 screen for known and unknown mutations, including single base changes, in the human p53 gene. Methods are provided which allow for the identification of genetic mutations in the human p53 gene in a sample.
132 Citations
25 Claims
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1. A method for treating nucleic acid comprising an oligonucleotide containing human p53 gene sequences, comprising:
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a) providing; i) an enzymatic cleavage means; and ii) a nucleic acid substrate containing human p53 gene sequences; b) treating said nucleic acid substrate under conditions such that said substrate forms one or more cleavage structures, said cleavage structures formed by intra-strand hydrogen bonding in the absence of a primer oligonucleotide; and c) reacting said cleavage means with said cleavage structures so that a plurality of cleavage products are produced. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method, comprising:
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a) providing i) an enzymatic cleavage means in a solution containing manganese; and ii) a nucleic acid substrate containing human p53 gene sequences; 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, said cleavage structures formed by intra-strand hydrogen bonding in the absence of a primer oligonucleotide; d) reacting said cleavage means with said cleavage structures so that a plurality of cleavage products are produced; and e) detecting said plurality cleavage products. - View Dependent Claims (12, 13, 14, 15, 16)
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17. A method for treating nucleic acid, comprising:
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a) providing i) an enzymatic cleavage means in a solution containing manganese; and ii) a nucleic acid substrate containing human p53 gene sequences, said nucleic acid substrate comprising exons 5-8 of the human p53 gene; 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, said cleavage structures formed by intra-strand hydrogen bonding in the absence of a primer oligonucleotide; 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 (18, 19, 20, 21, 22)
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23. A method for detecting mutation in the human p53 gene, comprising:
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a) providing; i) an enzymatic cleavage means; ii) a test nucleic acid substrate containing human p53 gene sequences; and iii) control cleavage products produced by cleavage of a reference p53 gene sequence; b) treating said test nucleic acid substrate under conditions such that said test substrate forms one or more cleavage structures; c) reacting said cleavage means with said cleavage structures so that a plurality of test cleavage products are produced; and d) comparing said test cleavage products to said control cleavage products. - View Dependent Claims (24, 25)
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Specification