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US 6,440,673 B1Granted
Methods for identifying nucleic acid mutations using mismatch modification
AssigneeThe Ohio State University
InventorsChristopher Mark Weghorst, Altaf Ahmad Wani
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Abstract
The present invention provides methods for specifically detecting DNA mismatches between heteroduplex strands produced between wildtype and mutation containing nucleic acid species. Kits for performing the methods of the invention are also provided.
Claims32
A method for detecting a nucleotide sequence mismatch in a heteroduplex nucleic acid molecule, the method comprising the following steps:
- a) preparing a heteroduplex between a first nucleic acid fragment and a second, complementary nucleic acid fragment having a nucleotide sequence that forms at least one nucleotide sequence mismatch when annealed with the first nucleic acid fragment;
- b) treating the heteroduplex formed in subpart (a) with an exonuclease for a time and at an activity sufficient to digest single stranded DNA;
- c) chemically modifying the heteroduplex with a reagent that forms a covalent linkage to a mismatched nucleotide in the heteroduplex;
- and
- d) detecting the chemically modified mismatched nucleotide heteroduplex under conditions wherein chemical modification of a homoduplex is not detected.
2 more elements
A method for isolating a heteroduplex nucleic acid molecule comprising a nucleotide sequence mismatch, the method comprising the following steps:
- a) preparing a heteroduplex between a first nucleic acid fragment and a second, complementary nucleic acid fragment having a nucleotide sequence that forms at least one nucleotide sequence mismatch when annealed with the first nucleic acid fragment;
- b) treating the heteroduplex formed in subpart (a) with an exonuclease for a time and at an activity sufficient to digest single stranded DNA;
- c) chemically modifying the heteroduplex with a reagent that forms a covalent linkage to a mismatched nucleotide in the heteroduplex;
- and
- d) isolating the chemically modified mismatched nucleotide heteroduplex under conditions wherein chemical modification of a homoduplex is not isolated.
2 more elements
Dependencies are inferred from the claim text.