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US 6,548,247 B1Granted
Detection and mapping of point mutations using partial digestion
AssigneeTrevigen, Inc.
InventorsJack G. Chirikjian, Leonard S. Bazar
Life of this patent
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Abstract
According to the present invention there is provided a method for detecting the presence of at least two point mutations in a target polynucleotide, as well as their relative positions and specific nucleotide positions, involving partial digestion and the use of mismatch repair enzymes.
Claims25
A method of detecting the presence of and determining the relative positions of at least two point mutations in a target polynucleotide, comprising:
- (a) hybridizing single-stranded oligonucleotide probes to a target polynucleotide to form hybrid, double-stranded polynucleotides such that mismatches occur at the sites of said point mutations, wherein said probes are complementary to a non-mutated sequence of said target polynucleotide and are labelled at one end but not both ends, and wherein said target polynucleotide is not labelled;
- (b) partially digesting the probe strand of said hybrid polynucleotide with a nucleic acid repair enzyme, wherein all possible recognition sites within the oligonucleotide probe are not cleaved and probe fragments of differing lengths are generated;
- (c) separating said probe fragments by size in a medium suitable for visualizing the separated probe fragments;
- and then
- (d) visualizing said separated probe fragments in said medium, whereby the presence and relative positions of said point mutations are determined.
2 more elements
A method of detecting the presence of and determining the relative positions of at least two point mutations in a target polynucleotide, comprising:
- (a) hybridizing a single-stranded oligonucleotide probe to a target polynucleotide to form a hybrid, double-stranded polynucleotide such that mismatches occur at the sites of said point mutations, wherein said probe is complementary to a non-mutated sequence of said target polynucleotide and is labelled at one end but not both ends, and wherein said target polynucleotide is not labelled;
- (b) partially digesting the probe strand of said hybrid polynucleotide with a nucleic acid repair enzyme producing oligonucleotide fragments, wherein said oligonucleotide probe is designed such that said oligonucleotide fragments dissociate from said target polynucleotide spontaneously at a predetermined temperature;
- (c) repeating steps (a) and (b) such that probe fragments of differing lengths are generated;
- (d) separating said probe fragments by size in a medium suitable for visualizing the separated probe fragments;
- and then
- (e) visualizing said separated probe fragments in said medium, whereby the presence and relative positions of said point mutations are determined.
3 more elements
Dependencies are inferred from the claim text.