Method of detecting a nucleotide change in nucleic acids
First Claim
1. An assay for a biologically derived denatured DNA or RNA test substance, which has a known normal nucleotide sequence and a known possible mutation at at least one target nucleotide position in said sequence, which assay determines whether the test substance has said normal nucleotide sequence or said possible mutation, said assay comprising the steps of(a) annealing a target oligonucleotide probe of predetermined sequence to a first sequence of said test substance so that said target nucleotide position is aligned with a nucleotide in an end region of said target probe,(b) annealing an adjacent oligonucleotide probe of predetermined sequence to a second sequence of said test substance contiguous to said first sequence, so that the terminal nucleotide in said end region of said target probe and one end of said adjacent probe are directly adjacent to each other,(c) contacting said annealed target probe and adjacent probe with a linking agent under conditions such that the directly adjacent ends of said probes covalently bond to form a linked probe product unless there is nucleotide base pair mismatching between said target probe and said test substance at the target nucleotide position,(d) separating said test substance and linked probe product, if formed, and(e) detecting whether or not said linked probe product is formed as an indication of nucleotide base pair matching or mismatching at said target nucleotide position.
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Accused Products
Abstract
Assay for determing the nucleic acid sequence in a region of a nucleic acid test substance having a known normal sequence and a known possible mutation at at least one target nucleotide position. Oligonucleotide probes are selected to anneal to immediately adjacent segments of a substantially complementary test DNA or RNA molecule. The target probe has an end region wherein one of the end region nucleotides is complementary to the normal or abnormal nucleotide at the corresponding target nucleotide position. A linking agent is added under conditions such that when the target nucleotide is correctly base paired, the probes are covalently joined and if not correctly base paired, the probes are incapable of being covalently joined under such conditions. The presence or absence of linking is detected as an indication of the sequence of the target nucleotide.
788 Citations
28 Claims
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1. An assay for a biologically derived denatured DNA or RNA test substance, which has a known normal nucleotide sequence and a known possible mutation at at least one target nucleotide position in said sequence, which assay determines whether the test substance has said normal nucleotide sequence or said possible mutation, said assay comprising the steps of
(a) annealing a target oligonucleotide probe of predetermined sequence to a first sequence of said test substance so that said target nucleotide position is aligned with a nucleotide in an end region of said target probe, (b) annealing an adjacent oligonucleotide probe of predetermined sequence to a second sequence of said test substance contiguous to said first sequence, so that the terminal nucleotide in said end region of said target probe and one end of said adjacent probe are directly adjacent to each other, (c) contacting said annealed target probe and adjacent probe with a linking agent under conditions such that the directly adjacent ends of said probes covalently bond to form a linked probe product unless there is nucleotide base pair mismatching between said target probe and said test substance at the target nucleotide position, (d) separating said test substance and linked probe product, if formed, and (e) detecting whether or not said linked probe product is formed as an indication of nucleotide base pair matching or mismatching at said target nucleotide position.
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20. A kit for assaying a biologically derived denatured DNA or RNA test substance which has a known normal nucleotide sequence and a known possible mutation at at least one target nucleotide position in said sequence, said kit being useful for determining whether the test substance has said known normal nucleotide sequence or said possible mutation, said kit comprising
(a) a first oligonucleotide probe capable of serving as a target probe of predetermined nucleotide sequence capable of annealing to a first sequence of said test substance, said target probe having at least one nucleotide position in an end region of said target probe which is complementary to the normal nucleotide or said possible mutation at said target nucleotide position, and (b) a second oligonucleotide probe capable of serving as an adjacent probe of predetermined sequence capable of annealing to a second sequence of said test substance contiguous to said first sequence such that the terminal nucleotide in said end region of said target probe and one end of said adjacent probe are directly adjacent to each other, said target or adjacent probes being labeled.
Specification