Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample
DCFirst Claim
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1. A method for detecting the presence of a clonal subpopulation of transformed cells in a biological sample obtained from an organism, comprising the steps of:
- a) determining from the biological sample a number X of a first wild-type polynucleotide characteristic of a genomic region of said organism that is not mutated in said subpopulation of transformed cells;
b) determining from the biological sample a number Y of a second wild-type polynucleotide in a genomic region of said organism suspected of being mutated in said subpopulation of transformed cells; and
c) determining whether a difference exists between number X and number Y,the presence of a statistically-significant difference being indicative of a clonal subpopulation of transformed cells in said biological sample.
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Abstract
Methods are provided for detecting the presence of mutant sequences in a subpopulation of gene sequences in a biological sample. These methods are particularly useful for identifying individuals with gene mutations indicative of early colorectal cancer.
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Citations
38 Claims
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1. A method for detecting the presence of a clonal subpopulation of transformed cells in a biological sample obtained from an organism, comprising the steps of:
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a) determining from the biological sample a number X of a first wild-type polynucleotide characteristic of a genomic region of said organism that is not mutated in said subpopulation of transformed cells; b) determining from the biological sample a number Y of a second wild-type polynucleotide in a genomic region of said organism suspected of being mutated in said subpopulation of transformed cells; and c) determining whether a difference exists between number X and number Y, the presence of a statistically-significant difference being indicative of a clonal subpopulation of transformed cells in said biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for detecting the presence of a colorectal cancer or precancerous lesion in a mammalian tissue or body fluid sample, comprising the steps of:
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(a) exposing the sample to a plurality of a first oligonucleotide probe and to a plurality of a second oligonucleotide probe under hybridization conditions, thereby to hybridize (1) said first oligonucleotide probes to copies of a first polynucleotide segment characteristic of wild-type cells of the organism, and (2) said second oligonucleotide probes to copies of a second polynucleotide segment characteristic of a wild-type genomic region suspected to be deleted or mutated in colorectal cancer cells; (b) detecting a first number of duplexes formed between said first probe and said first segment and a second number of duplexes formed between said second probe and said second segment; and (c) determining whether there is a difference between the number of duplexes formed between said first probe and said first segment and the number of duplexes formed between said second probe and said second segment, the presence of a statistically-significant difference being indicative of the presence in said sample of a colorectal cancer or precancerous lesion. - View Dependent Claims (12, 13, 14, 15, 16, 17)
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18. A method for detecting a nucleic acid sequence change in a target allele in a subpopulation of cells in a biological sample, comprising the steps of:
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(a) determining (i) an amount of wild-type target allele in the biological sample, and (ii) an amount of a reference allele in the biological sample; and (b) detecting a nucleic acid sequence change in the target allele in a subpopulation of cells in the biological sample as a statistically significant difference in the amount of wild-type target allele and the amount of reference allele obtained in said determining step. - View Dependent Claims (19, 20, 21, 22)
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23. A method for detecting a change in the nucleotide sequence in a subpopulation of a target allele in a heterogeneous sample of cellular material, comprising the steps of:
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a) exposing the heterogeneous sample, under hybridization conditions, to a plurality of isolation probes, each of which hybridizes to at least a portion of only one member selected from a first group consisting of a coding strand of said target allele and complement of a coding strand of said target allele; b) exposing the heterogeneous sample, under hybridization conditions, to a plurality of second isolation probes, each of which hybridizes to at least a portion of only one member selected from a second group consisting of a coding strand of a reference allele and a complement of a coding strand of said reference allele; c) contacting said heterogeneous sample, under hybridization conditions, with a plurality of first hybridization probes, each of which hybridizes to at least a portion of the member of said first group to which said first isolation probe does not hybridize; d) contacting the heterogeneous sample, under hybridization conditions, with a plurality of second hybridization probes, each of which hybridizes with at least a portion of the member of said second group to which said second isolation probe does not hybridize; e) removing non-hybridizing first and second hybridization probes from said heterogeneous sample; f) determining an amount of each of said first and second hybridization probes remaining in the heterogeneous sample after said removing step; and g) detecting allelic loss in a subpopulation of target allele as a statistically-significant difference in the amount of said first hybridization probe and said second hybridization probe obtained in said determining step. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31)
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32. A method for detecting a deletion in polymorphic locus in a subpopulation of cells in a biological sample, comprising the steps of:
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a) detecting an amount of a maternal allele at a polymorphic locus in the biological sample; b) detecting an amount of a paternal allele at the polymorphic locus in the biological sample; and c) determining whether a statistically-significant difference exists between the amount of maternal allele and the amount of paternal allele at the polymorphic locus, The presence of a statistically-significant difference being indicative of a deletion at the polymorphic locus in a subpopulation of cells in the biological sample. - View Dependent Claims (33, 34, 35, 36, 37)
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38. A method for detecting heterozygosity at a single-nucleotide polymorphic locus in a biological sample, comprising the steps of:
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a) hybridizing probes to a sequence immediately adjacent to a single-base polymorphism; b) exposing the sample to a plurality of different labeled dideoxy nucleotides c) washing the sample; d) determining which of said dideoxy nucleotides are incorporated into said probes; and e) detecting heterozygosity at the single-nucleotide polymorphic site as the detection of two dideoxy nucleotides having been incorporated into the probe.
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Specification