Methods for the detection of loss of heterozygosity
First Claim
Patent Images
1. A method for detecting the presence of a mutant DNA in a pooled biological sample, comprising the steps of:
- a) pooling biological samples collected from a plurality of members of a patient population;
b) determining from said pooled samples a number X of a first wild-type polynucleotide characteristic of a genomic region of said members of said patient population that is not mutated in said subpopulation of transformed cells;
c) determining from said pooled biological sample a number Y of a second wild-type polynucleotide in a genomic region of one or more members of said patient population suspected of being mutated in said subpopulation of transformed cells; and
d) determining whether a difference exists between number X and Y, the presence of a statistically-significant difference being indicative of a clonal subpopulation of transformed cells in said pooled biological sample.
4 Assignments
0 Petitions
Accused Products
Abstract
Methods are provided for detecting loss of heterozygosity in a pooled nucleic acid sample obtained from a patient population. These methods are particularly useful for identifying populations or individuals within a population with gene mutations indicative of early colorectal cancer.
164 Citations
38 Claims
-
1. A method for detecting the presence of a mutant DNA in a pooled biological sample, comprising the steps of:
-
a) pooling biological samples collected from a plurality of members of a patient population; b) determining from said pooled samples a number X of a first wild-type polynucleotide characteristic of a genomic region of said members of said patient population that is not mutated in said subpopulation of transformed cells; c) determining from said pooled biological sample a number Y of a second wild-type polynucleotide in a genomic region of one or more members of said patient population suspected of being mutated in said subpopulation of transformed cells; and d) determining whether a difference exists between number X and Y, the presence of a statistically-significant difference being indicative of a clonal subpopulation of transformed cells in said pooled biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
-
-
11. A method for detecting the presence of a colorectal cancer or precancerous lesion in pooled mammalian tissue or body fluid samples obtained from a patient population, comprising the steps of:
-
a) pooling said tissue or said body fluid samples collected from a plurality of members of a patient population; b) exposing said pooled biological 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 said members of said population, 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; c) 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 d) 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 statistically-significant difference being indicative of the presence in said pooled biological sample of a colorectal cancer or precancerous lesion. - View Dependent Claims (12, 13, 14, 15, 16, 17)
-
-
18. A method for detecting a nucleic acid sequence change in a target allele in a subpopulation of cells in a pooled biological sample compromising the steps of:
-
a) pooling biological samples collected from a plurality of members of a patient population, b) determining (i) an amount of wild-type target allele in said aliquot, and (ii) an amount of a reference allele in said aliquot; and c) detecting a nucleic acid sequence change in the target allele in a subpopulation of cells in said aliquot 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)
-
-
23. A method for detecting a change in the nucleotide sequence in a subpopulation of a target allele in a pooled heterogeneous sample of cellular material, comprising the steps of:
-
a) pooling biological samples collected from a plurality of members of a patient population; b) exposing said pooled 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; c) exposing the pooled 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; d) contacting said pooled 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; e) contacting said pooled samples 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 first isolation probe does not hybridize; f) removing non-hybridizing first and second hybridization probes from said pooled sample; g) determining an amount of each of said first and second hybridization probes remaining in said pooled sample after said removing step; and h) 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)
-
-
32. A method for detecting a deletion in polymorphic locus in a subpopulation of cells in a pooled biological sample, comprising the steps of:
-
a) pooling biological samples collected from a plurality of members of a patient population; b) detecting an amount of maternal allele at a polymorphic locus in said pooled sample; c) detecting an amount of a paternal allele at the polymorphic locus in said pooled sample; and d) 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 said pooled biological sample obtained from a patient population. - View Dependent Claims (33, 34, 35, 36, 37)
-
-
38. A method for detecting heterozygosity at a single-nucleotide polymorphic locus in a pooled biological sample, comprising the steps of:
-
a) pooling biological samples collected from a plurality of members of a patient population; b) hybridizing probes to a sequence immediately adjacent to a single-based polymorphism; c) exposing said pooled samples to a plurality of different labeled dideoxy nucleotides; d) washing said sample; g) 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.
-
Specification