Methods for stool sample preparation
First Claim
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1. A method for analyzing DNA extracted from stool, comprising:
- homogenizing a stool sample in a solvent for DNA in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;
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enriching said homogenized sample for human DNA; and
analyzing said human DNA for characteristics of disease.
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Abstract
The present invention provides methods for the preparation of stool samples to increase the yield of relevant DNA, and further provides methods for isolating and analyzing target DNA for characteristics indicative of colorectal cancer. Methods for screening patients for the presence of cancerous or pre-cancerous colorectal lesions are also provided.
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Citations
48 Claims
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1. A method for analyzing DNA extracted from stool, comprising:
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homogenizing a stool sample in a solvent for DNA in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;
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enriching said homogenized sample for human DNA; and
analyzing said human DNA for characteristics of disease.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 36, 37, 44, 45, 46, 47, 48)
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9. A method of screening for the presence of a colorectal cancerous or pre-cancerous lesion in a patient, the method comprising the steps of:
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obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;
homogenizing the sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;
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enriching said sample for a target human DNA; and
analyzing the target human DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion.- View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 38, 39)
a) detecting an amount of a maternal allele at a polymorphic locus in the sample;
b) detecting an amount of a paternal allele at the polymorphic locus in the sample; and
c) determining whether a difference exists between the amounts of maternal and paternal allele, the presence of a statistically-significant difference being indicative of a deletion at the polymorphic locus in a subpopulation of cells in the sample and the potential presence of a lesion.
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18. The method of claim 17 wherein said polymorphic locus is a single base polymorphism and is heterozygous between said maternal and paternal alleles.
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19. The method of claim 17 wherein said detecting steps comprise,
a) hybridizing probe to a portion of said polymorphic locus on both maternal and paternal alleles that is immediately adjacent to said single-base polymorphism; -
b) exposing said sample to a mixture of detectably-labeled dideoxy nucleoside triphosphates under conditions which allow appropriate binding of said dideoxy nucleoside triphosphates to said single-base polymorphism;
c) washing the sample; and
d) counting an amount of each detectably-labeled dideoxy nucleoside triphosphate remaining for the sample.
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20. The method of claim 19 wherein said detectable label is selected from the group consisting of radioisotopes, fluorescent compounds, and particles.
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21. The method of claim 9 wherein said analyzing step comprises a method for detecting heterozygosity at a single-nucleotide polymorphic locus, 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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22. The method of claim 9 wherein said analyzing step comprises:
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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.
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23. The method of claim 22 wherein said first and second oligonucleotide probes each are coupled to a distinct detectable label.
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24. The method of claim 22 wherein
said first oligonucleotide probes are attached to a first particle in a ratio of one first oligonucleotide probe to one particle and said second oligonucleotide probes are attached to a second particle detectably distinct from said first particle in a ratio of one second oligonucleotide probe to one second particle, wherein said detecting step comprises separating hybridized from unhybridized first and second oligonucleotide probes and subsequently passing hybridized first and second oligonucleotide probes through a detector to determine said first and second numbers. -
25. The method of claim 24 wherein said first and second particles are of detectably different sizes.
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26. The method of claim 24 wherein said first and second particles are of detectably different colors.
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27. The method of claim 22 further comprising, prior to step a) the steps of converting double-stranded DNA in said sample to single-stranded DNA and removing complement to said first and second polynucleotide segments.
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28. The method of claim 27 wherein said removing step comprises hybridizing said complement to a nucleic acid probe attached to a magnetic particle and subsequently removing said magnetic particle from the sample.
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29. The method of claim 9 wherein said analyzing step comprises a method for detecting a nucleic acid sequence change in a target allele in the sample, comprising the steps of:
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(a) determining (i) an amount of wild-type target allele in the sample, and (ii) an amount of a reference allele in the sample; and
(b) detecting a nucleic acid sequence change in the target allele in the sample, a statistically significant difference in the amount wild-type target allele and the amount of reference allele obtained in said determining step being indicative of a nucleic acid sequence change.
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30. The method according to claim 29 wherein said determining step comprises exposing said sample to a first oligonucleotide probe capable of hybridizing with a portion of said wild-type allele and to a second oligonucleotide probe capable of hybridizing to a portion of said reference allele, and removing from said sample any unhybridized first or second oligonucleotide probe.
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38. The method of claim 9 wherein the solvent comprises between about 10 mM EDTA and about 20 mM EDTA.
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39. The method of claim 9 wherein the solvent comprises about 16 mM EDTA.
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31. A method for screening for the presence of a colorectal cancerous or precancerous lesion in a patient, the method comprising the steps of:
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obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;
homogenizing the sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;
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determining if said sample has at least a minimum number N of total DNA molecules to provide for detection of a low-frequency target DNA molecule;
analyzing the target DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion. - View Dependent Claims (33, 40, 41)
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32. A method for screening for the presence of a colorectal cancerous or precancerous lesion in a patient, the method comprising the steps of:
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obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;
homogenizing the sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5;
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enriching said homogenized sample for target human DNA;
determining if said sample has at least a minimum number N of total DNA molecules to provide for detection of a low-frequency target DNA molecule;
analyzing the target human DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion. - View Dependent Claims (34, 35, 42, 43)
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Specification