Chip-based species identification and phenotypic characterization of microorganisms
First Claim
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1. A method for assigning a first organism to a particular group or species, comprising:
- (a) providing an array of oligonucleotides at known locations on a substrate, said array comprising probes complementary to reference DNA or RNA sequences from a second organism;
(b) obtaining a first hybridization pattern by hybridizing a target nucleic acid sequence from the first organism to the array; and
(c) comparing the first hybridization pattern of the first organism to a second hybridization pattern obtained by hybridizing a target nucleic acid sequence from the second organism to the array, wherein differences between the first and second hybridization patterns are indicative that the first organism is assigned to a particular group or species.
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Abstract
This invention provides oligonucleotide based arrays and methods for speciating and phenotyping organisms, for example, using oligonucleotide sequences based on the Mycobacterium tuberculosis rpoB gene. The groups or species to which an organism belongs may be determined by comparing hybridization patterns of target nucleic acid from the organism to hybridization patterns in a database.
170 Citations
97 Claims
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1. A method for assigning a first organism to a particular group or species, comprising:
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(a) providing an array of oligonucleotides at known locations on a substrate, said array comprising probes complementary to reference DNA or RNA sequences from a second organism;
(b) obtaining a first hybridization pattern by hybridizing a target nucleic acid sequence from the first organism to the array; and
(c) comparing the first hybridization pattern of the first organism to a second hybridization pattern obtained by hybridizing a target nucleic acid sequence from the second organism to the array, wherein differences between the first and second hybridization patterns are indicative that the first organism is assigned to a particular group or species. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 75, 77, 78, 79, 80, 87, 88)
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14. A method of identifying the presence of a nucleic acid polymorphism in a patient sample, comprising the steps of:
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(a) determining the difference between the hybridization intensities of a nucleic acid sequence from the patient sample and a corresponding nucleic acid sequence from a wild type sample to an array of reference nucleic acid probes;
(b) deriving ratios of the difference in (a) to the hybridization intensity of the wild type sample for each base position corresponding to each reference nucleic acid probe; and
(c) identifying the presence of a polymorphism at a base position corresponding to a reference probe if the ratio in (b) for the base position corresponding to the reference probe is greater than or equal to an assigned value. - View Dependent Claims (15, 16, 17, 40, 41, 59)
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18. A computerized method of assigning an organism to a group comprising the steps of:
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inputting groups of a plurality of known nucleic acid sequences, the plurality of known nucleic acid sequences being from known organisms;
inputting hybridization patterns for the plurality of known nucleic acid sequences, each hybridization pattern indicating hybridization of subsequences of the known nucleic acid sequence to subsequences of a reference nucleic acid sequence;
inputting a hybridization pattern for a sample nucleic acid sequence from the organism indicating hybridization of subsequences of the sample nucleic acid sequence to subsequences of the reference nucleic acid sequence;
comparing the hybridization pattern for the sample nucleic acid sequence to the hybridization patterns for the plurality of known nucleic acid sequences; and
assigning a particular group to which the organism belongs according to the group of at least one of the known nucleic acid sequences that has a hybridization pattern that most closely matches the hybridization pattern of the sample nucleic acid sequence at specific locations. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A computer program product that assigns an organism to a group, comprising:
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computer code that receives as input groups of a plurality of known nucleic acid sequences, the plurality of known nucleic acid sequences being from known organisms;
computer code that receives as input hybridization patterns for the plurality of known nucleic acid sequences, each hybridization pattern indicating hybridization of subsequences of the known nucleic acid sequence to subsequences of a reference nucleic acid sequence;
computer code that receives as input a hybridization pattern for a sample nucleic acid sequence from the organism indicating hybridization of subsequences of the sample nucleic acid sequence to subsequences of the reference nucleic acid sequence;
computer code that compares the hybridization pattern for the sample nucleic acid sequence to the hybridization patterns for the plurality of known nucleic acid sequences;
computer code that assigns a particular group to which the organism belongs according to the groups of at least one of the known nucleic acid sequences that has a hybridization pattern that most closely matches the hybridization pattern of the sample nucleic acid sequence at specific locations; and
a computer readable medium that stores the computer codes.
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32. A computerized method of assigning groups to which organisms belong utilizing a generic probe array, comprising the steps of:
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inputting hybridization intensities for a plurality of isolates, the hybridization intensities indicating hybridization affinity between the isolate and the generic probe array;
selecting hybridization intensities that have the most variance across the plurality of isolates; and
assigning each of the plurality of isolates to a group according to the selected hybridization intensities.- View Dependent Claims (33, 34, 35, 36, 37, 38)
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39. A computer program product that assigns groups to which organisms belong utilizing a generic probe array, comprising:
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computer code that receives as input hybridization intensities for a plurality of isolates, the hybridization intensities indicating hybridization affinity between the isolate and the generic probe array;
computer code that selects hybridization intensities that have the most variance across the plurality of isolates;
computer code that assigns a group to each of the plurality of isolates according to the selected hybridization intensities; and
a computer readable medium that stores the computer codes.
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42. A method of assigning a probability that at least a subset of a sample hybridization pattern is associated with one or more species, comprising:
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providing a first array of polynucleotides at known locations on a substrate;
hybridizing a first nucleic acid to said first array to obtain a first hybridization pattern;
providing a second array of polynucleotides at known locations on a substrate, wherein at least a subset of said polynucleotides of said second array comprises a subset of said polynucleotides of said first array;
hybridizing a second nucleic acid to said second array to obtain a second hybridization pattern;
comparing at least a portion of said first and second hybridization patterns and assigning a probability, based on said comparing, that the appearance of said portion in a sample hybridization pattern is associated with one or more species. - View Dependent Claims (43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
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60. A computer program product that identifies the presence of a nucleic acid polymorphism in a patient sample, comprising:
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computer code that determines the difference between the hybridization intensities of a nucleic acid sequence from the patient sample and a corresponding nucleic acid sequence from a wild type sample to an array of reference nucleic acid probes;
computer code that derives ratios of the difference to the hybridization intensity of the wild type sample for each base position corresponding to each reference nucleic acid probe;
computer code that identifies the presence of a polymorphism at a base position corresponding to a reference probe if the ratio for the base position corresponding to the reference probe is greater than or equal to an assigned value; and
a computer readable medium that stores the computer codes.
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61. A method for assigning a first organism to a particular group or species, comprising:
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(a) providing an array of oligonucleotides at known locations on a substrate, said array comprising probes complementary to reference DNA or RNA sequences from a second organism;
(b) obtaining a first hybridization pattern by hybridizing a target nucleic acid sequence from the first organism to the array;
(c) comparing the first hybridization pattern of the first organism to a second hybridization pattern obtained by hybridizing a target nucleic acid sequence from the second organism to the array;
(d) deriving one or more sets of differences between the first and second hybridization patterns;
(e) comparing the set of differences to a data base comprising sets of differences correlated with speciation of organisms to assign the first organism to a particular group or species. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 81, 82, 83, 84)
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85. A method for assigning phenotypic information to first organism group or species, comprising:
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(a) providing an array of oligonucleotides at known locations on a substrate, said array comprising probes complementary to reference DNA or RNA sequences from a second organism;
(b) obtaining a first hybridization pattern by hybridizing a target nucleic acid sequence from the first organism to the array; and
(c) comparing the first hybridization pattern of the first organism to a second hybridization pattern obtained by hybridizing a target nucleic acid sequence from the second organism to the array, wherein differences between the first and second hybridization patterns are indicative of phenotypic information assigned to the first organism. - View Dependent Claims (86)
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89. A computerized method of assigning an organism to a group, comprising the steps of:
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inputting hybridization patterns for a plurality of organisms, each of the organisms belonging to a known group, each hybridization pattern indicating hybridization of subsequences of the organism belonging to a known group to subsequences of a reference nucleic acid sequence;
inputting a hybridization pattern for a sample nucleic acid sequence from an organism not assigned to a group indicating hybridization of subsequences of the sample nucleic acid sequence to subsequences of the reference nucleic acid sequence;
comparing the hybridization pattern for the sample nucleic acid sequence to the hybridization patterns for the plurality of organisms, each of the organisms belonging to known group; and
assigning a particular group to which the organism belongs according to the group of at least one of the organisms that has a hybridization pattern that most closely matches the hybridization pattern of the sample nucleic acid sequence at specific locations. - View Dependent Claims (90, 91, 92, 93, 94, 95, 96)
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97. A computer program product that assigns an organism to a group, comprising:
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computer code that receives as input hybridization patterns for a plurality of organisms, each of the organisms belonging to a known group, each hybridization pattern indicating hybridization of subsequences of the organism belonging to a known group to subsequences of a reference nucleic acid sequence;
computer code that receives as input a hybridization pattern for a sample nucleic acid sequence from an organism not assigned to a group indicating hybridization of subsequences of the sample nucleic acid sequence to subsequences of the reference nucleic acid sequence;
computer code that compares the hybridization pattern for the sample nucleic acid sequence to the hybridization patterns for the plurality of organisms, each organism belonging to a known group;
computer code that assigns a particular group to which the organism belongs according to the group of at least one of the organisms that has a hybridization pattern that most closely matches the hybridization pattern of the sample nucleic acid sequence at specific locations; and
a computer readable medium that stores the computer codes.
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Specification