Methods for reducing complexity of nucleic acid samples
First Claim
1. A method of analyzing a subset of nucleic acids within a nucleic acid population, comprising:
- (a) providing a population of single-stranded nucleic acid fragments wherein at least some of said fragments have sequences that are repeated;
(b) incubating said population of nucleic acid fragments under conditions to produce a double-stranded subset of said population of nucleic acid fragments and a single-stranded subset of said population of nucleic acid fragments, wherein under said incubating conditions nucleic acid fragments of said population having repeat sequences preferentially anneal with each other relative to nucleic acid fragments of said population lacking repeat sequences;
(c) separating said single-stranded subset from said double-stranded subset;
(d) hybridizing said separated single-stranded subset to probes on a microarray; and
(e) determining which of said probes on said array hybridize to said single-stranded subset, thereby analyzing said single-stranded subset of said population of nucleic acid fragments.
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Abstract
The invention provides several methods for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids on a nucleic acid probe array. The methods result in a subset of the initial population enriched for a desired property, or lacking nucleic acids having an undesired property. The resulting nucleic acids in the subset are then applied to the array for various types of analysis. The methods are particularly useful for analyzing populations having a high degree of complexity, for example, chromosomal-derived DNA, or whole genomic DNA, or mRNA population. In addition, such methods allow for analysis of pooled samples.
59 Citations
45 Claims
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1. A method of analyzing a subset of nucleic acids within a nucleic acid population, comprising:
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(a) providing a population of single-stranded nucleic acid fragments wherein at least some of said fragments have sequences that are repeated;
(b) incubating said population of nucleic acid fragments under conditions to produce a double-stranded subset of said population of nucleic acid fragments and a single-stranded subset of said population of nucleic acid fragments, wherein under said incubating conditions nucleic acid fragments of said population having repeat sequences preferentially anneal with each other relative to nucleic acid fragments of said population lacking repeat sequences;
(c) separating said single-stranded subset from said double-stranded subset;
(d) hybridizing said separated single-stranded subset to probes on a microarray; and
(e) determining which of said probes on said array hybridize to said single-stranded subset, thereby analyzing said single-stranded subset of said population of nucleic acid fragments. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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15. A method of analyzing a subset of nucleic acids within a nucleic acid population, comprising:
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(a) providing a single-stranded driver population of nucleic acids and a single-stranded tester population of nucleic acids;
(b) annealing said driver population of nucleic acids to said tester population of nucleic acids;
(c) immobilizing said driver population of nucleic acids;
(d) separating said unimmobilized subset of nucleic acids from said immobilized nucleic acids;
(e) hybridizing said unimmobilized subset of nucleic acids to probes on a microarray; and
(f) determining which of said probes on said microarray hybridize to said unimmobilized subset of nucleic acids, thereby analyzing said unimmobilized subset of nucleic acids.
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25. A method of analyzing a subset of nucleic acids within a nucleic acid population, comprising:
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(a) providing a single-stranded driver population of nucleic acids and a single-stranded tester population of nucleic acids;
(b) annealing said driver population of nucleic acids to said tester population of nucleic acids;
(c) immobilizing said driver population of nucleic acids;
(d) separating said unimmobilized nucleic acids from said immobilized nucleic acids;
(e) dissociating tester nucleic acids annealed to immobilized driver nucleic acids to produce a subset of complementary tester nucleic acids;
(f) separating said subset of complementary tester nucleic acids from said immobilized driver nucleic acids;
(g) hybridizing said subset of complementary tester nucleic acids to probes on a microarray;
(h) determining which of said probes on said microarray hybridize to said subset of complementary tester nucleic acids, thereby analyzing said subset of complementary tester nucleic acids.
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Specification