Complexity management of genomic DNA
First Claim
1. A method of reducing the complexity of a nucleic acid sample comprising:
- fragmenting the nucleic acid sample using a first and second restriction enzyme to produce fragments;
ligating adaptors to the fragments; and
selectively amplifying the fragments that were cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme.
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Accused Products
Abstract
The presently claimed invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample by providing non-gel based methods for amplification of a subset of the sequences in a sample. In a preferred embodiment, amplification of a subset can be accomplished by digesting a sample with two or more restriction enzymes and ligating adaptors to the fragments so that only a subset of the fragments can be amplified. The invention further provides for analysis of the above amplified sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism.
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Citations
34 Claims
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1. A method of reducing the complexity of a nucleic acid sample comprising:
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fragmenting the nucleic acid sample using a first and second restriction enzyme to produce fragments;
ligating adaptors to the fragments; and
selectively amplifying the fragments that were cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method for analyzing a nucleic acid sample comprising:
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fragmenting the nucleic acid sample using a first and second restriction enzyme to produce fragments;
ligating adaptors to the fragments;
selectively amplifying the fragments that were cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme;
providing a nucleic acid array;
hybridizing the amplified fragments to the array; and
analyzing a hybridization pattern resulting from the hybridization. - View Dependent Claims (21, 22, 23, 24)
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25. A method of screening for DNA sequence variations in an individual comprising:
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providing a nucleic acid sample from the individual;
fragmenting the nucleic acid sample using a first and second restriction enzyme to produce fragments;
ligating adaptors to the fragments; and
selectively amplifying the fragments that were cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme;
providing a nucleic acid array;
hybridizing the amplified fragments to the array;
generating a hybridization pattern resulting from the hybridization; and
determining the presence or absence of DNA sequence variations in the individual based upon an analysis of the hybridization pattern. - View Dependent Claims (26, 27, 28, 31, 32)
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29. A method of screening for DNA sequence variation in a population of individuals comprising:
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providing a first nucleic acid sample from each of the individuals;
providing a second nucleic acid sample by;
fragmenting the first nucleic acid sample using a first and second restriction enzyme to produce fragments;
ligating adaptors to the fragments; and
selectively amplifying the fragments that were cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme;
providing a plurality of identical nucleic acid arrays wherein the arrays comprise probes designed to interrogate for DNA sequence variations;
hybridizing each of the second nucleic acid samples to one of the plurality of identical arrays;
generating a plurality of hybridization patterns resulting from the hybridizations; and
analyzing the hybridization patterns to determine the presence or absence of sequence variation in the population of individuals. - View Dependent Claims (30)
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33. A method of genotyping an individual comprising:
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providing a first nucleic acid sample from the individual;
providing a second nucleic acid sample by;
fragmenting the first nucleic acid sample using a first and second restriction enzyme to produce fragments;
ligating adaptors to the fragments; and
selectively amplifying the fragments that were cut on one end by the first restriction enzyme and on the other end by the second restriction enzyme;
hybridizing the second nucleic acid sample to an array designed to determine the presence or absence of one or more alleles of a collection of SNPs;
generating a hybridization pattern resulting from the hybridization; and
determining the presence or absence of the one or more alleles of the collection of SNPs.
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34. A kit for reducing the complexity of a nucleic acid sample comprising:
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buffers and restriction enzymes for fragmenting a nucleic acid sample, a ligase and adaptors to be ligated to the fragments, the adaptors being designed for the selective amplification of the fragments that were cut on one end by a first restriction enzyme and on the other end by a second restriction enzyme, primers for the selective amplification, and instructions for the use of the kit.
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Specification