Complexity management of genomic DNA
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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 size fractionation. In a preferred embodiment, size fractionation can be accomplished by varying conditions or reagents of a PCR reaction to amplify fragments of specific size ranges. The invention further provides for analysis of the above 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
56 Claims
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1-36. -36. (canceled)
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37. A method for analyzing a first nucleic acid sample comprising:
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obtaining a second nucleic acid sample by;
fragmenting said first nucleic acid sample to produce fragments;
ligating one or more adaptors to said fragments; and
,amplifying the fragments by a polymerase chain reaction (PCR), wherein fragments of a specific size range are preferentially amplified by varying one or more of the reaction conditions or reagents chosen from the group consisting of;
extension time, annealing time, primer concentration, primer length, presence or absence of a 3′
to 5′
exonuclease activity, and concentration of nucleotide analogues;
providing a nucleic acid array;
hybridizing said second nucleic acid sample to said array; and
analyzing a hybridization pattern resulting from said hybridization. - View Dependent Claims (38, 39, 40, 41, 42)
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43. A method of screening for DNA sequence variations in an individual comprising:
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providing a first nucleic acid sample from said individual;
obtaining a second nucleic acid sample by;
fragmenting said first nucleic acid sample to produce fragments;
ligating adaptor sequences to said fragments; and
amplifying a subset of the fragments by a polymerase chain reaction (PCR) wherein one or more reaction conditions or reagents are varied to favor amplification of a subset of fragments of a specific size range;
providing a nucleic acid array wherein said array comprises probes designed to interrogate for DNA sequence variations;
hybridizing said second nucleic acid sample to said array;
generating a hybridization pattern resulting from said 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 (44, 45, 46)
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47. A method for screening for DNA sequence variations in a population of individuals comprising:
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providing a first nucleic acid sample from each of said individuals;
providing a second nucleic acid sample by;
fragmenting said first nucleic acid sample to produce fragments;
ligating adaptor sequences to said fragments; and
amplifying a subset of the fragments by a polymerase chain reaction (PCR) wherein one or more reaction conditions or reagents are varied to favor amplification of a subset of fragments of a specific size range;
providing a plurality of nucleic acid arrays wherein said arrays comprise probes designed to interrogate for DNA sequence variations;
hybridizing each of said second nucleic acid samples to one of said plurality of arrays;
generating a plurality of hybridization patterns resulting from said hybridizations; and
analyzing the hybridization patterns to determine the presence or absence of sequence variation in the population of individuals. - View Dependent Claims (48)
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50-54. -54. (canceled)
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55. A method of genotyping an individual comprising:
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identifying a collection of SNPs that are found on fragments of a selected size range resulting from digestion with one or more selected restriction enzymes;
designing an array to interrogate said collection of SNPs;
providing a first nucleic acid sample from said individual;
fragmenting said first nucleic acid sample with the said one or more selected restriction enzymes;
amplifying said fragments by PCR wherein a subset of fragments of said selected size range are preferentially amplified;
hybridizing the PCR product to an array; and
analyzing the hybridization pattern to determine the presence or absence of the collection of SNPs.
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56. (canceled)
Specification