Complexity management of genomic DNA by locus specific amplification

Abstract

The present invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences. In one embodiment complexity reduction can be accomplished by extension of a locus specific capture probe followed by amplification of the extended capture probe using common primers. The locus specific capture probes may be attached to a solid support. Multiple DNA sequences may be amplified simultaneously to produce a reduced complexity sample. The invention further provides for analysis of the above sample to interrogate sequences of interest such as polymorphisms. The amplified sample may be hybridized to an array, which may be specifically designed to interrogate the desired fragments for the presence or absence of a polymorphism.

1 - 85 . (canceled)  
     
     
         86 . A method of amplifying a collection of target sequences from a nucleic acid sample, wherein each target sequence comprises a polymorphic position of interest, comprising: 
 generating a collection of capture probes comprising a plurality of different species of primer sequences, wherein each species comprises a first priming sequence that is common to the capture probes in the collection and each species further comprises a 3′ target specific region that is complementary to a different target sequence in the collection of target sequences, wherein the target specific region of each capture probe hybridizes to a target sequence at a region that is between 1 and 1000 bases 3′ of a polymorphic position of interest in the target sequence, and wherein each capture probe in said collection of capture probes is attached to a solid support and the 3′ end of the capture probes is available for extension;    fragmenting the nucleic acid sample with a restriction enzyme that cleaves the target sequences 5′ of the polymorphic position of interest;    ligating an adapter to the fragments, said adapter comprising a second priming sequence;    hybridizing the adapter-ligated fragments to the collection of capture probes, extending the capture probes through the polymorphic position in the target sequence; and    amplifying the extended capture probes with primers to said first and second priming sequences.    
     
     
         87 . The method of  claim 86  further comprising releasing the extended capture probes from the solid support prior to amplification.  
     
     
         88 . The method of  claim 87  wherein prior to releasing the extended capture probes from the solid support, the solid support is washed to remove nucleic acids that are not covalently attached to the solid support.  
     
     
         89 . The method of  claim 86  wherein said capture probes are attached to the solid support through a covalent interaction.  
     
     
         90 . The method of  claim 86  wherein the capture probes further comprise a tag sequence that is unique for each species of capture probe.  
     
     
         91 . The method of  claim 86  wherein each species of capture probe is attached to a solid support in a discrete and determinable location.  
     
     
         92 . The method of  claim 86  wherein prior to amplification, the extended capture probes are enriched in the sample to be amplified, wherein the method of enrichment comprises depletion of non-extended products or positive selection of extended products.  
     
     
         93 . The method of  claim 86  wherein labeled nucleotides are incorporated into the extended capture probes and extended capture probes are isolated by affinity chromatography.  
     
     
         94 . The method of  claim 93  wherein said labeled nucleotides are labeled with biotin, wherein avidin, streptavidin or an anti-biotin antibody is used to isolate extended capture probes.  
     
     
         95 . The method of  claim 86  wherein prior to amplification, the extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested.  
     
     
         96 . The method of  claim 95  wherein the single stranded nucleic acid in the sample is digested with a nuclease.  
     
     
         97 . The method of  claim 96  wherein the nuclease is Exonuclease I.  
     
     
         98 . The method of  claim 87  wherein prior to amplification, the extended capture probes that have been released from the solid support are circularized and uncircularized nucleic acid in the sample is digested.  
     
     
         99 . The method of  claim 98  wherein extended capture probes are circularized by a method comprising: 
 hybridizing an oligonucleotide splint to the extended capture probes, wherein the oligonucleotide splint is complementary to the first and second priming sequences, thereby juxtaposing the 5′ and 3′ ends of extended capture probes; and    ligating the ends of the extended capture probes to form circular extended capture probes.    
     
     
         100 . The method of  claim 98  wherein the uncircularized nucleic acid remaining in the sample is digested with a nuclease.  
     
     
         101 . The method of  claim 100  wherein the nuclease is Exonuclease III.  
     
     
         102 . The method of  claim 86  wherein the nucleic acid sample is fragmented by digestion with one or more restriction enzymes.  
     
     
         103 . The method of  claim 86  wherein said capture probes are synthesized on a solid support.  
     
     
         104 . The method of  claim 86  wherein there are 100 to 1500 different target sequences in the collection of target sequences.  
     
     
         105 . The method of  claim 86  wherein there are 1,000 to 5,000 different target sequences in the collection of target sequences.  
     
     
         106 . The method of  claim 86  wherein there are 2,000 to 10,000 different target sequences in the collection of target sequences.  
     
     
         107 . The method of  claim 86  wherein there are 10,000 to 1,000,000 different target sequences in the collection of target sequences.  
     
     
         108 . A method of analyzing a nucleic acid sample comprising: 
 amplifying a collection of target sequences from said nucleic acid sample according to the method of  claim 86;     hybridizing the amplified collection of target sequences to an array; and    analyzing the hybridization pattern to detect the presence or absence of target sequences from the collection of target sequences.    
     
     
         109 . A method of genotyping one or more polymorphic positions in a sample comprising: 
 amplifying a collection of target sequences from the sample according to the method of  claim 86;     hybridizing the amplified collection of target sequences to an array designed to interrogate at least one polymorphic position in the collection of target sequences; and    analyzing the hybridization pattern to determine the identify of the allele or alleles present at one or more polymorphic position in the collection of target sequences.    
     
     
         110 . A method for analyzing sequence variations in a population of individuals comprising; 
 obtaining a nucleic acid sample from each individual;    amplifying a collection of target sequences from each nucleic acid sample according to the method of  claim 86;     hybridizing each amplified collection of target sequences to an array designed to interrogate sequence variation in the collection of target sequences to generate a hybridization pattern for each sample; and    analyzing the hybridization patterns to determine the presence or absence of sequence variation in the population of individuals.    
     
     
         111 . The method of  claim 86  wherein the target specific region of each capture probe hybridizes to a target sequence so that the 3′ end of the capture probe hybridizes to a base that is from 1 to 100 bases 3′ of the polymorphic position in that target sequence.