Compositions and methods for representational selection of nucleic acids from complex mixtures using hybridization

Abstract

The invention provides a method of selecting a representational sample of nucleic acid sequences from a complex mixture. The method includes: (a) contacting a complex mixture of nucleic acids under conditions sufficient for hybridization with a population of capture probes complementary to one or more nucleic acids comprising a predetermined portion of the sequence collectively present in the complex mixture to form hybridization complexes of the one or more nucleic acids with the population of probes, the population of capture probes being attached to a solid support, and (b) removing unhybridized nucleic acids to select a representational sample of nucleic acids having a complexity of less than 10% but more than 0.001% of the complex mixture, wherein the representational sample comprises a nucleic acid copy having a proportion of each sequence in the copy relative to all other sequences in the copy substantially the same as the proportions of the sequences in the predetermined portion of one or more nucleic acids within the complex mixture. A method of selecting a representational sample of genomic sequences from a complete genome also is provided. The invention further provides a nucleic acid population that includes a representational sample having a complexity of less than 10% but more than 0.001% of a complex mixture, the representational sample comprising a nucleic acid copy having a proportion of each sequence in the copy relative to all other sequences in the copy substantially the same as the proportions of sequences in a predetermined portion of a sequence collectively present in one or more nucleic acids within the complex mixture.

1 . A method of selecting a representational sample of nucleic acid sequences from a complex mixture, comprising:
 (a) contacting a complex mixture of nucleic acids with a population of solid support-attached capture probes under conditions sufficient for hybridization of said nucleic acids with said capture probes,   wherein said capture probes are complementary to nucleic acids comprising a predetermined portion of the sequence collectively present in said complex mixture, and   wherein said complex mixture of nucleic acids comprises genomic DNA sequence having a complexity of at least 1.7 Gbp; and   (b) removing unhybridized nucleic acids to select a representational sample of nucleic acids having a complexity of at least 0.001% and at most 49% of said complex mixture, wherein the proportion of each sequence in said representational sample relative to all other sequences in said representational sample is substantially the same as the proportions of the sequences in said complex mixture.   
     
     
         2 . The method of  claim 1 , wherein said genomic DNA comprises human genomic DNA. 
     
     
         3 . The method of  claim 1 , wherein said predetermined portion comprises:
 (a) at least 100 kb of genomic DNA sequence;   (b) a region of genomic DNA selected from a chromosomal arm, a chromosome or a complete genome; and/or   (c) a plurality of exon sequences, a plurality of gene sequences or a plurality of intron sequences.   
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the copy number of each sequence in said representational sample of nucleic acid sequences substantially correlates with the copy number for each said sequence in said predetermined portion. 
     
     
         7 . The method of  claim 1 , wherein said representational sample comprises an unbiased representation of non-repetitive DNA. 
     
     
         8 . The method of  claim 1 , wherein said population of capture probes:
 (a) comprises oligonucleotides having a length of at least about 35 nucleotides;   (b) consists essentially of oligonucleotides having substantially similar melting temperatures (Tm);   (c) comprises an amount in molar excess compared to complementary sequences within said predetermined portion of nucleic acids; and/or   (d) comprises sequences having complementarity to both strands of said genomic DNA.   
     
     
         9 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said complex mixture of nucleic acids comprises fragments smaller than 5000 nucleotides in length. 
     
     
         13 . The method of  claim 1 , wherein said solid support comprises microspheres or a chip. 
     
     
         14 . The method of  claim 1 , further comprising amplifying said representational sample of nucleic acids. 
     
     
         15 . The method of  claim 1 , further comprising eluting said representational sample of nucleic acids from said solid support. 
     
     
         16 . The method of  claim 15 , further comprising attaching the eluted nucleic acids to a second solid support. 
     
     
         17 . The method of  claim 15 , further comprising attaching each of said eluted nucleic acids at a separate identifiable location of an array. 
     
     
         18 . A method of analyzing a complex mixture of nucleic acids comprising:
 (a) contacting a complex mixture of nucleic acids with a population of solid support-attached capture probes under conditions sufficient for hybridization of said nucleic acids with said capture probes,   wherein said capture probes are complementary to nucleic acids comprising a predetermined portion of the sequence collectively present in said complex mixture, and   wherein said complex mixture of nucleic acids comprises genomic DNA sequence having a complexity of at least 1.7 Gbp; and   (b) removing unhybridized nucleic acids to select a representational sample of nucleic acids having a complexity of at least 0.001% and at most 49% of said complex mixture, wherein the proportion of each sequence in said representational sample relative to all other sequences in said representational sample is substantially the same as the proportions of the sequences in said complex mixture; and   (c) detecting individual nucleic acids in said representational sample to determine a sequence characteristic of said predetermined portion of the sequence collectively present in said complex mixture.   
     
     
         19 . The method of  claim 18 , wherein said sequence characteristic comprises:
 (a) the nucleotide sequence for said predetermined portion of the sequence collectively present in said complex mixture;   (b) the copy number for sequences in said predetermined portion of the sequence collectively present in said complex mixture;   (c) loss of heterozygosity in said predetermined portion of the sequence collectively present in said complex mixture;   (d) genotype for said predetermined portion of the sequence collectively present in said complex mixture; and/or   (e) methylation analysis for said predetermined portion of the sequence collectively present in said complex mixture.   
     
     
         20 .- 23 . (canceled) 
     
     
         24 . The method of  claim 18 , wherein step (c) comprises amplifying said representational sample and detecting targets in an amplified population of the representational sample, thereby detecting individual nucleic acids in said representational sample to determine a sequence characteristic of said predetermined portion of the sequence collectively present in said complex mixture. 
     
     
         25 . The method of  claim 18 , further comprising eluting said representational sample of nucleic acids from said solid support before step (c). 
     
     
         26 . The method of  claim 25 , further comprising attaching the eluted nucleic acids to a second solid support before step (c) or attaching each of said eluted nucleic acids at a separate identifiable location of an array before step (c). 
     
     
         27 . (canceled) 
     
     
         28 . A nucleic acid population, comprising a representational sample having a complexity of at least 0.001% and at most 49% of a genome having a complexity of at least 1.7 Gbp, wherein the proportion of each sequence in said representational sample relative to all other sequences in said representational sample is substantially the same as the proportions of the sequences in said complex mixture. 
     
     
         29 . The nucleic acid population of  claim 28 , wherein said representational sample is attached to at least one solid support. 
     
     
         30 . The nucleic acid population of  claim 28 , wherein said representational sample:
 (a) comprises at least 1000 different nucleic acids;   (b) comprises at least 5×10 5  different nucleic acids;   (c) comprises fragments smaller than 5000 nucleotides in length;   (d) comprises a plurality of exon sequences;   (e) excludes introns sequences; and/or   (f) comprises a plurality of gene sequences, optionally wherein said representational sample excludes intervening sequences between said gene sequences.   
     
     
         31 .- 36 . (canceled)