Element Defined Sequence Complexity Reduction

Abstract

A method for providing defined mixtures of nucleic acids is described. In certain embodiments, the method uses oligonucleotide probes attached to a solid support as a sequence-specific affinity agent to isolate and facilitate the amplification of defined nucleic acid fragment mixtures.

1 - 32 . (canceled) 
     
     
         33 . A method of nucleic acid sample analysis, comprising:
 a) fragmenting a sample of genomic DNA from a cell to obtain fragmented double-stranded genomic nucleic acids;   b) ligating a double-stranded adaptor duplex comprising a primer site to both ends of said fragmented double-stranded genomic nucleic acids to produce adaptor-ligated nucleic acids;   c) hybridizing said adaptor-ligated nucleic acids with nucleic acid probes to result in complementary fragments being retained on a solid support via probes bound to the solid support;   d) washing the solid support to provide a washed solid support comprising said complementary fragments; and   e) subjecting said washed solid support to conditions sufficient to separate all complementary fragments retained on said washed solid support via said probes from the washed solid support to produce a reduced complexity mixture that is made up of said complementary fragments; and   f) sequencing said complementary fragments of said mixture.   
     
     
         34 . The method of  claim 33 , wherein the adaptor duplex comprises a reactive moiety for binding the fragmented nucleic acids to a substrate having a surface-bound complementary active moiety for binding with the reactive moiety. 
     
     
         35 . The method of  claim 33 , wherein the contacting is performed under stringent conditions. 
     
     
         36 . The method of  claim 33 , wherein the solid support is a microarray having the nucleic acid probes bound thereto. 
     
     
         37 . The method of  claim 36 , wherein the solid support comprises a planar support comprising one or more substrate materials selected from glass, silicas, metals, teflons, and polymeric materials. 
     
     
         38 . The method of  claim 33 , wherein the solid support comprises beads. 
     
     
         39 . The method of  claim 38 , wherein the beads comprises one or more substrate materials selected from nitrocellulose, glass, silicas, teflons, metals, and polymeric materials. 
     
     
         40 . The method of  claim 36 , wherein said solid support comprises from 2 to 2,000,000 probes. 
     
     
         41 . The method of  claim 36 , wherein the nucleic acid probes are bound to said solid support in features that are at a density of up to 100,000 per 1 mm2. 
     
     
         42 . The method of  claim 33 , wherein 10 to 100,000 different probes are bound to the solid support. 
     
     
         43 . The method of  claim 33 , wherein between 100 and 500,000 different probes are bound to the solid support. 
     
     
         44 . The method of  claim 33 , further comprising enriching said adaptor-ligated nucleic acids. 
     
     
         45 . The method of  claim 33 , further comprising amplifying said complementary fragments. 
     
     
         46 . The method of  claim 45 , wherein amplifying the complementary fragments comprises performing an amplification reaction selected from PCR, LCR, or isothermal amplification. 
     
     
         47 . The method of  claim 33 , wherein said conditions of step e) comprise elution of the complementary fragments retained on said washed solid support. 
     
     
         48 . The method of  claim 47 , wherein said elution comprises washing in a buffer at a pH of 8 to 9.5 and a temperature of 70° C. to 95° C. 
     
     
         49 . The method of  claim 33 , wherein said conditions of step e) are sufficient to cleave said probes from the washed solid support.