Complexity Management of Genomic DNA

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.

We 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.   
     
     
         2 . The method of  claim 1  wherein the amplified fragments comprise at least 0.01% of the first nucleic acid sample. 
     
     
         3 . The method of  claim 1  wherein the amplified fragments comprise at least 0.5% of the first nucleic acid sample. 
     
     
         4 . The method of  claim 1  wherein the amplified fragments comprise at least 3% of the first nucleic acid sample. 
     
     
         5 . The method of  claim 1  wherein the amplified fragments comprise at least 12% of the first nucleic acid sample. 
     
     
         6 . The method of  claim 1  wherein the amplified fragments comprise at least 30% of the first nucleic acid sample. 
     
     
         7 . The method of  claim 1  wherein the amplified fragments comprise at least 50% of the first nucleic acid sample. 
     
     
         8 . The method of  claim 1  wherein the first restriction enzyme has a six base pair recognition sequence and the second restriction enzyme has a four base pair recognition sequence. 
     
     
         9 . The method of  claim 1  wherein the first restriction enzyme has an eight base pair recognition sequence and the second restriction enzyme has a four base pair recognition sequence. 
     
     
         10 . The method of  claim 1  wherein the fragments are amplified by PCR. 
     
     
         11 . The method of  claim 1  wherein the nucleic acid sample is genomic DNA, DNA, cDNA derived from RNA, total RNA or mRNA. 
     
     
         12 . The method of  claim 1  wherein ligation of one strand of each adaptor is blocked. 
     
     
         13 . The method of  claim 12  wherein ligation is blocked by introducing a gap of at least one nucleotide between one strand of the adaptor and one strand of the fragment. 
     
     
         14 . The method of  claim 12  wherein ligation is blocked by the absence of a phosphate at the 5′ end of an adaptor strand. 
     
     
         15 . The method of  claim 12  wherein ligation is blocked by the presence of a modified nucleotide at the 5′ or 3′ end of an adaptor strand. 
     
     
         16 . The method of  claim 12  wherein ligation is blocked by a terminal modification in one strand of an adaptor. 
     
     
         17 . The method of  claim 12  wherein ligation is blocked at the 5′ end of one strand of one adaptor and at the 3′ end of one strand of the other adaptor. 
     
     
         18 . The method of  claim 12  wherein ligation is blocked at the 5′ end of one adaptor and at the 3′ end of the other adaptor. 
     
     
         19 . The method of  claim 1  wherein one adaptor comprises a 5′ overhang comprising a primer binding site and the other adaptor comprises a 3′ overhang comprising a primer binding site.