Normalized nucleic acid libraries and methods of production thereof

Abstract

The present invention relates generally to methods for producing normalized nucleic acid libraries in which each member of the library can be isolated with approximately equivalent probability. In particular, the present methods comprise subtractive hybridization of a nucleic acid library with haptenylated (e.g., biotinylated, avidinated or streptavidinated) nucleic acid molecules that are complementary to one or more of the nucleic acid molecules of the library, such that the variation in the abundances of the individual nucleic acid molecules in the library is reduced. The invention also relates to production of normalized nucleic acid libraries (particularly cDNA libraries) in which contaminating nucleic acid molecules have been reduced or eliminated, and to normalized nucleic acid libraries produced by such methods.

1 - 52 . (canceled) 
     
     
         53 . A method for normalizing a nucleic acid library comprising:
 (a) incubating a nucleic acid library to be normalized with haptenylated nucleic acid molecules complementary to all or a portion of the nucleic acid molecules of the library under conditions favoring the hybridization of the more highly abundant molecules of the library with the haptenylated nucleic acid molecules; and   (b) removing the hybridized molecules, thereby producing a normalized library.   
     
     
         54 . The method of  claim 53 , wherein the nucleic acid library is a cDNA library. 
     
     
         55 . The method of  claim 54 , wherein the nucleic acid molecules of the cDNA library are single-stranded. 
     
     
         56 . The method of  claim 54 , wherein the nucleic acid molecules of the cDNA library are double-stranded. 
     
     
         57 . The method of  claim 53 , wherein the haptenylated nucleic acid molecules are RNA molecules. 
     
     
         58 . The method of  claim 53 , further comprising reduction or removal of contaminating nucleic acid molecules from the normalized library. 
     
     
         59 . The method of  claim 58 , wherein the contaminating nucleic acid molecules are vectors. 
     
     
         60 . The method of  claim 58 , wherein said reduction or removal comprises incubating the normalized library with at least one haptenylated probe. 
     
     
         61 . The method of  claim 60 , wherein the haptenylated probe hybridizes to nucleic acid molecules of the normalized library. 
     
     
         62 . The method of  claim 61 , wherein the haptenylated probe is not capable of hybridizing to the vector sequences of said library. 
     
     
         63 . The method of  claim 60 , wherein at least one of the haptenylated probe is used to isolate a normalized library having substantially reduced contaminating nucleic acid molecules, thereby producing a selected normalized library. 
     
     
         64 . The method of  claim 63 , wherein at least one haptenylated probe comprises avidin, streptavidin, protein A, protein G, a cell-surface Fc receptor, an antibody-specific antigen, an enzyme-specific substrate, polymyxin B, endotoxin-neutralizing protein (ENP), Fe. sup.+++, a transferrin receptor, an insulin receptor, a cytokine receptor, CD4, spectrin, fodrin, ICAM-1, ICAM-2, C3bi, fibrinogen, Factor X, ankyrin, an integrin, vitronectin, fibronectin, collagen, laminin, glycophorin, Mac-1, LFA-1, beta.-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody, or biotin or combinations thereof. 
     
     
         65 . The method of  claim 64 , wherein at least one haptenylated probe comprises biotin. 
     
     
         66 . The method of  claim 63 , wherein the isolation comprises the use of a solid support comprising at least one ligand that binds the at least one hapten. 
     
     
         67 . The method of  claim 63 , wherein the selected normalized library is single-stranded. 
     
     
         68 . The method of  claim 67 , further comprising incubating the single-stranded selected normalized library under conditions sufficient to render the molecules double stranded. 
     
     
         69 . The method of  claim 68 , wherein the conditions comprise incubating the single-stranded selected normalized library with one or more nucleotides, one or more polypeptides having polymerase activity and one or more primers. 
     
     
         70 . The method of  claim 69 , wherein the one or more nucleotides are nucleotide analogues which confer nuclease resistance on the double stranded molecules. 
     
     
         71 . The method of  claim 70 , further comprising digesting a sample comprising the double stranded molecules with the nuclease. 
     
     
         72 . The method of  claim 68 , further comprising transforming the double stranded molecules into one or more host cells. 
     
     
         73 . The method of  claim 69 , wherein the primers are target specific primers. 
     
     
         74 . The method of  claim 57 , wherein the RNA molecules are produced by one or more RNA polymerases. 
     
     
         75 . The method of  claim 74 , wherein the RNA polymerases are selected from the group consisting of SP6, T7 and T3 RNA polymerases. 
     
     
         76 . The method of  claim 57 , wherein the RNA molecules are produced with one or more promoters. 
     
     
         77 . The method of  claim 76 , wherein the one or more promoters are provided by one or more vectors or by one or more adapters. 
     
     
         78 . The method of  claim 53 , wherein the hybridized molecules are removed by hapten-ligand interactions and/or extraction. 
     
     
         79 . The method of  claim 78 , wherein the removal comprises the use of a solid support comprising at least one ligand. 
     
     
         80 . The method of  claim 53 , wherein the hybridization conditions are selected from the group consisting of:
 (a) a COT equal to or greater than 25;   (b) a COT equal to or greater than 50;   (c) a COT equal to or greater than 100;   (d) a COT from about 10 to 10,000;   (e) a COT from about 25 to 10,000;   (f) a COT from about 50 to 10,000;   (g) a COT from about 100 to 10,000; and   (h) a COT of less than 10,000.   
     
     
         81 . The method of  claim 58 , wherein the reduction or removal comprises incubating the library with at least one primer and at least one nucleotide which confers nuclease resistance under condition sufficient to make double stranded nucleic acid molecules. 
     
     
         82 . The method of  claim 81 , wherein the primer hybridizes to nucleic acid molecules of the library. 
     
     
         83 . The method of  claim 81 , wherein the primer is not capable of hybridizing to vector sequences of the library. 
     
     
         84 . The method of  claim 81 , wherein the nucleotide is a nucleotide analog. 
     
     
         85 . The method of  claim 84 , wherein the nucleotide analog is a methylated nucleotide. 
     
     
         86 . The method of  claim 85 , wherein the methylated nucleotide is 5-methyldeoxycytosine. 
     
     
         87 . The method of  claim 81 , further comprising digesting the double-stranded nucleic acid molecules with one or more nucleases. 
     
     
         88 . The method of  claim 87 , further comprising transforming the digested molecules into one or more host cells.