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 composition comprising: 
 (a) a nucleic acid library to be normalized, wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 2-fold; and    (b) at least one haptenylated nucleic acid molecule comprising: 
 (i) a nucleotide sequence complementary to all or a portion of at least one nucleic acid molecule of said library; and  
 (ii) at least one hapten.  
   
     
     
         54 . The composition of  claim 53 , wherein said at least one haptenylated nucleic acid molecule is hybridized to at least one nucleic acid molecule of said library.  
     
     
         55 . The composition of  claim 53 , further comprising at least one ligand specific for said at least one hapten.  
     
     
         56 . The composition of  claim 55 , wherein said at least one ligand is bound to a solid support.  
     
     
         57 . The composition of  claim 53 , wherein said nucleic acid library is a cDNA library.  
     
     
         58 . The composition of  claim 53 , wherein said hapten is selected from the group consisting of 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 +++ , 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, β-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody and combinations thereof.  
     
     
         59 . The composition of  claim 53 , wherein said hapten is biotin.  
     
     
         60 . A composition comprising: 
 (a) a nucleic acid library to be normalized, wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 2-fold; and    (b) at least one haptenylated target-specific probe comprising a nucleotide sequence that hybridizes to all or a portion of at least one nucleic acid molecule of said library, but does not hybridize to contaminating nucleic acid molecules.    
     
     
         61 . The composition of  claim 60 , wherein said nucleic acid library is a cDNA library.  
     
     
         62 . The composition of  claim 60 , wherein said nucleic acid library is single stranded.  
     
     
         63 . The composition of  claim 60 , wherein said at least one target-specific probe is hybridized to at least one nucleic acid molecule of said library.  
     
     
         64 . The composition of  claim 60 , wherein said contaminating nucleic acid molecules are vector molecules which do not contain nucleic acid molecules of the library.  
     
     
         65 . The composition of  claim 60 , further comprising at least one ligand specific for said at least one hapten.  
     
     
         66 . The composition of  claim 65 , wherein said at least one ligand is bound to a solid support.  
     
     
         67 . The composition of  claim 60 , further comprising one or more polypeptides having polymerase activity.  
     
     
         68 . The composition of  claim 67 , further comprising at least one nucleotide that confers nuclease resistance.  
     
     
         69 . The composition of  claim 68 , wherein said at least one nucleotide is 5-methyldeoxycytosine, 3-methyldeoxyadenosine, or 7-methylguanine.  
     
     
         70 . The composition of  claim 60 , wherein said hapten is selected from the group consisting of 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 +++ , 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, β-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody and combinations thereof.  
     
     
         71 . The composition of  claim 60 , wherein said hapten is biotin.  
     
     
         72 . A kit comprising a nucleic acid library to be normalized, wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 2-fold, and one or more haptenylated primers for normalizing said nucleic acid library.  
     
     
         73 . The kit of  claim 72 , further comprising one or more components selected from the group consisting of one or more polymerases, one or more adapters, one or more vectors, one or more nucleotides, one or more target-specific probes, one or more endonucleases, one or more exonucleases, and one or more restriction enzymes.  
     
     
         74 . The kit of  claim 73 , wherein said one or more nucleotides are nucleotides which confer nuclease resistance.  
     
     
         75 . The kit of  claim 72 , wherein said haptenylated primer comprises a nucleotide sequence complementary to all or a portion of at least one nucleic acid molecule of said library.  
     
     
         76 . The kit of  claim 72 , wherein said hapten is selected from the group consisting of 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 +++ , 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, β-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody and combinations thereof.  
     
     
         77 . The kit of  claim 72 , wherein said hapten is biotin.  
     
     
         78 . The composition of  claim 53 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 3-fold.  
     
     
         79 . The composition of  claim 53 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 10-fold.  
     
     
         80 . The composition of  claim 53 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 20-fold.  
     
     
         81 . The composition of  claim 53 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 25-fold.  
     
     
         82 . The composition of  claim 60 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 3-fold.  
     
     
         83 . The composition of  claim 60 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 10-fold.  
     
     
         84 . The composition of  claim 60 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 20-fold.  
     
     
         85 . The composition of  claim 60 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 25-fold.  
     
     
         86 . The kit of  claim 72 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 3-fold.  
     
     
         87 . The kit of  claim 72 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 10-fold.  
     
     
         88 . The kit of  claim 72 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 20-fold.  
     
     
         89 . The kit of  claim 72 , wherein the relative variation in abundance of the nucleic acid molecules in said library is greater than about 25-fold.