Methods for detection and quantification of analytes in complex mixtures

Abstract

The invention provides a diverse population of uniquely labeled probes, containing about thirty or more target specific nucleic acid probes each attached to a unique label bound to a nucleic acid. Also provided is a method of producing a population of uniquely labeled nucleic acid probes. The method consists of (a) synthesizing a population of target specific nucleic acid probes each having a different specifier; (b) synthesizing a corresponding population of anti-genedigits each having a unique label, the population having a diversity sufficient to uniquely hybridize to genedigits within the specifiers, and (c) hybridizing the populations of target nucleic acid probes to the anti-genedigits, to produce a population in which each of the target specific probes is uniquely labeled. Also provided is a method of detecting a nucleic acid analyte. The method consists of (a) contacting a mixture of nucleic acid analytes under conditions sufficient for hybridization with a plurality of target specific nucleic acid probes each having a different specifier; (b) contacting the mixture under conditions sufficient for hybridization with a corresponding plurality of anti-genedigits each having a unique label, the plurality of anti-genedigits having a diversity sufficient to uniquely hybridize to genedigits within the specifiers, and (c) uniquely detecting a hybridized complex between one or more analytes in the mixture, a target specific probe, and an anti-genedigit.

1 .- 15 . (canceled) 
   
   
       16 . A method of producing a population of labels, comprising synthesizing a population of nucleic acids, each of said nucleic acids within said synthesized population having bound a predetermined ratio of at least two different label monomers, wherein said population of labels has a diversity of predetermined ratios of about 30 or more. 
   
   
       17 . The method of  claim 16 , wherein each of said nucleic acids bound by a predetermined ratio of at least two labels further comprises about the same unit signal. 
   
   
       18 . The method of  claim 16 , wherein said diversity is selected from the group consisting of 40, 60, 80, 100, 120, 140, and 150. 
   
   
       19 . The method of  claim 16 , wherein said diversity is selected from the group consisting of 200, 500, 2,000, 5,000, 1×10 4 , 3×10 4  and 1×10 5 . 
   
   
       20 . The method of  claim 16 , wherein said labels are fluorescent. 
   
   
       21 . The method of  claim 16 , wherein each of said nucleic acids within said synthesized population further comprise an anti-genedigit. 
   
   
       22 . The method of  claim 16 , wherein each of said nucleic acids within said synthesized population further comprise a target specific probe. 
   
   
       23 .- 33 . (canceled) 
   
   
       34 . A method of producing a target specific nucleic acid probe, comprising: (a) producing a first nucleic acid comprising a target specific probe, wherein said target specific probe comprises a target specific region and a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have different nucleotide sequences; (b) producing a second nucleic acid comprising two or more different anti-genedigits, wherein each anti-genedigit hybridizes to a genedigit in said target specific probe, and (c) hybridizing said first and second nucleic acids to produce a target specific nucleic acid probe. 
   
   
       35 . (canceled) 
   
   
       36 . The method of  claim 34 , wherein said target specific nucleic acid probe comprises four or more genedigits. 
   
   
       37 . The method of  claim 34 , wherein said target specific nucleic acid probe comprise five or more genedigits. 
   
   
       38 .- 40 . (canceled) 
   
   
       41 . The method of  claim 34 , wherein at least one of said anti-genedigits hybridizes to its corresponding genedigit through a sequence having a complexity less than the number of hybridized base pairs. 
   
   
       42 . The method of  claim 41 , wherein said number of hybridized base pairs is about 24. 
   
   
       43 .- 44 . (canceled) 
   
   
       45 . The method of  claim 34 , wherein said anti-genedigits comprise a label monomer or combination of label monomers. 
   
   
       46 . The method of  claim 45 , wherein each of said different anti-genedigits comprises a unique label monomer or combination of label monomers. 
   
   
       47 .- 48 . (canceled) 
   
   
       49 . A method of producing a population of uniquely labeled nucleic acid probes, comprising: (a) synthesizing a population of target specific nucleic acid probes each having a different specifier; (b) synthesizing a corresponding population of anti-genedigits each having a unique label monomer or combination of label monomers, said population having a diversity sufficient to uniquely hybridize to genedigits within said specifiers, and (c) hybridizing said populations of target nucleic acid probes to said anti-genedigits, to produce a population wherein each of said target specific probes is uniquely labeled. 
   
   
       50 .- 84 . (canceled) 
   
   
       85 . The method of  claim 45 , wherein said label monomers are selected from radioisotopes, fluorochromes, dyes, enzymes, nanoparticles, chemiluminescent markers, and biotin. 
   
   
       86 . The method of  claim 45 , wherein each of said target specific nucleic acid probes comprises a mixture of two or more different label monomers. 
   
   
       87 . The method of  claim 45 , wherein at least one label monomer is a quantum dot. 
   
   
       88 . The method of  claim 45 , wherein at least one anti-genedigit is a dendrimer. 
   
   
       89 . The method of  claim 45 , wherein each said anti-genedigit is covalently attached to each said at least one label monomer.