Conjugates of nucleotides and methods for their application

Abstract

Methods for enzymatic synthesis of nucleic acid chains are disclosed in which a nucleotide-macromolecular conjugate is incorporated into the complementary strand of a target sequence.

What is claimed is: 
     
         1 . A kit for labeling nucleic acid chains comprising at least one nucleotide-conjugate and at least one component selected from the group consisting of at least one polymerase and a solid phase for binding labeled nucleic acid chains,
 wherein the nucleotide-conjugate has the structure (Nuc-Linker)n-Marker   wherein:   Nuc is a nucleotide (nuc-component);   Linker is a linker component, wherein the linker links the nuc-component to a macromolecular marker component;   Marker is a macromolecular marker component which comprises at least one target domain having an oligonucleotide sequence complementary to a portion of the target sequence or equivalents thereof; and   n is a positive integer from 1 to 1000; and   wherein binding of the at least one target domain of the nucleotide-conjugate to the complementary portion of the target sequence facilitates selective incorporation of the said nucleotide-conjugate into the synthesized strand of the one or more nucleic acid chains.   
     
     
         2 . The kit according to  claim 1 , further comprising at least one component selected from the group consisting of
 at least one solution for carrying out an enzymatic reaction;   a composition for a nucleotide incorporation reactioncomprising at least one nucleoside triphosphate;   a composition for binding labeled nucleic acid chains to the solid phase;   a composition for washing the solid phase following the nucleotide incorporation reaction; and   a composition for detection of optical signals bound to the solid phase.   
     
     
         3 . The kit according to  claim 1 , which further comprises at least one component selected from the group consisting of:
 one or more primers for amplification of nucleic acid chains;   at least one solution for carrying out an enzymatic reaction;   a composition comprising four dNTPs or NTPs;   a composition for binding labeled nucleic acid chains to the solid phase;   a composition for washing the solid phase after the nucleotide incorporation reaction; and   a composition for detection of optical signals bound tothe solid phase.   
     
     
         4 . The kit according to  claim 1 , wherein the at least one polymerase is selected from the group of polymerases consisting of:
 Reverse Transcriptases selected from M-MLV, RSV, AMV, RAV, MAV, and HIV; and   DNA Polymerases selected from Klenow Fragment DNA Polymerase, Klenow Fragment exo minus DNA Polymerase, T7 DNA Polymerase, Sequenase 2, Vent DNA Polymerase, Vent exo minus DNA Polymerase, Deep Vent DNA Polymerase, Deep Vent exo minus DNA Polymerase, Taq DNA Polymerase, Tli DNA Polymerase, Pwo DNA Polymerase, Thermosequenase DNA Polymerase, and Pfu DNA Polymerase.   
     
     
         5 . The kit according to  claim 1 , wherein the Marker of the at least one nucleotide conjugate comprises at least one moiety selected from the group consisting of dye, fluorescent dye, quencher, antigen, hapten, biotin, oligonucleotide, protein, sugar moiety, hormone, nanocrystals, nanoparticles, microparticles and water soluble polymers. 
     
     
         6 . The kit according to  claim 1 , wherein the kit comprises a plurality of nucleotide-conjugates each comprising a distinct nuc-component and wherein the marker of each of the nucleotide-conjugates comprises at least one characteristic moiety for a particular target domain of the nucleotide-conjugate and wherein each Marker is distinguishable from each other Marker by the characteristic moieties. 
     
     
         7 . The kit according to  claim 1 , wherein the nuc-component of the nucleotide-conjugate comprises at least one base selected independently from the group consisting of purine nucleobases, pyrimidine nucleobases and analogs of purine or pyrimidine nucleobases each of which is capable of forming Watson-Crick-base pairing. 
     
     
         8 . The kit according to  claim 1 , wherein the nuc-component of the nucleotide-conjugate comprises at least one ribose moiety, wherein the 2′-position of the ribose moiety comprises a chemical group selected independently from the group consisting of H, OH, halogen, NH2 and SH. 
     
     
         9 . The kit according to  claim 1 , wherein the nuc-component of the nucleotide-conjugate comprises at least one ribose moiety, wherein the 3′-position of the ribose moiety comprises a chemical group is selected independently from the group consisting of H, OH, halogen, —O—PO3H, SH, N3, NH2, O—CH3, O—CH2-O—CH3 and O—CH2-CH═CH2, 
     
     
         10 . The kit according to  claim 1 , wherein the nuc-component of the nucleotide conjugate comprises at least one ribose moiety, wherein the 5′-position of the ribose moiety comprises a chemical group selected independently from the group consisting of triphosphate group, tetraphosphate group, alpha thiotriphosphate group and pharmaceutically acceptable salts thereof.