Modified Nucleotides and Methods for Making and Use Same

Abstract

Labeled nucleotide triphosphates are disclosed having a label bonded to the gamma phosphate of the nucleotide triphosphate. Methods for using the gamma phosphate labeled nucleotide are also disclosed where the gamma phosphate labeled nucleotide are used to attach the labeled gamma phosphate in a catalyzed (enzyme or man-made catalyst) reaction to a target biomolecule or to exchange a phosphate on a target biomolecule with a labeled gamme phosphate. Preferred target biomolecules are DNAs, RNAs, DNA/RNAs, PNA, polypeptide (e.g., proteins enzymes, protein, assemblages, etc.), sugars and polysaccharides or mixed biomolecules having two or more of DNAs, RNAs, DNA/RNAs, polypeptide, sugars and polysaccharides moieties.

1 - 43 . (canceled) 
     
     
         44 . The method of  claim 80 , wherein the nucleotides are the same or different. 
     
     
         45 . The method of  claim 44 , wherein the gamma labeled nucleotide or nucleotide analog comprises a nucleotides-selected from the group consisting of adenine triphosphate (ATP), cytosine triphosphate (CTP), guanine triphosphate (GTP), inosine triphosphate (ITP), thymine triphosphate (TTP), uridine triphosphate (UTP), pseudouridine triphosphate (YTP), xanthine triphosphate (XTP), Orotidine triphosphate (OTP), 5-bromouridine triphosphate (BTP), thiouridine triphosphate (STP), 5,6-dihydrouridine triphosphate (DTP), dATP, dGTP, dTTP, dUTP, dCTP and analogs thereof. 
     
     
         46 - 48 . (canceled) 
     
     
         49 . The method of  claim 80 , wherein the gamma label of the gamma labeled nucleotide or nucleotide analog comprises a linker and a fluorescent dye. 
     
     
         50 . The method of  claim 49 , wherein each linker has the same or different properties, where the properties are selected from the group consisting of chain length, bulk or size, rigidity, polarity and combinations thereof. 
     
     
         51 . The method of  claim 50 , wherein the linker has the general formula -E-R-E-, where E is a main group element selected from the group consisting of C, N, O, P, S and combinations thereof and R is a carbon-containing group having between 1 and about 20 main group atoms selected from the group consisting of B, C, N, O, P, S and combinations thereof, with the valency being completed by H. 
     
     
         52 . The method of  claim 80 , wherein the detecting comprises detecting a detectable property of the gamma label of the gamma labeled nucleotide or nucleotide analog. 
     
     
         53 . The method of  claim 80 , wherein the detectable property is selected from the group consisting of light emission, emission frequency, emission duration, emission intensity, quenching, electron spin, radio-activity, nuclear spin, color, absorbance, near IR absorbance, UV absorbance, and far UV absorbance. 
     
     
         54 . The method of  claim 52 , wherein the analytical technique comprises an analytical chemical or physical instrument for detecting and/or monitoring the property. 
     
     
         55 . The method of  claim 54 , wherein the instrument is selected from the group consisting of a camera, an electron spin resonance spectrometry instrument, a nuclear magnetic resonance (NMR) spectrometry instrument, a UV and visible light spectrometry instrument, a far IR, IR or near IR spectrometry instrument, and an X-ray spectrometromety instrument. 
     
     
         56 - 74 . (canceled) 
     
     
         75 . The method of  claim 80 , wherein the phosphatase comprises an alkaline phosphatase. 
     
     
         76 . The method of  claim 75 , wherein the intestinal phosphatase is selected from the group consisting calf intestinal alkaline phosphatase, shrimp alkaline phosphatase and mixtures or combinations thereof. 
     
     
         77 . The method of  claim 80 , wherein the solution is contained in a container. 
     
     
         78 . The method of  claim 55 , wherein the solution is viewable within a viewing field of a camera, the detectable property is fluorescence and the fluorescence is detectable in a single pixel or pixel-bin of the viewing field of the camera. 
     
     
         79 . The method of  claim 80 , wherein the-label of the gamma labeled nucleotide or nucleotide analog comprises a fluorescent dye. 
     
     
         80 . A method comprising:
 purifying one or more gamma labeled nucleotides or nucleotide analogs from a solution comprising both unlabeled and gamma labeled nucleotides or nucleotide analogs by contacting the solution with a phosphatase to produce one or more purified gamma labeled nucleotides or nucleotide analogs;   using the one or more purified gamma labeled nucleotides or nucleotide analogs in a polymerase reaction;   detecting each of one or more incorporations of a gamma labeled nucleotide or nucleotide analog into a nascent nucleic acid strand by the polymerase; and   determining the base identity of the at least one incorporated nucleotide or nucleotide analog.   
     
     
         81 . The method of  claim 80 , wherein one or more gamma labeled nucleotides or nucleotide analogs comprise a deoxynucleotide or analog thereof. 
     
     
         82 . The method of  claim 80 , wherein one or more gamma labeled nucleotides or nucleotide analogs comprise a fluorescent or fluorogenic label attached to or associated with the γ-(gamma) phosphate of the nucleotide or nucleotide analog.