Isometric primer extension method and kit for detection and quantification of polynucleotides

Abstract

Methods, compositions, and kits for the detecting and/or quantification of polynucleotides are provided. An exemplary method provides hybridizing a first primer to the target polynucleotide and forming equal length primer extension products using a nucleotide reaction mixture consisting of X, Y, and Z, wherein X and Y are different purine non-terminator nucleotides, and Z is a pyrimidine non-terminator nucleotide, or X and Y are different pyrmidine non-terminator nucleotides, and Z is a purine non-terminator nucleotide. A portion of the extension products are hybridized to a second primer that is immobilized on a solid support, and the second primer is extended with at least one nucleotide having a detectable marker. The signal from the detectable marker can be correlated to the amount of target polynucleotide. Kits for performing this method are also provided.

What is claimed is:  
     
         1 . A method for detecting or quantifying a known target polynucleotide having a known nucleotide sequence comprising: 
 (a) hybridizing a first primer to the known target polynucleotide and extending the primer using a non-terminator nucleotide mixture formulated to produce equal length primer extension products;    (b) hybridizing the equal length extension products to a second primer;    (c) producing extension products from the second primer; and    (d) detecting the extension products from the second primer.    
     
     
         2 . The method of  claim 1 , wherein the extension products of the first primer comprise a primer portion and an extended portion.  
     
     
         3 . The method of  claim 2 , wherein the second primer hybridizes to the extended portion of the extension products of the first primer.  
     
     
         4 . The method of  claim 2 , wherein the second primer is not complementary to the first primer.  
     
     
         5 . The method of  claim 1 , wherein the amount of detectable extension product correlates to the amount of target polynucleotide.  
     
     
         6 . The method of  claim 1 , wherein the hybridization of the first and second primers occurs under high stringency.  
     
     
         7 . The method of  claim 1 , wherein the extension products from the second primer are detected using fluorescence spectroscopy or mass spectroscopy.  
     
     
         8 . The method of  claim 1 , wherein the extension products from the second primer comprise a detectable label.  
     
     
         9 . The method of  claim 8 , wherein the labeled comprises an epitope, fluorophore, metal particle, enzyme, carbohydrate, polypeptide, radioactive isotope, dye, biotin, or digitonin.  
     
     
         10 . The method of  claim 1 , wherein the primer comprises deoxyribonucleic acid, ribonucleic acid, or a combination thereof.  
     
     
         11 . The method of  claim 1 , wherein the nucleic acid of interest comprises deoxyribonucleic acid, ribonucleic acid, or a combination thereof.  
     
     
         12 . The method of  claim 1 , wherein the extension products are enzymatically produced.  
     
     
         13 . The method of  claim 12 , wherein the enzyme is template-dependent.  
     
     
         14 . The method of  claim 13 , wherein the template-dependent enzyme is DNA polymerase, RNA polymerase or reverse transcriptase, or a combination thereof.  
     
     
         15 . The method of  claim 1 , wherein the target polynucleotide is synthesized enzymatically in vivo, in vitro, or synthesized non-enzymatically.  
     
     
         16 . The method of  claim 1 , wherein the target polynucleotide is synthesized by polymerase chain reaction.  
     
     
         17 . The method of  claim 1 , wherein the target polynucleotide comprises genomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.  
     
     
         18 . The method of  claim 17 , wherein the organism is a plant, microorganism, bacteria, virus.  
     
     
         19 . The method of  claim 17 , wherein the organism is a vertebrate or invertebrate.  
     
     
         20 . The method of  claim 17 , wherein the organism is a mammal.  
     
     
         21 . The method of  claim 20 , wherein the organism is a human being.  
     
     
         22 . The method of  claim 1 , wherein an amplification step is performed on the target polynucleotide.  
     
     
         23 . The method of  claim 1 , wherein the first primer comprises one or more moieties that permit affinity separation of the primer from unincorporated reagent and/or the polynucleotide of interest.  
     
     
         24 . The method of  claim 1 , wherein the second primer comprises one or more moieties that allows immobilization of the second primer onto a solid support to produce an immobilized second primer sequence.  
     
     
         25 . The method of any one of  claim 23  or  24 , wherein the moieties comprises a biotin, digitonin, a phosphate group, or amine group.  
     
     
         26 . The method of  claim 1 , wherein the second primer is synthesized directly on a solid support to produce an immobilized second primer sequence.  
     
     
         27 . The method of  claim 26 , wherein the synthesis is accomplished enzymatically, chemically, or physically.  
     
     
         28 . The method of  claim 1 , the first or second primer is immobilized onto a solid support to produce an immobilized target nucleic acid sequence.  
     
     
         29 . The method of  claim 28 , wherein the first or second primer can be cleaved from the solid support by a chemical, enzymatic or physical process.  
     
     
         30 . The method of  claim 28 , wherein immobilization is accomplished via a photocleavable bond.  
     
     
         31 . The method of  claim 28 , wherein the solid support comprises beads, flat surfaces, chips, capillaries, pins, combs or wafers.  
     
     
         32 . The method of  claim 28 , wherein the immobilization of the first primer is accomplished by hybridization between a complementary capture nucleic acid molecule, which has been previously immobilized to a solid support.  
     
     
         33 . The method of  claim 28 , wherein said immobilization is accomplished via direct bonding between the solid support and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.  
     
     
         34 . A kit for quantification of a target nucleic acid comprising: 
 (a) a first primer complementary to a polynucleotide sequence of the target nucleic acid;    (b) a second primer complementary to an extension product formed from the first primer;    (c) one or more enzymes for performing a primer extension reaction; and    (e) a non-terminator nucleotide mixture formulated to produce equal length primer extension products.    
     
     
         35 . The kit of  claim 34 , wherein at least one non-terminator nucleotide of the non-terminator nucleotide mixture comprises a detectable label.  
     
     
         36 . The kit of  claim 34 , wherein the one or more enzymes comprise a polymerase or reverse transcriptase.  
     
     
         37 . The kit of  claim 34 , where in said non-terminator nucleotide mixture is formulated to prevent at least one canonical Watson-Crick base pair during the extension reaction.  
     
     
         38 . The kit of  claim 34 , wherein the enzyme is a template dependent enzyme.  
     
     
         39 . The kit of  claim 38 , wherein the template dependent enzyme is DNA polymerase, RNA polymerase, or reverse transcriptase.  
     
     
         40 . The kit of  claim 34 , wherein the second primer is immobilized on a solid support.  
     
     
         41 . The kit of  claim 35 , wherein the detectable label comprises an enzyme, protein moieties, radioactive isotope, dye, fluorescent moieties, biotin, or digitonin.  
     
     
         42 . The kit of  claim 34 , further comprising a buffer solution.  
     
     
         43 . The kit of  claim 34 , wherein the non-terminator nucleotide mixture includes nucleotides consisting of X, Y, and Z, wherein X and Y are different purine non-terminator nucleotides, and Z is a pyrimidine non-terminator nucleotide; or X and Y are different pyrmidine non-terminator nucleotides, and Z is a purine non-terminator nucleotide.  
     
     
         44 . A method of detecting a target polynucleotide comprising: 
 (a) hybridizing a first primer to the target polynucleotide;    (b) forming equal length primer extension products using a nucleotides consisting of X, Y, and Z, wherein X and Y are different purine non-terminator nucleotides, and Z is a pyrimidine non-terminator nucleotide; or X and Y are different pyrmidine non-terminator nucleotides, and Z is a purine non-terminator nucleotide;    (c) hybridizing a portion of the extension products of (b) to a second primer;    (d) extending the second primer with at least one nucleotide having a detectable marker using a portion of the equal length extension products of (b) as a template; and    (e) correlating the amount of detectable marker in the extension products of (d) with the amount of target polynucleotide.    
     
     
         45 . The method of  claim 44 , wherein the second primer hybridizes to the non-primer portion of the extension product of (b).  
     
     
         46 . The method of  claim 44 , wherein a primer portion of the extension products of (b) serves as a template strand for extending the second primer  
     
     
         47 . A method for diagnosing a host, comprising: 
 (a) obtaining from the host a sample comprising a polynucleotide;    (b) contacting the sample with a first primer, the first primer comprising a nucleotide sequence complementary to a portion of a known target polynucleotide;    (c) extending the primer using a non-terminator nucleotide mixture formulated to produce equal length primer extension products;    (d) hybridizing the equal length extension products to a second primer;    (e) producing extension products from the second primer; and    (f) detecting the extension products from the second primer, wherein the detection of an extension product from the second primer is indicative of a pathology or predisposition to a pathology of the host.    
     
     
         48 . The method of  claim 47 , wherein the known target polynucleotide is an oncogene or variant thereof.