Universal nucleotides for nucleic acid analysis

Abstract

The invention includes methods and kits for making and analyzing primer extension products incorporating one or more universal bases, including methods and kits for nucleic acid sequencing and microsatellite analysis.

1 . A method of sequencing at least one target nucleic acid template comprising: 
 (a) forming a reaction composition comprising at least one target nucleic acid template, at least one primer, at least one polymerase, at least one universal nucleotide, and at least one specific terminator comprising a label;    (b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides and one or more of the at least one specific terminators;    (c) separating one or more of the at least one primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and    (d) detecting one or more of the at least one primer extension products.    
     
     
         2 . The method of  claim 1 , wherein the reaction composition further comprises at least one specific extendable nucleotide.  
     
     
         3 . The method of  claim 1 , wherein the reaction composition does not include a specific extendable nucleotide.  
     
     
         4 . The method of  claim 1 , wherein the at least one universal nucleotide is one type of universal nucleotide.  
     
     
         5 . The method of  claim 1 , wherein the at least one universal nucleotide are two types of universal nucleotides.  
     
     
         6 . The method of  claim 1 , wherein the at least one universal nucleotide are three types of universal nucleotides.  
     
     
         7 . The method of  claim 1 , wherein one or more of the at least one universal nucleotides is individually selected from 2′-deoxy-7-azaindole-5′-triphosphate (d7AITP), 2′-deoxy-isocarbostyril-5′-triphosphate (dlCSTP), 2′-deoxy-propynylisocarbostyril-5′-triphosphate (dPICSTP), 2′-deoxy-6-methyl-7-azaindole-5′-triphosphate (dM7AITP), 2′-deoxy-imidizopyridine-5′-triphosphate (dlmPyTP), 2′-deoxy-pyrrollpyrizine-5′-triphosphate (dPPTP), 2′-deoxy-allenyl-7-azaindole-5′-triphosphate (dA7AITP), and 2′-deoxy-propynyl-7-azaindole-5′-triphosphate (dP7AITP).  
     
     
         8 . The method of  claim 1 , wherein the at least one specific terminator comprises an A terminator comprising a first label, a T terminator comprising a second label, a G terminator comprising a third label, and a C terminator comprising a fourth label, wherein the first, second, third, and fourth labels are detectably different.  
     
     
         9 . The method of  claim 1 , wherein the MDAT comprises at least one of electrophoresis, chromatography, HPLC, mass spectroscopy, sedimentation, field-flow fractionation, or multi-stage fractionation.  
     
     
         10 . The method of  claim 1 , wherein the MDAT comprises electrophoresis.  
     
     
         11 . The method of  claim 1 , wherein the label comprises a fluorescent dye.  
     
     
         12 . The method of  claim 11 , wherein the label comprises an energy-transfer fluorescent dye.  
     
     
         13 . A method of sequencing at least one target nucleic acid template comprising: 
 (a) forming at least one reaction composition comprising at least one target nucleic acid template, at least one primer comprising a label, at least one polymerase, at least one universal nucleotide, and at least one specific terminator;    (b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides and one or more of the at least one specific terminators;    (c) separating one or more of the at least one primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and    (d) detecting one or more of the at least one primer extension products.    
     
     
         14 . The method of  claim 13 , wherein the reaction composition further comprises at least one specific extendable nucleotide.  
     
     
         15 . The method of  claim 13 , wherein the reaction composition does not include a specific extendable nucleotide.  
     
     
         16 . The method of  claim 13 , wherein the at least one universal nucleotide is one type of universal nucleotide.  
     
     
         17 . The method of  claim 13 , wherein the at least one universal nucleotide are two types of universal nucleotides.  
     
     
         18 . The method of  claim 13 , wherein the at least one universal nucleotide are three types of universal nucleotides.  
     
     
         19 . The method of  claim 13 , wherein one or more of the at least one universal nucleotides is individually selected from d7AITP, dlCSTP, dPICSTP, dM7AITP, dlmPyTP, dPPTP, dA7AITP, and dP7AITP.  
     
     
         20 . The method of  claim 13 , wherein the label comprises a fluorescent dye.  
     
     
         21 . The method of  claim 20 , wherein the label comprises an energy-transfer fluorescent dye.  
     
     
         22 . The method of  claim 13 , wherein the at least one reaction composition comprises a first reaction composition comprising a primer comprising a first label and a first specific terminator, a second reaction composition comprising a primer comprising a second label and a second specific terminator, a third reaction composition comprising a primer comprising a third label and a third specific terminator, and a fourth reaction composition comprising a primer comprising a fourth label and a fourth specific terminator, wherein the first, second, third, and fourth labels are different, and wherein the first, second, third, and fourth terminators are different.  
     
     
         23 . The method of  claim 13 , wherein the MDAT comprises at least one of electrophoresis, chromatography, HPLC, mass spectroscopy, sedimentation, field-flow fractionation, or multi-stage fractionation.  
     
     
         24 . The method of  claim 13 , wherein the MDAT comprises electrophoresis.  
     
     
         25 . A method of detecting a plurality of primer extension products comprising: 
 (a) forming a reaction composition comprising at least one target nucleic acid template, at least one primer comprising a label, at least one polymerase, and at least one universal nucleotide;    (b) incubating the reaction composition under appropriate conditions to generate at least one primer extension product comprising one or more of the at least one universal nucleotides;    (c) releasing one or more of the at least one primer extension products from one or more of the at least one target nucleic acid templates;    (d) repeating (b) and (c) to generate a plurality of primer extension products;    (e) separating one or more of the plurality of primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and    (f) detecting one or more of the plurality of primer extension products.    
     
     
         26 . The method of  claim 25 , wherein one or more of the at least one target nucleic acid templates has been cut with at least one restriction endonuclease.  
     
     
         27 . The method of  claim 25 , wherein one or more of the at least one target nucleic acid templates comprises a short tandem repeat.  
     
     
         28 . The method of  claim 25 , wherein the reaction composition further comprises at least one specific extendable nucleotide.  
     
     
         29 . The method of  claim 25 , wherein the reaction composition does not include a specific extendable nucleotide.  
     
     
         30 . The method of  claim 25 , wherein the at least one universal nucleotide is one type of universal nucleotide.  
     
     
         31 . The method of  claim 25 , wherein the at least one universal nucleotide are two types of universal nucleotides.  
     
     
         32 . The method of  claim 25 , wherein the at least one universal nucleotide are three types of universal nucleotides.  
     
     
         33 . The method of  claim 25 , wherein one or more of the at least one universal nucleotides is individually selected from d7AITP, dlCSTP, dPICSTP, dM7AITP, dlmPyTP, dPPTP, dA7AITP and dP7AITP.  
     
     
         34 . The method of  claim 25 , wherein the label comprises a fluorescent dye.  
     
     
         35 . The method of  claim 34 , wherein the label comprises an energy-transfer fluorescent dye.  
     
     
         36 . The method of  claim 25 , wherein the at least one target nucleic acid template comprises a first target nucleic acid template and a second target nucleic acid template, wherein the first and second target nucleic acid templates are different, and wherein the at least one primer comprises (i) a first primer that is specific for the first target nucleic acid template and that comprises a first label and (ii) a second primer that is specific for the second target nucleic acid template and that comprises a second label, and wherein the first and second labels are detectably different.  
     
     
         37 . A method of detecting a plurality of second primer extension products comprising: 
 (a) forming a first reaction composition comprising at least one target nucleic acid template, at least one first primer, at least one specific nucleotide, and at least one first polymerase, wherein the first reaction composition does not include a universal nucleotide;    (b) incubating the reaction composition under appropriate conditions to generate at least one first primer extension product;    (c) releasing the one or more of at least one first primer extension products from one or more of the at least one target nucleic acid templates;    (d) repeating (b) and (c) to generate a plurality of first primer extension products;    (e) forming a second reaction composition comprising one or more of the plurality of first primer extension products, at least one second primer comprising a label, at least one second polymerase, and at least one universal nucleotide;    (f) incubating the reaction composition under appropriate conditions to generate at least one second primer extension product;    (g) releasing one or more of the at least one second primer extension products from one or more of the at least one first primer extension products;    (h) repeating (f) and (g) to generate a plurality of second primer extension products;    (i) separating one or more of the plurality of second primer extension products, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT); and    (j) detecting one or more of the plurality of second primer extension products.    
     
     
         38 . The method of  claim 37 , wherein one or more of the at least one target nucleic acid templates comprises a short tandem repeat.  
     
     
         39 . The method of  claim 37 , wherein the second reaction composition further comprises at least one specific extendable nucleotide.  
     
     
         40 . The method of  claim 37 , wherein the second reaction composition does not include a specific extendable nucleotide.  
     
     
         41 . The method of  claim 37 , wherein the at least one universal nucleotide is one type of universal nucleotide.  
     
     
         42 . The method of  claim 37 , wherein the at least one universal nucleotide are two types of universal nucleotides.  
     
     
         43 . The method of  claim 37 , wherein the at least one universal nucleotide are three types of universal nucleotides.  
     
     
         44 . The method of  claim 37 , wherein one or more of the at least one universal nucleotides is individually selected from d7AITP, dlCSTP, dPICSTP, dM7AITP, dlmPyTP, dPPTP, dA7AITP, and dP7AITP.  
     
     
         45 . The method of  claim 37 , wherein the label comprises a fluorescent dye.  
     
     
         46 . The method of  claim 45 , wherein the label comprises an energy-transfer fluorescent dye.  
     
     
         47 . The method of  claim 37 , further comprising removing the target nucleic acid template prior to forming the second reaction composition.  
     
     
         48 . The method of  claim 1 , wherein (b) further comprises releasing one or more of the at least one primer extension products from one or more of the at least one target nucleic acid templates, and said incubating and releasing are repeated at least one additional time.