Compositions and methods for polynucleotide sequence determination

Abstract

The present invention relates to a method for identifying a nucleotide at a predetermined location on a target polynucleotide. The method involves single nucleotide extension reaction comprising an oligonucleotide primer comprising a first sequence and a second sequence or a tag. The method may further comprises a probe which hybridizes to the second sequence or an anti-tag molecule which interacts with the tag, where the hybridization or interaction causes a detectable signal transfer which is indicative of the identity of the nucleotide base at the predetermined location. The invention further provides compositions and kits for performing the subject method of the invention.

1 . A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising: 
 (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide, an oligonucleotide probe which hybridizes to said oligonucleotide primer and labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of said pair of interactive labels, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer, and permits said oligonucleotide probe to hybridize to said oligonucleotide primer to permit said pair of interactive labels to generate a signal; and    (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.    
     
     
         2 . A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising the steps: 
 (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide and a polynucleotide chain terminator labeled with a second member of a pair of interactive labels, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer;    (b) incubating the oligonucleotide primer comprising said second member of said pair of interactive labels with an oligonucleotide probe labeled with a first member of said pair of interactive labels, such that formation of a hybrid between said oligonucleotide probe and said primer permits said pair of interactive labels to a generate a signal; and    (c) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.    
     
     
         3 . The method of  claim 1  or  2 , wherein said signal is generated by fluorescent resonance energy transfer.  
     
     
         4 . The method of  claim 1  or  2 , wherein said oligonucleotide primer comprises a first sequence which hybridizes to said target polynucleotide and a second sequence which does not hybridize to said target polynucleotide in the presence of a third sequence.  
     
     
         5 . The method of  claim 4 , wherein said oligonucleotide probe comprises said third sequence which hybridizes to said second sequence of said oligonucleotide primer.  
     
     
         6 . The method of  claim 1  or  2 , wherein said polynucleotide chain terminator is incorporated by a polynucleotide synthesis enzyme.  
     
     
         7 . The method of  claim 1  or  2 , wherein said reaction mixture further comprises one or more of a second, a third and/or a fourth polynucleotide chain terminator, wherein said first, second, third and fourth polynucleotide terminators are not identical.  
     
     
         8 . The method of  claim 6 , wherein said polynucleotide synthesis enzyme is a JDF-3 DNA polymerase.  
     
     
         9 . The method of  claim 4 , wherein said second sequence is at the 5′ terminal of said first sequence.  
     
     
         10 . The method of  claim 1  or  2 , wherein said oligonucleotide primer comprises a separation moiety that permits separation of said oligonucleotide primer from said reaction mixture.  
     
     
         11 . The method of  claim 10 , wherein a target moiety is provided for said separation moiety to form a specific binding pair for separation.  
     
     
         12 . The method of  claim 11 , wherein said target moiety is attached to a solid support.  
     
     
         13 . A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising: 
 (a) incubating said target polynucleotide in a reaction mixture comprising an anti-tag molecule labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of said pair of interactive labels, and an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of the nucleotide, said oligonucleotide primer covalently coupled to a tag molecule, wherein said incubating permits said polynucleotide chain terminator to be incorporated into said oligonucleotide primer, and said incubating also permits said anti-tag molecule to interact with said tag molecule on said oligonucleotide primer, so that said pair of interactive labels generate a signal; and    (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.    
     
     
         14 . The method of  claim 13 , wherein said signal is generated by fluorescent resonance energy transfer.  
     
     
         15 . The method of  claim 13 , wherein said tag molecule is at 5′ terminal of said oligonucleotide primer.  
     
     
         16 . The method of  claim 15 , wherein said tag molecule comprises a first member of a specific binding pair which comprises said first member and a second member.  
     
     
         17 . The method of  claim 16 , wherein said anti-tag molecule comprises said second member of said specific binding pair.  
     
     
         18 . The method of  claim 17 , wherein said specific binding pair is a biotin-streptavidin binding pair.  
     
     
         19 . The method of  claim 1  or  2 , wherein one member of the pair of interactive labels is a quencher molecule.  
     
     
         20 . The method of  claim 1 ,  2 , or  13 , wherein said chain terminator is one selected from the group consisting of: a dideoxynucleotide triphosphate, a ribofuranose analog, a reversible nucleotide terminator, and an acyclic terminator.  
     
     
         21 . The method of  claim 1 ,  2 , or  13 , wherein the target polynucleotide presents in a sample.  
     
     
         22 . A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising: 
 (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide, an oligonucleotide probe which hybridizes to said oligonucleotide primer and labeled with a first member of a pair of interactive labels, a conventional deoxynucleotide labeled with a second member of said pair of interactive labels, wherein said incubating permits said labeled conventional deoxynucleotide to be incorporated into said oligonucleotide primer at a position corresponding to the predetermined position of the target polynucleotide, and permits said oligonucleotide probe to hybridize to said oligonucleotide primer to permit said pair of interactive labels to generate a signal; and    (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.    
     
     
         23 . A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising the steps: 
 (a) incubating said target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of said nucleotide and a conventional deoxynucleotide labeled with a second member of a pair of interactive labels, wherein said incubating permits said conventional deoxynucleotide to be incorporated into said oligonucleotide primer at a position corresponding to the predetermined position of the target polynucleotide;    (b) incubating the oligonucleotide primer comprising said second member of said pair of interactive labels with an oligonucleotide probe labeled with a first member of said pair of interactive labels, such that formation of a hybrid between said oligonucleotide probe and said primer permits said pair of interactive labels to a generate a signal; and    (c) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.    
     
     
         24 . A method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, said method comprising: 
 (a) incubating said target polynucleotide in a reaction mixture comprising an anti-tag molecule labeled with a first member of a pair of interactive labels, a conventional deoxynucleotide labeled with a second member of said pair of interactive labels, and an oligonucleotide primer which hybridizes to said target polynucleotide immediately 3′ of the nucleotide, said oligonucleotide primer covalently coupled to a tag molecule, wherein said incubating permits said conventional deoxynucleotide to be incorporated into said oligonucleotide primer at a position corresponding to the predetermined position of the target polynucleotide, and said incubating also permits said anti-tag molecule to interact with said tag molecule on said oligonucleotide primer, so that said pair of interactive labels generate a signal; and    (b) detecting said signal, wherein said detection is indicative of the presence of said nucleotide in said target polynucleotide.    
     
     
         25 . The method of  claim 22 ,  23 , or  24 , wherein the reaction mixture further comprising at least one unlabeled chain terminator.