Polynucleotide sequencing method

Abstract

Disclosed is a method for determining a sequence of one or more nucleotides in a target polynucleotide. In the method, a target-specific primer is contacted with a polynucleotide sample under conditions effective for the primer to anneal specifically to a primer-complementary region in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein each target-specific primer contains (i) a target binding segment and (ii) a mobility-reducing moiety which does not bind the target. The hybrid(s) are reacted with a primer extension reagent in the presence of at least one labeled 3′-nucleotide terminator complementary to a selected nucleotide base-type, optionally in the presence of one or more extendable nucleoside monomers, under conditions effective to form one or more primer extension products which terminate with at least one labeled 3′-nucleotide terminator. The primer extension products are then separated electrophoretically in a sieving matrix under denaturing conditions such that smaller extension products migrate more rapidly than larger extension products, and the desired sequence information is determined.

1 . A method for determining a sequence of one or more nucleotides in a target polynucleotide, the method comprising: 
 (a) contacting a target-specific primer with a polynucleotide sample under conditions effective for the primer to anneal specifically to a primer-complementary region in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein each target-specific primer contains (i) a target binding segment and (ii) a mobility-reducing moiety which does not bind the target,    (b) reacting said hybrid(s) with a primer extension reagent in the presence of at least one labeled 3′-nucleotide terminator complementary to a selected nucleotide base-type, under conditions effective to form one or more primer extension products which terminate with at least one labeled 3′-nucleotide terminator,    separating electrophoretically the primer extension product(s) under denaturing conditions in a sieving matrix such that smaller extension products migrate more rapidly than larger extension products, and    determining a target sequence on the basis of the mobilities and terminator labels of the separated extension product(s).    
     
     
         2 . The method of  claim 1 , wherein primer extension is performed in the presence of at least one extendable nucleoside monomer under conditions effective to extend specifically hybridized primers by addition of target-complementary polynucleotide monomers and terminator(s), to form said one or more extension products.  
     
     
         3 . The method of  claim 1 , wherein primer extension is performed in the absence of extendable nucleoside monomers, under conditions effective to append the at least one labeled 3-nucleotide terminator to at least one specifically annealed primer only when the terminator is complementary to a base in the target polynucleotide that is immediately adjacent the extendable end of the annealed primer.  
     
     
         4 . The method of  claim 1 , wherein at least one 3′-nucleotide terminator contains a fluorescent label.  
     
     
         5 . The method of  claim 4 , wherein said reacting is conducted in the presence of four different 3′-nucleotide terminators which (i) are each complementary to a different nucleotide base-type and (ii) each contain a distinguishable fluorescent label that identifies the base-type of the terminator.  
     
     
         6 . The method of  claim 1 , wherein said separating is performed by slab gel electrophoresis.  
     
     
         7 . The method of  claim 1 , wherein said separating is performed by capillary electrophoresis.  
     
     
         8 . The method of  claim 1 , wherein said separating is performed in a microchannel.  
     
     
         9 . The method of  claim 1 , wherein at least one 3′-nucleotide terminator contains a radioactive label.  
     
     
         10 . The method of  claim 1 , wherein said mobility-reducing moiety comprises a polymer segment that does not substantially bind to the target polynucleotide(s).  
     
     
         11 . The method of  claim 10 , wherein said polymer segment comprises a polynucleotide segment that does not substantially bind to the target polynucleotide(s).  
     
     
         12 . The method of  claim 1 , wherein said mobility-reducing moiety is effective to decrease the mobility rate of each primer extension product in an amount equivalent to at least 10 additional nucleotide bases, relative to the mobility of a corresponding primer extension product lacking the mobility-reducing moiety.  
     
     
         13 . The method of  claim 12 , wherein the amount of said decrease in mobility is equivalent to the addition of at least 15 additional nucleotide bases.  
     
     
         14 . The method of  claim 13 , wherein the amount of said decrease in mobility is equivalent to the addition of at least 20 additional nucleotide bases.