Stabilizing a nucleic acid for nucleic acid sequencing

Abstract

The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer/target nucleic acid duplex on a substrate. Methods of the invention generally contemplate the use of a dual-anchored primer/target nucleic acid duplex, or a stabilizing molecule in a single molecule sequencing reaction.

1 . A method for stabilizing a nucleic acid duplex on a surface, the method comprising the steps of: 
 exposing a nucleic acid duplex, wherein each member of said duplex contains a member of a binding pair such that each of said members is oriented in the same direction, to a surface comprising a binding partner for each member of said binding pair, thereby to stabilize said duplex on said surface.    
     
     
         2 . The method of  claim 1 , wherein each of said members is the same molecular species.  
     
     
         3 . The method of  claim 1 , wherein each of said members is a different molecular species.  
     
     
         4 . The method of  claim 1 , wherein said binding pair is selected from the group consisting of a ligand/receptor pair, a carbohydrate/lectin pair, and an antigen/antibody pair.  
     
     
         5 . The method of  claim 1 , wherein said binding pair is selected from the group consisting of biotin/avidin, biotion/streptavidin, digoxigenin/anti-digoxigenin, and dinitrophenol/anti-dinitrophenol.  
     
     
         6 . The method of  claim 1 , wherein said duplex is a template/primer duplex.  
     
     
         7 . The method of  claim 1 , wherein said member is located at the 5′ terminus of said template and the 3′ terminus of said primer.  
     
     
         8 . The method of  claim 1 , wherein said member is located at the 3′ terminus of said template and the 5′ terminus of said primer.  
     
     
         9 . The method of  claim 6 , further comprising the steps of exposing a surface-bound duplex to a nucleotide base and a polymerase under conditions sufficient for said base to be incorporated into said primer if it is complementary to a corresponding base in said template.  
     
     
         10 . The method of  claim 9 , further comprising the step of compiling a nucleic acid sequence of said template by detecting sequential incorporations of nucleotides into said primer.  
     
     
         11 . The method of  claim 6 , wherein said primer comprises a locked nucleic acid base.  
     
     
         12 . The method of  claim 6 , wherein said primer comprising a peptide nucleic acid base.  
     
     
         13 . A method for performing a nucleic acid sequencing reaction, the method comprising the steps of: 
 exposing a mixture comprising a nucleic acid template, a polymerase, and a primer, wherein said primer comprises a locked nucleic acid, to a nucleotide under conditions wherein said nucleotide is capable of incorporation into said primer.    
     
     
         14 . The method of  claim 1 , wherein a plurality of said duplex is attached to a substrate such that each duplex is individually optically resolvable.  
     
     
         15 . A surface for nucleic acid sequencing, said surface comprising a nucleic acid duplex composed of a template and a primer, each of said template and primer being attached to a member of a binding pair, such that each of said members is oriented toward said surface.  
     
     
         16 . The surface of  claim 15 , wherein each duplex is individually optically resolvable.  
     
     
         17 . The surface of  claim 14 , wherein said surface is a polyelectrolyte multilayer.  
     
     
         18 . The surface of  claim 14 , wherein said surface is an epoxide surface.  
     
     
         19 . The surface of  claim 14 , wherein said surface is deposited on a substrate selected from the group consisting of glass and silica.  
     
     
         20 . The surface of  claim 14 , wherein binding partners of said members are covalently attached to said surface.  
     
     
         21 . The surface of  claim 14 , wherein said binding pair is selected from the group consisting of a ligand/receptor pair, an affinity binding pair, an antigen/antibody pair, and a carbohydrate/lectin pair.  
     
     
         22 . The surface of  claim 21 , wherein said pair is selected from the group consisting of biotin/avidin, digoxigenin/anti-digoxigenin, and dinitrophenol/anti-dinitrophenol.