Compositions and techniques for nucleic acid primer extension

Abstract

A method of characterizing a nucleic acid that includes steps of (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and to form a stabilized ternary complex including the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of the first base type has a reversible terminator, and wherein nucleotide cognates of the second, third and fourth base types are extendable; (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet including the first base type followed by the next base.

What is claimed is: 
     
         1 . A method of characterizing a nucleic acid, comprising
 (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides under conditions to produce an extended primer hybrid,   wherein the mixture comprises nucleotide cognates of first, second, third and fourth different base types, wherein the nucleotide cognate of the first base type comprises a reversible terminator, and wherein at least one of the nucleotide cognates of the second, third or fourth base types is extendable;   (b) contacting the extended primer hybrid with a nucleotide cognate for at least one of the different base types and a polymerase to form a stabilized ternary complex comprising the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template;   (c) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and   (d) determining the presence of a base multiplet in the template nucleic acid comprising the first base type followed by the next base.   
     
     
         2 . The method of  claim 1 , further comprising
 (e) repeating steps (a) through (c) using the extended primer hybrid as the primer-template nucleic acid hybrid and   (f) determining the presence of a series of at least two base multiplets in the template nucleic acid.   
     
     
         3 . The method of  claim 2 , wherein the nucleotide cognates of the second, third and fourth base types are extendable in step (a). 
     
     
         4 . The method of  claim 3 , further comprising
 (g) repeating steps (a) through (f), wherein the nucleotide cognate of the second base type comprises a reversible terminator, and wherein nucleotide cognates of the first, third and fourth base types in the mixture are extendible, and wherein the base multiplet determined in step (c) comprises the second base type followed by the next base,   thereby determining the presence of two series of base multiplets in the template nucleic acid.   
     
     
         5 . The method of  claim 4 , further comprising
 (h) repeating steps (a) through (f), wherein the nucleotide cognate of the third base type comprises a reversible terminator, and wherein nucleotide cognates of the first, second and fourth base types in the mixture are extendible, and wherein the base multiplet determined in step (c) comprises the third base type followed by the next base; and   (i) repeating steps (a) through (f), wherein the nucleotide cognate of the fourth base type comprises a reversible terminator, and wherein nucleotide cognates of the first, second and third base types in the mixture are extendible, and wherein the base multiplet determined in step (c) comprises the fourth base type followed by the next base,   thereby determining the presence of four series of base multiplets in the template nucleic acid.   
     
     
         6 . The method of  claim 5 , further comprising a computer assisted process of aligning the four series of base multiplets to determine a sequence of the nucleic acid. 
     
     
         7 . The method of  claim 2 , wherein the nucleotide cognates of the first and second base types comprise a reversible terminator, and wherein the nucleotide cognates of the third and fourth base types are extendable in step (a). 
     
     
         8 . The method of  claim 7 , further comprising
 (g) repeating steps (a) through (f), wherein the nucleotide cognate of the third and fourth base types comprise a reversible terminator, and wherein nucleotide cognates of the first and second base types in the mixture are extendible.   
     
     
         9 . The method of  claim 8 , further comprising
 (h) repeating steps (a) through (f), wherein the nucleotide cognates of the first and third base types comprise a reversible terminator, and wherein nucleotide cognates of the second and fourth base types in the mixture are extendible.   
     
     
         10 . The method of  claim 2 , wherein steps (a) through (f) are repeated using the template nucleic acid after removing the extended primer from the extended primer hybrid and then hybridizing a second primer to the template. 
     
     
         11 . The method of  claim 2 , wherein steps (a) through (f) are repeated using a second template nucleic acid molecule having the same sequence as the template nucleic acid used in step (a). 
     
     
         12 . The method of  claim 2 , wherein the primer-template nucleic acid hybrid of step (a) is the product of polymerase extension during a sequencing process, wherein the sequencing process detects signals indicative of a sequence of the template that is 3′ to the series of at least two base multiplets. 
     
     
         13 . The method of  claim 12 , further comprising a computer assisted process of aligning the sequence of the template to a region of a reference genome, wherein the aligning is carried out by aligning the region of the reference genome to the sequence of the template that is 3′ to the series of at least two base multiplets. 
     
     
         14 . The method of  claim 13 , further comprising (g) performing polymerase extension of the extended primer in a second sequencing process, wherein the second sequencing process detects signals indicative of a sequence of the template that is 5′ to the series of at least two base multiplets. 
     
     
         15 . The method of  claim 14 , further comprising the computer assisted process of aligning the sequence of the template to a region of the reference genome, wherein the aligning is carried out by aligning the region of the reference genome to the sequence of the template that is 3′ to the series of at least two base multiplets, the series of at least two base multiplets, and the sequence of the template that is 5′ to the series of at least two base multiplets. 
     
     
         16 . The method of  claim 1 , wherein the nucleotide cognates of first, second, third and fourth different base types comprise exogenous labels that are detected in step (c). 
     
     
         17 . The method of  claim 1 , wherein the nucleotide cognates of first, second, third and fourth different base types do not comprise exogenous labels that are detected in step (c). 
     
     
         18 . The method of  claim 1 , wherein the polymerase comprises an exogenous label that is detected in step (c). 
     
     
         19 . The method of  claim 1 , wherein step (b) is performed after removing the mixture from contact with the extended primer hybrid. 
     
     
         20 . A method of characterizing a nucleic acid, comprising
 (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides under conditions to produce an extended primer hybrid and to form a stabilized ternary complex comprising the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template,   wherein the mixture comprises nucleotide cognates of first, second, third and fourth different base types, wherein the nucleotide cognate of the first base type comprises a reversible terminator, and wherein at least one of the nucleotide cognates of the second, third or fourth base types is extendable;   (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and   (c) determining the presence of a base multiplet in the template nucleic acid comprising the first base type followed by the next base.   
     
     
         21 . A method of characterizing a nucleic acid, comprising
 (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides under conditions to produce an extended primer hybrid, wherein the mixture comprises nucleotide cognates of no more than three of four different base types;   (b) further extending the extended primer hybrid with a nucleotide cognate of a fourth of the four different base types in the absence of the nucleotide cognates of (a), thereby producing a further extended primer hybrid;   (c) forming a stabilized ternary complex comprising the further extended primer hybrid, a polymerase and a nucleotide cognate of the next base in the template;   (d) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and   (e) determining the presence of a base multiplet in the template nucleic acid comprising the fourth of the four different base types followed by the next base.   
     
     
         22 . A method of characterizing a template nucleic acid, wherein the template comprises a linker region that is adjacent to and between first and second regions of the template nucleic acid, comprising steps of
 (a) obtaining a single base resolution sequence of the first region by extending a primer along the first region of the template nucleic acid, thereby producing an extended primer-template hybrid;   (b) obtaining a signature for the linker region by
 (i) further extending the extended primer of the extended primer-template hybrid using a mixture of nucleotides, wherein the mixture of nucleotides comprises nucleotide cognates of first, second, third and fourth different base types, wherein the nucleotide cognate of the first base type comprises a reversible terminator, and wherein at least one of the nucleotide cognates of the second, third or fourth base types is extendable, and 
 (ii) detecting a stabilized ternary complex, the stabilized ternary complex comprising the further extended primer-template hybrid, a polymerase and a nucleotide cognate of the next base in the template, wherein the signature comprises a base multiplet comprising the first base type followed by the next base; and 
   (c) obtaining a single base resolution sequence of the second region by extending the further extended primer of the further extended primer-template hybrid.   
     
     
         23 . A method of characterizing a nucleic acid, wherein the template comprises a linker region that is adjacent to and between first and second regions of the template nucleic acid, comprising steps of
 (a) obtaining a single base resolution sequence of the first region by extending a primer along the first region of the template nucleic acid, thereby producing an extended primer-template hybrid;   (b) obtaining a signature for the linker region by
 (i) further extending the extended primer of the extended primer-template hybrid using a mixture of nucleotides, wherein the mixture of nucleotides comprises nucleotide cognates of no more than three of four different base types, 
 (ii) further extending the extended primer-template hybrid of step (i) with a nucleotide cognate of a fourth of the four different base types in the absence of the nucleotide cognates of step (i), 
 (iii) detecting a stabilized ternary complex comprising the further extended primer-template hybrid of step (ii), a polymerase and a nucleotide cognate of the next base in the template, wherein the signature comprises a base multiplet comprising the fourth of the four different base types followed by the next base; and 
   (c) obtaining a single base resolution sequence of the second region by extending the further extended primer of the further extended primer-template hybrid of step (iii).   
     
     
         24 . A method of characterizing a nucleic acid, wherein the template comprises a linker region that is adjacent to and between first and second regions of the template nucleic acid, comprising steps of
 (a) obtaining a single base resolution sequence of the first region by extending a primer along the first region of the template nucleic acid, thereby producing an extended primer-template hybrid;   (b) obtaining a signature for the linker region by further extending the extended primer of the extended primer-template hybrid without distinguishing the types of nucleotides incorporated into the extended primer, wherein the signature comprises a count of the nucleotides in the linker region; and   (c) obtaining a single base resolution sequence of the second region by extending the primer of the extended primer-template hybrid of step (b).