Pyrophosphorolytic sequencing

Abstract

A method for determining the sequence of a target nucleic acid, including steps of contacting a target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and distinguishing the different base moieties for the nucleotide triphosphates that are removed. Also provided is a apparatus including a nanopore positioned in a fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from a first fluid reservoir to a second fluid reservoir, and a reaction mix in the first fluid reservoir that includes a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.

What is claimed is: 
     
         1 . A method for determining the sequence of a target nucleic acid, comprising
 (a) providing a target nucleic acid having two strands;   (b) contacting the target nucleic acid with a polymerase under conditions to sequentially remove nucleotides from the first of the two strands by pyrophosphorolysis, thereby sequentially producing nucleotide triphosphates having a variety of different base moieties; and   (c) distinguishing the different base moieties for the sequentially produced nucleotide triphosphates, thereby determining the sequence of the target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the distinguishing of the different base moieties for the sequentially produced nucleotide triphosphates comprises passing the nucleotide triphosphates through a nanopore. 
     
     
         3 . The method of  claim 2 , wherein the polymerase is attached to the nanopore. 
     
     
         4 . The method of  claim 2 , wherein the nanopore comprises a protein nanopore that is embedded in a membrane. 
     
     
         5 . The method of  claim 1 , wherein the second of the two strands of the target nucleic acid is attached to the membrane. 
     
     
         6 . The method of  claim 2 , wherein the nanopore comprises a solid state nanopore. 
     
     
         7 . The method of  claim 1 , wherein the conditions to sequentially remove nucleotides from one of the two strands by pyrophosphorolysis comprise contacting the polymerase with a pyrophosphorolytic concentration of pyrophosphate 
     
     
         8 . The method of  claim 7 , wherein the pyrophosphorolytic concentration of pyrophosphate comprises a concentration of at least 100 μM. 
     
     
         9 . The method of  claim 7 , wherein the method further comprises a step of pausing the sequential production of the nucleotide triphosphates by removing pyrophosphate from contact with the polymerase and then resuming the sequential production of the nucleotide triphosphates by contacting the polymerase with pyrophosphate. 
     
     
         10 . The method of  claim 1 , wherein the variety of different base moieties comprises at least two different species of base moieties and at most four different species of base moieties. 
     
     
         11 . The method of  claim 10 , wherein the sequence that is determined is longer than four nucleotides. 
     
     
         12 . The method of  claim 1 , wherein the base moieties comprise naturally occurring adenine, guanine, cytosine or thymine. 
     
     
         13 . The method of  claim 1 , wherein at least one of the base moieties comprises a moiety that is non-naturally occurring in DNA or RNA. 
     
     
         14 . A apparatus, comprising
 (a) a fluid impermeable barrier separating a first fluid reservoir from a second fluid reservoir;   (b) a nanopore positioned in the fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from the first fluid reservoir to the second fluid reservoir; and   (c) a reaction mix in the first fluid reservoir, the reaction mix comprising a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.   
     
     
         15 . The apparatus of  claim 14 , further comprising electrodes positioned to create difference in potential for the first fluid reservoir compared to the second fluid reservoir. 
     
     
         16 . The apparatus of  claim 14 , wherein the polymerase is attached to the nanopore. 
     
     
         17 . The apparatus of  claim 14 , wherein the fluid impermeable barrier comprises a membrane. 
     
     
         18 . The apparatus of  claim 17 , wherein the nanopore comprises a protein nanopore that is embedded in the membrane. 
     
     
         19 . The apparatus of  claim 17 , wherein a strand of the target nucleic acid is attached to the membrane. 
     
     
         20 . The apparatus of  claim 19 , wherein the target nucleic acid includes at least one base moiety that is non-naturally occurring in DNA or RNA. 
     
     
         21 . The apparatus of  claim 14 , wherein the nanopore comprises a solid state nanopore. 
     
     
         22 . The apparatus of  claim 14 , wherein the pyrophosphorolytic concentration comprises at least 100 μM pyrophosphate. 
     
     
         23 . The apparatus of  claim 14 , wherein the polymerase lacks 3′ to 5′ exonuclease activity. 
     
     
         24 . A method for determining the sequence of a target nucleic acid, comprising
 (a) providing a target nucleic acid;   (b) contacting the target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and   (c) distinguishing the different base moieties for the nucleotide triphosphates that are removed, thereby determining the sequence of the target nucleic acid.