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 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;   (c) a reaction mix in the first fluid reservoir, the reaction mix comprising a polymerase, a target nucleic acid having two strands, and a pyrophosphorolytic concentration of pyrophosphate; and   (d) at least one pump fluidly connected to the first fluid reservoir for adding and/or removing a component of the reaction mix.   
     
     
         2 . The apparatus of  claim 1 , further comprising electrodes positioned to create difference in potential for the first fluid reservoir compared to the second fluid reservoir. 
     
     
         3 . The apparatus of  claim 1 , wherein the polymerase is attached to the nanopore. 
     
     
         4 . The apparatus of  claim 1 , wherein the fluid impermeable barrier comprises a membrane. 
     
     
         5 . The apparatus of  claim 4 , wherein the nanopore comprises a protein nanopore that is embedded in the membrane. 
     
     
         6 . The apparatus of  claim 4 , wherein a strand of the target nucleic acid is attached to the membrane. 
     
     
         7 . The apparatus of  claim 6 , wherein the target nucleic acid includes at least one base moiety that is non-naturally occurring in DNA or RNA. 
     
     
         8 . The apparatus of  claim 1 , wherein the nanopore comprises a solid state nanopore. 
     
     
         9 . The apparatus of  claim 1 , wherein the pyrophosphorolytic concentration comprises at least 100 μM pyrophosphate. 
     
     
         10 . The apparatus of  claim 1 , wherein the polymerase lacks 3′ to 5′ exo nuclease activity. 
     
     
         11 . The apparatus of  claim 1 , wherein the at least one pump provides pyrophosphate to the first fluid reservoir. 
     
     
         12 . The apparatus of  claim 1 , wherein the target nucleic acid is DNA. 
     
     
         13 . The apparatus of  claim 12 , wherein the target nucleic acid comprises at least one base moiety that is non-naturally occurring in DNA. 
     
     
         14 . The apparatus of  claim 12 , wherein the at least one of the base moieties comprises 5-methyl cytosine or 5-hydroxymethylcytosine. 
     
     
         15 . The apparatus of  claim 14 , wherein the 5-methyl cytosine or 5-hydroxymethylcytosine is distinguished from cytosine, thereby facilitating an epigenetic analysis. 
     
     
         16 . The apparatus of  claim 1 , wherein the pump cycles pyrophosphate into and out of the first fluid reservoir under conditions to pause pyrophosphorolytic cleavage.