High throughput nucleic acid sequencing by expansion and related methods

Abstract

Nucleic acid sequencing methods and related products and methods for detection and presentation of the same are disclosed. Methods for sequencing a target nucleic acid comprise providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield a surrogate polymer of a length longer than the plurality of the subunits of the daughter strand, the surrogate polymer comprising the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the surrogate polymer are then detected, Disclosed methods for detecting the surrogate polymers comprise nanopore detection and other detection methods suitable for high-throughput DNA sequencing. Methods for presenting the surrogate polymer to the detector comprise presenting the surrogate polymers: 1) in flow, 2) tethered to a solid support, and 3) aligned on a substrate surface. Corresponding products, including surrogate polymers and oligomeric and monomeric substrate constructs are also disclosed.

1 - 73 . (canceled) 
     
     
         74 . A method of presenting at least one surrogate polymer for detection, comprising:
 a) providing a detector construct, wherein the detector construct comprises at least one detector element;   b) providing the at least one surrogate polymer, wherein the at least one surrogate polymer comprises one or more individual reporter elements; and   c) processing the at least one surrogate polymer to obtain a uniform spatial and temporal spacing of the one or more individual reporter elements.   
     
     
         75 . (canceled) 
     
     
         76 . The method of  claim 75 , wherein the detector construct comprises a regular array of nanopore channels. 
     
     
         77 . The method of  claim 74 , wherein processing the at least one surrogate polymer comprises tethering an end of the at least one surrogate polymer to a solid substrate having at least one binding site. 
     
     
         78 . (canceled) 
     
     
         79 . The method of  claim 75 , wherein processing the at least one surrogate polymer comprises attaching a charged, linear polymer having a low molecular weight to an end of the at least one surrogate polymer. 
     
     
         80 . The method of  claim 79 , wherein the charged, linear polymer is selected from polyglutamic acid and polyphosphate. 
     
     
         81 . The method of  claim 75 , wherein processing the at least one surrogate polymer comprises applying a voltage to the at least one nanopore channel, wherein the voltage is higher than a desired measurement voltage, and decreasing the voltage to the desired measurement voltage when a surrogate polymer is detected in the nanopore channel. 
     
     
         82 . The method of  claim 81 , wherein the voltage is manipulated such that only one surrogate polymer may occupy the at least one nanopore channel at a time. 
     
     
         83 . The method of  claim 75 , wherein processing the at least one surrogate polymer comprises attaching a stop to an end of the at least one surrogate polymer, wherein the stop prevents the at least one surrogate polymer from passing through the at least one nanopore channel and prevents multiple surrogate polymers from occupying the same nanopore channel, and prefilling the at least one surrogate polymer in the at least one nanopore channel. 
     
     
         84 - 87 . (canceled) 
     
     
         88 . The method of  claim 74 , wherein processing the at least one surrogate polymer comprises controlling the flow of the at least one surrogate polymer toward the detector construct. 
     
     
         89 - 91 . (canceled) 
     
     
         92 . The method of  claim 88 , wherein controlling the flow of the at least one surrogate polymer comprises:
 a) providing at least one gating construct, wherein the at least one gating construct comprises a first, second, and third electrode; and   b) manipulating an electric field applied independently to the first, second, and third electrodes to obtain a uniform spatial and temporal spacing of the one or more individual reporter elements.   
     
     
         93 . (canceled) 
     
     
         94 . The method of  claim 88 , wherein controlling the flow of the at least one surrogate polymer comprises:
 a) providing at least one gating construct, wherein the at least one gating construct comprises a first and second porous electrode and a gating element, wherein the first and second porous electrodes are affixed to a first and second side of the gating element, respectively;   b) applying an electric field to the first and second electrodes; and   c) transporting the at least one surrogate polymer though the gate toward the at least one detector element.   
     
     
         95 . The method of  claim 94 , wherein the gating element is selected from a porous membrane and a nanohole. 
     
     
         96 . The method of  claim 94 , wherein the electric field is manipulated to obtain a uniform spatial and temporal spacing of the one or more individual reporter elements. 
     
     
         97 . (canceled) 
     
     
         98 . The method of  claim 95 , wherein the gating element is a porous membrane. 
     
     
         99 . The method of  claim 98 , wherein the porous membrane comprises pores from about 20 nm to about 100 nm in diameter. 
     
     
         100 . The method of  claim 98 , wherein the porous membrane is selected from aluminum oxide and a polymer track-formed membrane. 
     
     
         101 - 127 . (canceled) 
     
     
         128 . The method of  claim 74 , wherein processing the at least one surrogate polymer comprises affixing the at least one surrogate polymer to a solid substrate comprising nanopore channels, wherein affixing the at least one surrogate polymer to the solid substrate comprises attaching a stop to an end of the at least one surrogate polymer, wherein the stop prevents the at least one surrogate polymer from passing through the at least one nanopore channel and prevents multiple surrogate polymers from occupying the same nanopore channel, and prefilling the at least one surrogate polymer in the at least one nanopore channel. 
     
     
         129 . The method of  claim 128 , wherein the stop is selected from a bulky dendrimer and a bead. 
     
     
         130 . The method of  claim 129 , wherein the bead is a magnetic bead.