Super-Resolution Sequencing

Abstract

A method for template-directed sequencing-by-synthesis of an array of target polynucleotide can include: (a) providing an array of target polynucleotides in a fluidic vessel; (b) contacting the array of polynucleotides with a solution comprising (i) polymerization complex and (ii) reversibly terminating and differently labeled A, C, G, and T/U nucleotides; (c) incorporating one of the differently labeled nucleotides, using the polymerization complex, into a chain complementary to at least one of the array of polynucleotides; (d) binding imaging tags to the differently labeled nucleotides of step (c); (e) imaging and storing the identity and position of the imaging tags of step (d); (f) reversing termination (b)-(e); (g) repeating steps (b)-(e) and assembling a sequence for each of the array of target polynucleotides from the stored identity and position of the imaging tags, optionally as a homogeneous or one pot reaction. Additional methods of sequencing target polynucleotides are described herein.

1 - 20 . (canceled) 
     
     
         21 . A method of sequencing a target polynucleotide comprising the steps of:
 (a) positioning the target polynucleotide onto a surface;   (b) contacting the target polynucleotide with a solution comprising
 (i) a polymerase comprising an initiation and extension complex, and 
 (ii) four types of reversible terminators, wherein each reversible terminator nucleotide comprises the structure: N-XT, wherein N is a nucleotide A, C, G or T/U, and X represents a cleavable group chemically linked to a terminator (T); 
 and incorporating one of the reversible terminator nucleotides A, C, G or T/U. 
   (c) incubating target polynucleotide with a polymerase under conditions to transiently bind a nucleotide complementary to the target polynucleotide adjacent to the reversible terminator nucleotides, wherein the nucleotide complementary to the target polynucleotide has the structure: N-L, wherein N is a nucleotide and L is a label;   (d) imaging under illumination with one or more wavelength of electromagnetic radiation;   (e) detecting a persistent signal from individual labeled nucleotides at specific locations on the surface; thereby identifying the identity of the differently labeled nucleotide bound at those locations;   (f) inducing or allowing cleavage to remove the cleavable group in step (b); and   (g) cycling by repeating steps (b) through (f) thereby sequencing the target polynucleotide by compiling the identity of the nucleotides identified in (e) in each cycle.   
     
     
         22 . The method of  claim 21 , wherein the reversible terminators are labelled. 
     
     
         23 . The method of  claim 22 , wherein the labelled reversible terminators provide an independent reading of the polynucleotide independent of the transient binding nucleotide. 
     
     
         24 . The method of  claim 22 , wherein the labelled reversible terminators act as a resonance energy transfer (RET) partner to the transiently binding nucleotide. 
     
     
         25 . The method of  claim 21 , wherein the cleavable group in step (g) is removed by illuminating the target polynucleotide with a second wavelength of electromagnetic radiation. 
     
     
         26 . The method of  claim 21 , wherein the cleavable group on step (g) is removed by exposing the target polynucleotide by chemical or biochemical means. 
     
     
         27 . The method of  claim 21 , wherein the transient binding comprises multiple, stochastic on/off binding events. 
     
     
         28 . The method of  claim 21 , wherein the target polynucleotide is stretched and/or elongated. 
     
     
         29 . The method of  claim 21 , further comprising sequencing two or more polynucleotides simultaneously. 
     
     
         30 . The method of  claim 21 , wherein the label comprises a fluorescent organic dye or fluorescent nanoparticle. 
     
     
         31 . The method of  claim 21 , wherein the nucleotide comprises a quencher. 
     
     
         32 . The method of  claim 21 , wherein the imaging step (d) comprises providing electromagnetic radiation via an evanescent wave. 
     
     
         33 . The method of  claim 21 , wherein the imaging step (d) comprises enhancing the fluorescence by proximity related effects with metals. 
     
     
         34 . The method of  claim 21 , wherein the imaging step (d) comprises controlling the attraction and repulsion of the labeled nucleotides using an electric field. 
     
     
         35 . The method of  claim 21 , wherein detecting the signal from the individual labeled nucleotides at specific locations on the surface in step (e) is conducted by a method comprising single molecule localization, stochastic optical reconstruction microscopy (STORM), points accumulation for imaging in nanoscale topography (PAINT), or stimulated emission depletion (STED). 
     
     
         36 . The method of  claim 21 , wherein the initiation and extension complex comprises a primer or a 3′ nick. 
     
     
         37 . The method of  claim 21 , wherein the target polynucleotide is DNA and the polymerase is DNA polymerase. 
     
     
         38 . The method of  claim 21 , wherein the target polynucleotide is RNA and the polymerase is RNA polymerase. 
     
     
         39 . The method of  claim 21 , wherein the nucleotide in step (b) and (c) are the same structure comprising a label at the terminal phosphate and a reversible terminator at 3′ end of the nucleotide. 
     
     
         40 . The method of  claim 21 , wherein the method is conducted in a homogenous or one-pot manner. 
     
     
         41 . A method of sequencing a target polynucleotide comprising the steps of:
 (a) positioning the target polynucleotide onto a surface;   (b) contacting the target polynucleotide with a solution comprising;
 (i) a polymerase comprising an initiation and extension complex, and 
 (ii) four types of labelled nucleotides, of the structure: N-L, wherein N is a nucleotide and L is a label in a buffer not comprising a divalent cation, 
   (c) incubating the polymerase under conditions to transiently bind a nucleotide complementary to the target polynucleotide adjacent to the 3′ end of a primer or nick;   (d) imaging under illumination with one or more wavelength of electromagnetic radiation;   (e) detecting a persistent signal from individual labeled nucleotides at specific locations on the surface; thereby identifying the identity of the differently labeled nucleotide bound at those locations;   (f) adding and removing one or more buffers, at least one of which comprises a divalent cation to enable incorporation of only one of the one of the four types of nucleotides;   (g) cycling by repeating steps (b) through (f) thereby sequencing the target polynucleotide by compiling the identity of the nucleotides identified in (e) in each cycle.   
     
     
         42 . The method of  claim 41 , wherein in step (b)(ii), the nucleotide is labeled at the terminal phosphate. 
     
     
         43 . The method of  claim 41 , wherein the persistent signal comprises, repetitive transient binding and unbinding.