Compositions, systems, and methods for detecting the presence of polymer subunits using chemiluminescence

Abstract

Under one aspect, a composition includes a substrate; a first polynucleotide coupled to the substrate; a second polynucleotide hybridized to the first polynucleotide; and a catalyst coupled to a first nucleotide of the second polynucleotide, the catalyst being operable to cause a chemiluminogenic molecule to emit a photon. Under another aspect, a method includes providing a catalyst operable to cause a first chemiluminogenic molecule to emit a photon; providing a substrate; providing a first polynucleotide coupled to the substrate; hybridizing a second polynucleotide to the first polynucleotide; coupling a first quencher to a first nucleotide of the second polynucleotide; and inhibiting, by the first quencher, photon emission by the first chemiluminogenic molecule.

1 .- 68 . (canceled) 
     
     
         69 . A method of sequencing a nucleic acid, comprising:
 (a) hybridizing a polynucleotide to a nucleic acid attached to a substrate;   (b) incorporating a nucleotide into the polynucleotide by contacting the polynucleotide with a plurality of nucleotides in the presence of a polymerase, wherein the plurality of nucleotides comprises:
 a first subset of nucleotides comprising a first moiety, 
 a second subset of nucleotides comprising a second moiety, 
 a third subset of nucleotide comprising a third moiety, and 
 a fourth subset of nucleotides comprising a fourth moiety; and 
   (c) determining the identity of the incorporated nucleotide by:
 (i) contacting the incorporated nucleotide with a first catalyst coupled to a fifth moiety, wherein the fifth moiety is capable of selectively coupling to one, two or three moieties selected from the first, second, third or fourth moieties, 
 (ii) detecting the presence or absence of the first catalyst coupled to the incorporated nucleotide, 
 (iii) contacting the incorporated nucleotide with a second catalyst coupled to a sixth moiety, wherein the sixth moiety is capable of selectively coupling to one, two or three moieties selected from the first, second, third or fourth moieties, 
 (iv) detecting the presence or absence of the second catalyst coupled to the incorporated nucleotide, 
 (v) contacting the incorporated nucleotide with a cleaving agent, wherein the cleaving agent is capable of selectively cleaving one or more linkages between the fifth moiety and any one of the first, second, third or fourth moieties, or between the sixth moiety and any one of the first, second, third or fourth moieties, and 
 (vi) detecting the presence or absence of the first or second catalyst coupled to the incorporated nucleotide, thereby determining the identity of the incorporated nucleotide. 
   
     
     
         70 . The method of  claim 69 , wherein step (i) further comprises contacting the first catalyst with a chemiluminogenic molecule. 
     
     
         71 . The method of  claim 70 , wherein step (ii) comprises detecting the emission of or absence of a photon from the chemiluminogenic molecule. 
     
     
         72 . The method of  claim 69 , wherein step (iii) further comprises contacting the second catalyst with a chemiluminogenic molecule. 
     
     
         73 . The method of  claim 72 , wherein step (iv) comprises detecting the emission of or absence of a photon from the chemiluminogenic molecule. 
     
     
         74 . The method of  claim 69 , wherein step (v) further comprises contacting the first or second catalyst with a chemiluminogenic molecule. 
     
     
         75 . The method of  claim 74 , wherein step (vi) comprises detecting the emission of or absence of a photon from the chemiluminogenic molecule. 
     
     
         76 . The method of  claim 69 , further comprising repeating steps (b) and (c). 
     
     
         77 . The method of  claim 70 , wherein the first catalyst comprises a luciferase. 
     
     
         78 . The method of  claim 77 , wherein the chemiluminogenic molecule comprises luciferin or coelenterazine. 
     
     
         79 . The method of  claim 70 , wherein the first catalyst includes a peroxide generator. 
     
     
         80 . The method of  claim 79 , wherein the peroxide generator comprises a peroxide catalyst selected from the group consisting of an enzyme, a metallic catalyst, an organic catalyst, and a metalorganic catalyst. 
     
     
         81 . The method of  claim 80 , wherein the chemiluminogenic molecule comprises a substituent selected from the group consisting of luminol, a luminol derivative, and acridinium. 
     
     
         82 . The method of  claim 69 , wherein the fifth moiety and sixth moiety are capable of selectively coupling to a different first, second, third or fourth moiety from one another. 
     
     
         83 . The method of  claim 69 , wherein the first catalyst and second catalyst are the same as one another. 
     
     
         84 . The method of  claim 69 , wherein the first catalyst and second catalyst are different from one another. 
     
     
         85 . The method of  claim 69 , wherein the cleaving agent is selected from dithioerythritol (DTE), dithiothreitol (DTT), β-mercaptoethanol (BME), glutathione, thioglycolic acid, 2,3-dimercaptopropanol, tris (2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THP), and β-[tris(hydroxymethyl)phosphine] propionic acid (THPP). 
     
     
         86 . A method of sequencing a first polynucleotide, comprising:
 a) providing the first polynucleotide coupled to a substrate;   b) hybridizing a second polynucleotide to the first polynucleotide;   c) providing a catalyst coupled sufficiently close to the second polynucleotide that a quencher coupled to the second polynucleotide can inhibit photon emission from chemiluminescent molecules that interact with the catalyst;   d) contacting the second polynucleotide with a polymerase and a plurality of nucleotides comprising:
 a first subset of nucleotides comprising a first moiety, 
 a second subset of nucleotides comprising a second moiety, 
 a third subset of nucleotides comprising a third moiety, and 
 a fourth subset of nucleotides comprising a fourth moiety or no moiety; 
   e) adding a nucleotide of the plurality of nucleotides to the second polynucleotide based on a sequence of the first polynucleotide, thereby obtaining an added nucleotide;   f) exposing the added nucleotide to a quencher coupled to a fifth moiety;   g) exposing the added nucleotide to chemiluminogenic molecules;   h) detecting emission of photons or an absence of photons from the chemiluminogenic molecules;   i) exposing the added nucleotide to a quencher coupled to a sixth moiety;   j) exposing the added nucleotide to chemiluminogenic molecules;   k) detecting emission of photons or an absence of photons from the chemiluminogenic molecules;   l) exposing the added nucleotide to a cleaver molecule;   m) exposing the added nucleotide to chemiluminogenic molecules;   n) detecting emission of photons or an absence of photons from the chemiluminogenic molecules; and   o) detecting the added nucleotide based on the detection of emission of photons or absence of photons from the chemiluminogenic molecules at step h, k, n, or a combination thereof.