Polynucleotide sequencing methods

Abstract

Methods for determining the sequence of a polynucleotide comprising the steps of (i) contacting a polynucleotide processive enzyme immobilised in a fixed position, with a target polynucleotide under conditions sufficient to induce enzyme activity; (ii) detecting an effect consequent on the interaction of the enzyme and the polynucleotide, wherein the effect is detected by measurement of a non-linear optical signal or a linear signal coupled to a non-linear signal.

1 . A method for determining the sequence of a polynucleotide, comprising the steps of:
 (i) contacting a polynucleotide processive enzyme immobilised in a fixed position, with a target polynucleotide under conditions sufficient to induce enzyme activity, wherein a dipolar molecule is positioned on or proximal to the enzyme, and wherein the enzyme activity occurs in the presence of the nucleoside triphosphates dATP, dTTP, dGTP, dCTP, or combinations thereof;   (ii) detecting an effect consequent on the interaction of the enzyme and polynucleotide, therein determining the sequence of the target polynucleotide;   wherein the dipolar molecule is attached to the individual bases of the target polynucleotide, wherein the effect is detected by measurement of a non-linear optical signal or a linear signal coupled to a non-linear signal, and wherein the detection method comprises Near-Field Scanning Optical Microscopy.   
     
     
         2 . The method according to  claim 1 , wherein the effect is detected by measurement of a non-linear signal. 
     
     
         3 . The method according to  claim 1 , wherein the non-linear optical detection is second or third harmonic generation imaging. 
     
     
         4 . The method according to  claim 1 , wherein the detection method further comprises Raman spectroscopy or surface enhanced Raman spectroscopy. 
     
     
         5 . The method according to  claim 1 , wherein the dipolar molecule is a styryl dye molecule. 
     
     
         6 . The method according to  claim 1 , wherein the dipolar molecule is green fluorescent protein. 
     
     
         7 . The method according to  claim 1 , wherein the enzyme is a polymerase. 
     
     
         8 . The method according to  claim 1 , wherein the enzyme is a helicase or primase enzyme. 
     
     
         9 . The method according to  claim 1 , wherein the enzyme activity occurs in the presence of the nucleoside triphosphates dATP, dTTP, dGTP and dCTP. 
     
     
         10 . The method according to  claim 9 , wherein the nucleoside triphosphates comprise one or more blocking groups which can be removed selectively by pulsed monochromatic light. 
     
     
         11 . The method according to  claim 1 , wherein a metal nanoparticle is positioned on or proximal to the enzyme. 
     
     
         12 . The method according to  claim 11 , wherein the nanoparticle is a gold or silver nanoparticle. 
     
     
         13 . The method according to  claim 11 , wherein the nanoparticle is incorporated onto one or more of the individual bases of the polynucleotide. 
     
     
         14 . The method according to  claim 1 , wherein the enzyme is immobilised on a solid support. 
     
     
         15 . The method according to  claim 14 , wherein there are a plurality of enzymes immobilised on the solid support. 
     
     
         16 . The method according to  claim 14 , wherein the solid support has a roughened metal surface. 
     
     
         17 . The method according to  claim 14 , wherein the support is silver or gold. 
     
     
         18 . The method according to  claim 1 , further comprising the application of localized surface plasmon resonance. 
     
     
         19 . The method according to  claim 1 , wherein the enzyme is immobilised in a fixed position within a cell. 
     
     
         20 . The method according to  claim 1 , wherein the nucleoside triphosphates comprise one or more blocking groups. 
     
     
         21 . A method for determining the sequence of a polynucleotide, comprising the steps of:
 (i) contacting a polynucleotide processive enzyme immobilised in a fixed position, with a target polynucleotide under conditions sufficient to induce enzyme activity, wherein a dipolar molecule is positioned on or proximal to the enzyme, and wherein the enzyme activity occurs in the presence of the nucleoside triphosphates dATP, dTTP, dGTP, dCTP, or combinations thereof;   (ii) detecting an effect consequent on the interaction of the enzyme and polynucleotide, therein determining the sequence of the target polynucleotide;   wherein a metal nanoparticle is positioned on or proximal to the enzyme, wherein the nanoparticle is a gold or silver nanoparticle and wherein the nanoparticle is incorporated onto one or more of the individual bases of the polynucleotide;   wherein the effect is detected by measurement of a non-linear optical signal or a linear signal coupled to a non-linear signal, and wherein the detection method comprises Near-Field Scanning Optical Microscopy.   
     
     
         22 . A method for determining the sequence of a polynucleotide, comprising the steps of:
 (i) contacting a polynucleotide processive enzyme immobilised in a fixed position, with a target polynucleotide under conditions sufficient to induce enzyme activity, wherein a dipolar molecule is positioned on or proximal to the enzyme, and wherein the enzyme activity occurs in the presence of the nucleoside triphosphates dATP, dTTP, dGTP, dCTP, or combinations thereof;   (ii) detecting an effect consequent on the interaction of the enzyme and polynucleotide, therein determining the sequence of the target polynucleotide;   wherein the enzyme is immobilised in a fixed position within a cell, wherein the effect is detected by measurement of a non-linear optical signal or a linear signal coupled to a non-linear signal, and wherein the detection method comprises Near-Field Scanning Optical Microscopy.