Sequencing method

Abstract

Disclosed is a single molecule sequencing method comprising the steps of: a) attaching a molecule to a solid phase; b) incubating the molecule with a first composition comprising a first reporter moiety; c) detecting incorporation of said first reporter moiety; d) performing a reaction to eliminate said first reporter moiety; e) incubating the molecule with a second composition comprising a second reporter moiety; and f) detecting incorporation of said second reporter moiety; characterized in that the first and second reporter moieties can be distinguished from each other.

1 . A single molecule sequencing method comprising the steps of 
 a) attaching a molecule to a solid phase    b) incubating the molecule with a first composition comprising a first reporter moiety    c) detecting incorporation of said first reporter moiety    d) performing a reaction to eliminate said first reporter moiety    e) incubating the molecule with a second composition comprising a second reporter moiety    f) detecting incorporation of said second reporter moiety characterised in that the first and second reporter moieties can be distinguished from each other.    
     
     
         2 . A method as claimed in  claim 1  wherein a plurality of molecules are attached to the solid phase in a random distribution.  
     
     
         3 . A method as claimed in  claim 1  wherein the molecule is a nucleic acid molecule, preferably a DNA molecule and, most preferably, a complex of a primer and a template.  
     
     
         4 . A method as claimed in  claim 3  wherein the first and second compositions comprise at least one nucleotide or polynucleotide and, preferably, wherein the first and second reporter moieties in the respective first and second compositions are incorporated into the at least one nucleotide or polynucleotide comprised therein.  
     
     
         5 . A method as claimed in  claim 4  wherein the first reporter moiety labels one nucleotide or polynucleotide in the first composition and the second reporter moiety labels a different nucleotide or polynucleotide in the second composition.  
     
     
         6 . A method as claimed in  claim 4  wherein the nucleotides are purines or pyrimidines and, preferably, any of the natural bases A, C, G and T, their analogues or modified variants thereof.  
     
     
         7 . A method as claimed in  claim 4  wherein the polynucleotides are adapter molecules.  
     
     
         8 . A method for nucleic acid sequencing comprising the steps of 
 a) attaching a primer/template complex to a solid phase    b) incubating the primer/template complex in the presence of a first composition comprising at least one reporter moiety and a polymerase    c) detecting incorporation of said first reporter moiety    d) performing a reaction to eliminate said first reporter moiety    e) incubating the primer/template complex in the presence of a second composition comprising a second reporter moiety and a polymerase    f) detecting incorporation of said second reporter moiety characterised in that the first and second reporter moieties can be distinguished from each other.    
     
     
         9 . A method as claimed in  claim 1  further comprising the steps 
 g) incubating the molecule in the presence of a further composition comprising a further reporter moiety; and    h) detecting incorporation of said further reporter moiety characterised in that the further reporter moiety can be distinguished from either the first or the second reporter moiety.    
     
     
         10 . A method as claimed in  claim 1  wherein each composition comprises at least two labelled nucleotides wherein different reporter moieties label different bases and the reporter moieties in the first composition are distinguishable from each other and from those in the second composition.  
     
     
         11 . A method as claimed in  claim 1  wherein each composition comprises all four natural bases, (their analogues or modified variants thereof), A, C, G and T, each labelled with a different reporter moiety.  
     
     
         12 . A method as claimed in  claim 1  wherein the molecule is labelled with a reporter moiety such that its location on the solid phase may be detected.  
     
     
         13 . A method as claimed in  claim 1  wherein incorporation of a reporter moiety is detected using an optical microscope linked to a sensitive detector, resulting in a distinct signal for each molecule.  
     
     
         14 . A method of detecting errors in a sequencing reaction comprising the steps of 
 a) attaching a molecule to a solid phase    b) incubating the molecule with a first composition comprising a first reporter moiety    c) detecting incorporation of said first reporter moiety    d) performing a reaction to eliminate said first reporter moiety    e) incubating the molecule with a second composition comprising a second reporter moiety    f) detecting incorporation of said second reporter moiety and detecting presence of said first reporter moiety as an indication of a false base call characterised in that the first and second reporter moieties can be distinguished from each other and wherein the presence of the first reporter moiety after the elimination step d) is indicative of an error.    
     
     
         15 . A kit for a nucleotide sequencing method as claimed in  claim 1 , said kit comprising a first set of nucleotides comprising at least one nucleotide labelled with a reporter moiety and a second set of nucleotides comprising at least one nucleotide labelled with a reporter moiety characterised in that the reporter moiety in the first set of nucleotides is distinguishable from that in the second set.