Labelled nucleotides

Abstract

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

1 . A nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: 
       
         
           
           
               
               
           
         
         (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10  substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10  substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). 
       
     
     
         2 . The nucleotide or nucleoside as claimed in  claim 1  wherein X is O or S. 
     
     
         3 . The nucleotide or nucleoside as claimed in  claim 1  or  claim 2  wherein Y is O or S. 
     
     
         4 . The nucleotide or nucleoside as claimed in any one of  claims 1  to  3  wherein Y is O. 
     
     
         5 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the moiety may be present in the nucleotide or nucleoside in either of two orientations. 
     
     
         6 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the base is a purine, or a pyrimidine. 
     
     
         7 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the linker is attached to the 5-position of a pyrimidine or 7-position of a purine. 
     
     
         8 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the base is a deazapurine. 
     
     
         9 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the nucleotide has a ribose or deoxyribose sugar moiety. 
     
     
         10 . The nucleotide or nucleoside as claimed in  claim 9  wherein the ribose or deoxyribose sugar comprises a hydroxyl protecting group attached to the 2′ or 3′ oxygen atom. 
     
     
         11 . The nucleotide or nucleoside as claimed in  claim 10  wherein the same chemical conditions may be used to effect cleavage of the cleavable linker and to remove the hydroxyl protecting group. 
     
     
         12 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the nucleotide is a deoxyribonucleotide triphosphate. 
     
     
         13 . The nucleotide or nucleoside as claimed in any one preceding claim wherein the detectable label is a fluorophore. 
     
     
         14 . An oligonucleotide comprising one or more nucleotides as defined in any one of  claims 1  to  13 . 
     
     
         15 . The oligonucleotide as claimed in  claim 14  wherein at least one nucleotide is present at a terminal position in said oligonucleotide. 
     
     
         16 . A method of cleaving a linker that contains a moiety selected from the groups comprising: 
       
         
           
           
               
               
           
         
         (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10  substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10  substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of a nucleotide or nucleoside), 
         said linker being present in a nucleotide or nucleoside and connecting the base thereof to a detectable label, said method comprising contacting the nucleotide or nucleoside with a water-soluble phosphine-based transition metal catalyst. 
       
     
     
         17 . The method as claimed in  claim 16  wherein the transition metal is selected from the group comprising platinum, palladium, rhodium, ruthenium, osmium and iridium. 
     
     
         18 . The method as claimed in  claim 16  wherein the transition metal is palladium. 
     
     
         19 . A method of cleaving a linker that contains a moiety selected from the groups comprising: 
       
         
           
           
               
               
           
         
         (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10  substituted or unsubstituted alkyl group, T is hydrogen or a C 1-10  substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of a nucleotide or nucleoside),
 said linker being present in a nucleotide or nucleoside and connecting the base thereof to a detectable label, said method comprising contacting the nucleotide or nucleoside with a water-soluble phosphine. 
 
       
     
     
         20 . The method as claimed in any one of  claims 16  to  19  wherein said phosphine is a derivatised triaryl phosphine or a derivatised trialkyl phosphine. 
     
     
         21 . The method as claimed in any one of  claims 16  to  20  wherein said phosphine is a triaryl phosphine derivatised with one or more functionalities selected from the group comprising amino, hydroxyl, carboxyl and sulfonate. 
     
     
         22 . The method as claimed in any one of  claim 16  to  21  wherein the water-soluble phosphine is selected from the group comprising 3,3′,3″-phosphinidynetris (benzenesulfonic acid) or tris(2-carboxyethyl)phosphine and their salts. 
     
     
         23 . The method as claimed in any one of  claims 16  to  19  wherein said phosphine contains one or more nitrogen atoms. 
     
     
         24 . The method as claimed in any one of  claims 16  to  23  wherein X is O or S. 
     
     
         25 . The method as claimed in any one of  claims 16  to  24  wherein Y is O or S. 
     
     
         26 . The method as claimed in any one of  claims 16  to  25  wherein Y is O. 
     
     
         27 . The method as claimed in any one of  claims 16  to  26  wherein the moieties may be present in the nucleoside or nucleotide in either of two orientations. 
     
     
         28 . The method of in any one of  claims 16  to  27  wherein said label is detected before said linker is cleaved. 
     
     
         29 . The method of  claim 28  wherein said method involves cleavage of the linker in a nucleotide which is incorporated into an oligonucleotide. 
     
     
         30 . The method of  claim 29  wherein said incorporated nucleotide is present at a terminal position in said oligonucleotide. 
     
     
         31 . The method as claimed in any one of  claim 16  to  30  wherein the base is a purine, or a pyrimidine. 
     
     
         32 . The method of  claim 31 , wherein the linker is attached to the 5-position of a pyrimidine or 7-position of a purine. 
     
     
         33 . The method as claimed in any one of  claims 16  to  32  wherein the base is a deazapurine. 
     
     
         34 . The method as claimed in any one of  claims 16  to  33  wherein the nucleotide has a ribose or deoxyribose sugar moiety. 
     
     
         35 . The method as claimed in  claim 34  wherein the ribose or deoxyribose sugar comprises a hydroxyl protecting group attached to the 2′ or 3′ oxygen atom. 
     
     
         36 . The method as claimed in any one of  claims 16  to  35  wherein the nucleotide is a deoxyribonucleotide triphosphate. 
     
     
         37 . The method as claimed in any one of  claims 16  to  36  wherein the detectable label is a fluorophore. 
     
     
         38 . The method as claimed in any one of  claims 29  to  37  wherein the incorporating step is effected by a reverse transcriptase, a terminal transferase or a polymerase. 
     
     
         39 . The method of  claim 38  wherein the polymerase is a  Thermococcus  sp. 
     
     
         40 . The method of  claim 39  wherein the  Thermococcus  sp is 9°N or a single mutant or double mutant thereof. 
     
     
         41 . The method of  claim 40  wherein the double mutant is −Y409V A485L. 
     
     
         42 . The method as claimed in any one of  claims 29  to  41  wherein the detectable label and/or the cleavable linker is of a size sufficient to prevent the incorporation of a subsequent nucleotide into the nascent oligonucleotide. 
     
     
         43 . The method as claimed in any one of  claims 29  to  42  wherein the incorporated nucleotide contains a 3′OH blocking group which serves to prevent incorporation of any further nucleotides. 
     
     
         44 . The method as claimed in  claim 43  wherein the same chemical conditions used to effect cleavage of the cleavable linker serve to remove the 3′OH blocking group. 
     
     
         45 . The method as claimed in any one of  claims 29  to  44  wherein the detecting step permits the identification of the incorporated nucleotide. 
     
     
         46 . A method for determining the identity of a nucleotide in a target single-stranded polynucleotide, comprising:
 (a) providing one or more of the nucleotides A, G, C and T or U in which each of said nucleotides has a base that is attached to a distinct detectable label via a linker, said linker being cleavable with a water-soluble phosphine; and a nascent polynucleotide complementary to the target polynucleotide, one of said provided nucleotides being suitable for incorporation into said nascent polynucleotide;   (b) incorporating the nucleotide suitable for incorporation into said nascent polynucleotide; and   (c) carrying out a method as defined in  claim 45 .   
     
     
         47 . The method as claimed in  claim 46  wherein steps (a) and (b) are repeated one or more times so as to determine the identity of a plurality of bases in the target polynucleotide. 
     
     
         48 . A method as claimed in  claim 46  or  claim 47  wherein step (a) comprises contacting the provided nucleotides with the target sequentially. 
     
     
         49 . A method as claimed in any one of  claims 46  to  48  wherein step (a) comprises at least one substep of providing one of the four said nucleotides. 
     
     
         50 . A method as claimed in  claim 49  wherein step (a) further comprises, after said substep, providing the other three nucleotides simultaneously or sequentially. 
     
     
         51 . A method as claimed in  claim 50  wherein said other three nucleotides are added sequentially, either by providing them one at a time; or two simultaneously and then the remaining one; or one of the three and then the remaining two simultaneously. 
     
     
         52 . A method as claimed in any one of  claims 46  to  48  wherein step (a) comprises at least a substep of providing two of the four said nucleotides. 
     
     
         53 . A method as claimed in  claim 52  wherein step (a) further comprises, after said substep, providing the other two nucleotides simultaneously or sequentially. 
     
     
         54 . A method as claimed in any one of  claims 46  to  48  wherein step (a) comprises at least a substep of providing three of the four said nucleotides. 
     
     
         55 . A method as claimed in  claim 54  wherein step (a) further comprises, after said substep, providing the remaining nucleotide of the four said nucleotides. 
     
     
         56 . A method as claimed in any one of  claims 46  to  48  wherein step (a) comprises providing all four of the said nucleotides and contacting them with the target simultaneously. 
     
     
         57 . A method as claimed in any one of  claims 46  to  56  wherein any unincorporated nucleotides are removed prior to the provision of further nucleotide(s) and/or the effecting of step (c). 
     
     
         58 . A method as claimed in  claim 57  wherein step (c) is effected without unsuitable nucleotides having been provided after provision of said suitable nucleotide. 
     
     
         59 . Use of a nucleotide as defined in any one of  claims 1  to  13  in a Sanger or Sanger-type sequencing method. 
     
     
         60 . A method of using a nucleotide of  claim 1  wherein said method includes a Sanger or Sanger-type sequencing method.