Photoprotective mixtures as imaging reagents in sequencing-by-synthesis

Abstract

The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of photoprotective mixture of compounds as imaging reagents to improve stability and storage of fluorescent compounds, including but not limited to, nucleotides with fluorescent labels.

We claim: 
     
         1 . A method of incorporating labeled nucleotides, comprising:
 a) providing;
 i) a plurality of nucleic acid primers and template molecules, 
 ii) a polymerase, 
 iii) an imaging reagent comprising a photoprotective mixture of compounds, and 
 iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable linker to the base; 
   b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;   c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue; and   d) imaging said incorporated labeled nucleotide analogue in the presence of said imaging reagent.   
     
     
         2 . The method of  claim 1 , wherein said photoprotective mixture comprises at least one fluorescence quenching inhibitor. 
     
     
         3 . The method of  claim 2 , wherein said at least one fluorescence quenching inhibitor is trolox. 
     
     
         4 . The method of  claim 1 , wherein said photoprotective mixture further comprises at least one antioxidant and at least one radical scavenger compound. 
     
     
         5 . The method of  claim 4 , wherein said at least one antioxidant is selected from the group consisting of gentisic acid, protocatechuic acid and protocatechuate ethyl ester. 
     
     
         6 . The method of  claim 4 , wherein said at least one radical scavenger compound is carnitine. 
     
     
         7 . The method of  claim 1 , further comprising step (e) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers. 
     
     
         8 . The method of  claim 1 , wherein said label is fluorescent. 
     
     
         9 . An imaging reagent comprising at least one antioxidant, at least one fluorescence quenching inhibitor and a buffer. 
     
     
         10 . The imaging reagent of  claim 9 , wherein said at least one antioxidant comprises compounds selected from the group consisting of gentisic acid, protocatechuic acid and protocatechuate ethyl ester. 
     
     
         11 . The imaging reagent of  claim 9 , further comprising at least one radical scavenger. 
     
     
         12 . The imaging reagent of  claim 11 , wherein said at least one radical scavenger is carnitine. 
     
     
         13 . The imaging reagent of  claim 9 , wherein said fluorescence quenching inhibitor is trolox. 
     
     
         14 . The imaging reagent of  claim 9 , wherein said buffer is a TRIS buffer. 
     
     
         15 . The imaging reagent of  claim 9 , wherein said buffer is a HEPES buffer. 
     
     
         16 . A kit, comprising:
 i) a first container comprising an imaging reagent comprising at least one antioxidant, at least one fluoresence quenching inhibitor and a buffer and   ii) a second container comprising a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable linker to the base.   
     
     
         17 . The kit of  claim 16 , wherein said first container further comprises at least one radical scavenger. 
     
     
         18 . The kit of  claim 17 , wherein said at least one radical scavenger is carnitine. 
     
     
         19 . The kit of  claim 16 , wherein said at least one fluorescence quenching inhibitor is trolox. 
     
     
         20 . The kit of  claim 16 , wherein said buffer is a TRIS buffer. 
     
     
         21 . The kit of  claim 16 , wherein said buffer is a HEPES buffer. 
     
     
         22 . A system comprising a solution of primers hybridized to a template comprising a plurality of nucleotide analogues attached to a cleavable label and an imaging reagent comprising at least one antioxidant, at least one fluorescent quenching inhibitor and a buffer. 
     
     
         23 . The system of  claim 22 , wherein said hybridized primers and said template are immobilized. 
     
     
         24 . The system of  claim 22 , wherein said hybridized primers and said template are in a flow cell. 
     
     
         25 . The system of  claim 22 , wherein said imaging agent further comprises at least one radical scavenger. 
     
     
         26 . The system of  claim 25 , wherein said at least one radical scavenger is carnitine. 
     
     
         27 . The system of  claim 22 , wherein said at least one fluorescence quenching inhibitor is trolox. 
     
     
         28 . The system of  claim 22 , wherein said buffer is a TRIS buffer. 
     
     
         29 . The system of  claim 22 , wherein said buffer is a HEPES buffer. 
     
     
         30 . An imaging reagent comprising:
 i) a TRIS HCl buffer;   ii) carnitine ranging in concentration between approximately 5-50 mM;   iii) trolox ranging in concentration between approximately 5-15 mM;   iv) 2,5 dihydroxybenzoic acid ranging in concentration between approximately 10 -50 mM; and   v) 3,4, dihydroxybenzoic acid ethyl ester ranging in concentration between approximately 10-20 mM.