Photoprotective mixtures as imaging reagents in sequencing-by-synthesis
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.
1. A method of incorporating labeled nucleotides, comprising:
a) providing;
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a first imaging reagent comprising a mixture of at least one fluorescence quenching inhibitor, at least one antioxidant, and at least one radical scavenger compound, 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 first imaging reagent.
2. The method of claim 1 , wherein said at least one fluorescence quenching inhibitor is 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
3. The method of claim 1 , wherein said at least one antioxidant is selected from the group consisting of gentisic acid, protocatechuic acid and protocatechuate ethyl ester.
4. The method of claim 1 , wherein said at least one radical scavenger compound is carnitine.
5. 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.
6. The method of claim 1 , wherein said label is fluorescent.
7. An imaging reagent comprising:
i) 2-Amino-2-hydroxymethyl-propane-1,3-diol (TRIS) HCl buffer;
ii) carnitine ranging in concentration between approximately 5-50 mM;
iii) 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 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.
8. The method of claim 1 , wherein said at least one antioxidant comprises gentisic acid, said at least one radical scavenger compound comprises carnitine, and said at least one fluorescence quenching inhibitor comprises 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
9. The method of claim 1 , wherein said at least one antioxidant comprises protocatechuic acid ethyl ester, said at least one radical scavenger compound comprises carnitine, and said at least one fluorescence quenching inhibitor comprises 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
10. The method of claim 1 , wherein said imaging delivers an average read length that is substantially similar to a read length obtained when said first imaging reagent is replaced by a second imaging reagent comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, glucose oxidase, glucose, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.