Polyphenolic additives 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 polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.
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 cleave reagent comprising a reducing agent and the polyphenolic compound gallic acid, 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 disulfide 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;
d) detecting said incorporated labeled nucleotide analogue; and
e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.
2. The method of claim 1 , wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).
3. The method of claim 1 , wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′-OH group.
4. The method of claim 2 , wherein the cleaving of step e) removes the removable chemical moiety capping the 3′-OH group.
5. The method of claim 4 , wherein the method further comprises:
f) 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. A kit, comprising i) a cleave reagent comprising i) a reducing agent, ii) a polyphenolic compound selected from the group consisting of gallic acid, gentisic acid, pryocatechol, and pyrogallol and iii) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base.
8. A system comprising primers hybridized to template in solution, said solution comprising a cleave reagent comprising i) a reducing agent, and ii) a polyphenolic compound selected from the group consisting of gallic acid, gentisic acid, pryocatechol, and pyrogallol.
9. The system of claim 8 , wherein said hybridized primers and template are immobilized.
10. The system of claim 8 , wherein said hybridized primers and template are in a flow cell.