Additives to improve sequencing by synthesis performance
The invention relates to methods, compositions, devices, systems and kits are described including, without limitation, reagents and mixtures, for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods.
1. A method of incorporating nucleotides, comprising:
a) providing i) a plurality of nucleic acid primers and template molecules, ii) an extend reagent comprising polymerase and a plurality of nucleotide analogues, and iii) a wash reagent comprising methylenediphosphonic acid;
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) exposing said hybridized primers to said extend reagent under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue;
d) washing said extended primers with said wash reagent.
2. The method of claim 1 , wherein said incorporated nucleotide analogue comprises a label attached through a cleavable disulfide linker to the base.
3. The method of claim 2 , wherein said label is fluorescent.
4. The method of claim 2 , wherein said extend reagent further comprises cystamine.
5. The method of claim 2 , further comprising:
e) detecting said label of a first labeled nucleotide analogue.
6. The method of claim 5 , wherein said detecting of step e) is performed in the presence of methylenediphosphonic acid.
7. The method of claim 1 , wherein the concentration of said methylenediphosphonic acid is between 0.1 mM and 4 mM.
8. The method of claim 1 , wherein the concentration of said methylenediphosphonic acid is between 0.25 mM and 1 mM.
9. The method of claim 1 , wherein the concentration of said methylenediphosphonic acid is between 0.35 mM and 0.65 mM.