METHODS AND SYSTEMS FOR NUCLEIC ACID SEQUENCING
The disclosure provides methods for sequencing nucleic acids using, including with nucleotide analogs and subsequently appended labels.
1 . A method for determining a sequence of a template nucleic acid molecule, comprising:
(a) generating a reaction mixture comprising said template nucleic acid molecule, a primer capable of hybridizing to said template nucleic acid molecule, and a plurality of free nucleotide analogs, wherein a given free nucleotide analog of said plurality of free nucleotide analogs comprises a functional group;
(b) subjecting said reaction mixture to conditions sufficient to conduct a primer extension reaction on said template nucleic acid molecule in presence of said primer, to incorporate said given free nucleotide analog comprising said functional group into a growing nucleic acid strand having sequence complementarity with said template nucleic acid molecule;
(c) upon incorporating said given free nucleotide analog into said growing nucleic acid strand, reacting said functional group with a labeling reagent comprising a label to generate a labeled functional group;
(d) detecting one or more signals indicative of said labeled functional group; and
(e) subjecting said labeled functional group to conditions sufficient to convert said labeled functional group to a moiety that is substantially unreactive with said labeling reagent.
2 . The method of claim 1 , wherein said template nucleic acid molecule is immobilized to a support.
3 . The method of claim 1 , wherein said support is a bead or a substantially planar surface.
4 . The method of claim 1 , wherein said functional group comprises sulfur or selenium.
5 . The method of claim 4 , wherein said plurality of nucleotide analogs comprises alpha-thio-deoxynucleotide triphosphates (α-S-dNTPs).
6 . The method of claim 1 , wherein the functional group comprises azido groups.
7 . The method of claim 1 , wherein said labeling reagent comprises a luminescent or an optically-active moiety.
8 . The method of claim 1 , wherein said labeling reagent comprises a self-quenching dye or a proximity quenching dye.
9 . The method of claim 1 , wherein (c) comprises contacting said functional group with a solution comprising said labeling reagent.
10 . The method of claim 9 , wherein said solution comprises a derivative of said label, wherein said derivative lacks a detectable moiety of said label.
11 . The method of claim 1 , wherein (c) comprises contacting said functional group with an antigen specific for said functional group and capable of coupling to said label.
12 . The method of claim 1 , wherein (c) comprises covalently coupling said label with at least a portion of said functional group.
13 . The method of claim 12 , further comprising, subsequent to (c), subjecting said template nucleic acid molecule to one or more washing cycles.
14 . The method of claim 1 , wherein (c) comprises conducting an alkylation reaction using said label and said functional group.
15 . The method of claim 1 , wherein said label is derived from Atto-647N-iodoacetamide, an S-pyridyl-containing reagent, monobromobimane, Cy5, Cy5-azide, Atto-633-iodoacetamide, Bodipy FL iodoacetamide, tetramethylrhodamine iodoacetamide or Atto-488 iodoacetamide.
16 . The method of claim 15 , wherein said label is derived from said S-pyridyl-containing reagent, and wherein (e) is conducted at a pH of about 5 to about 6.
17 . The method of claim 1 , wherein (c) comprises conducting a click reaction.
18 . The method of claim 17 , wherein said click reaction is conducted in the presence of an alkyne moiety, an azide moiety and copper(I).
19 . The method of claim 17 , wherein said click reaction is conducted without copper in the reaction.
20 . The method of claim 17 , wherein said click reaction is conducted in the presence of a reagent comprising dibenzocyclooctyne and azide moieties or trans-cycloocteyne and tetrazine moieties.