Use of Single-Stranded Nucleic Acid Binding Proteins In Sequencing
The invention provides methods for stabilizing a nucleic acid sequencing reaction. Generally, methods of the invention include exposing a target nucleic acid to a single-stranded nucleic acid binding protein and performing a sequencing reaction.
1 . A method for stabilizing a nucleic acid sequencing reaction, the method comprising the steps of:
exposing a mixture comprising a template, a polymerase, a primer, and at least one nucleotide to a single-stranded nucleic acid binding protein;
wherein said single-stranded nucleic acid binding protein binds to said template.
2 . The method of claim 1 , wherein said single-stranded nucleic acid binding protein increases the fidelity of said polymerase upon binding of said protein to said template.
3 . The method of claim 1 , wherein said nucleotide comprises a detectable label.
4 . The method of claim 2 , further comprising the detection of said nucleotide into said primer.
5 . The method of claim 1 , wherein said template is attached to a substrate such that it is individually optically resolvable.
6 . The method of claim 1 , wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.
7 . The method of claim 1 , wherein said nucleotide is labeled with a first label and wherein said polymerase comprises a second label.
8 . The method of claim 6 or 7 , wherein said first and second labels are fluorophores.
9 . The method of claim 8 , wherein said detecting step comprises detecting coincident fluorescence emission of said labeled nucleotide and said second label.
10 . The method of claim 1 , wherein said nucleotide is a nucleotide analog.
11 . The method of claim 10 , wherein said nucleotide analog is a chain terminating analog.
12 . A method for sequencing a polynucleotide, the method comprising the steps of:
(a) stabilizing a nucleic acid template/primer complex with a single-stranded nucleic acid binding protein;
(b) exposing said complex to a polymerase and at least one nucleotide capable of extending said primer;
(c) determining whether said nucleotide extends said primer;
(d) repeating said exposing and determining steps; and
(e) compiling a sequence of said polynucleotide based upon an order of nucleotides added to said primer.
13 . The method of claim 12 , wherein said template/primer complex is attached to a substrate such that it is individually optically resolvable.
14 . The method of claim 12 , further comprising the step of removing unincorporated nucleotide.
15 . The method of claim 12 , wherein said nucleotide comprises a detectable label.
16 . The method of claim 15 , further comprising the step of rendering said label undetectable subsequent to determining step.
17 . The method of claim 15 , wherein said nucleotide is labeled with a fluorophore.
18 . The method of claim 15 , wherein said determining step comprises optically detecting incorporation of said nucleotide.
19 . The method of claim 12 , wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.
20 . The method of claim 12 , wherein said nucleotide is labeled with a first label and wherein said polymerase comprises a second label.
21 . The method of claim 19 or 20 , wherein said first and second labels are fluorophores.
22 . The method of claim 21 , wherein said detecting step comprises detecting coincident fluorescence emission of said labeled nucleotide and said second label.
23 . The method of claim 12 , wherein said nucleotide is a nucleotide analog.
24 . The method of claim 23 , wherein said nucleotide analog is a chain terminating analog.
25 . A method for sequencing a nucleic acid template, the method comprising the steps of:
(a) exposing a nucleic acid template to a labeled nucleotide, a polymerase and a single-stranded nucleic acid binding protein under conditions that allow incorporation of said nucleotide into a primer attached to said template, wherein said single-stranded nucleic acid binding protein increases fidelity of said polymerase upon binding of said protein to said template;
(b) detecting incorporation of said nucleotide into said primer;
(c) repeating steps (a) and (b) at least once; and
(d) compiling a sequence of said template based upon an order of incorporated nucleotides.
26 . The method of claim 25 , wherein said template is attached to a substrate such that it is individually optically resolvable.
27 . The method of claim 25 , further comprising the step of removing unincorporated nucleotide.
28 . The method of claim 25 , further comprising the step of rendering said label undetectable subsequent to said detecting step.
29 . The method of claim 25 , wherein said nucleotide is labeled with a fluorophore.
30 . The method of claim 29 , wherein said detecting step comprises optically detecting incorporation of said nucleotide.
31 . The method of claim 25 , wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.
32 . The method of claim 25 , wherein said nucleotide is labeled with a first label and wherein said polymerase comprises a second label.
33 . The method of claim 31 or 32 , wherein said first and second labels are fluorophores.
34 . The method of claim 33 , wherein said detecting step comprises detecting coincident fluorescence emission of said labeled nucleotide and said second label.
35 . The method of claim 25 , wherein said nucleotide is a nucleotide analog.
36 . The method of claim 35 , wherein said nucleotide analog is a chain terminating analog.
37 . A method for sequencing a nucleic acid template, the method comprising the steps of:
(a) exposing a nucleic acid template to a nucleotide, a polymerase and a single-stranded nucleic acid binding protein under conditions that allow incorporation of said nucleotide into a primer attached to said template, said nucleotide comprising a first label, and said single-stranded nucleic acid binding protein comprising a second label,
wherein one of said first and second labels comprises a donor fluorophore and the other of said labels comprises an acceptor fluorophore; and
wherein, upon binding of said single-stranded nucleic acid binding protein to said template and incorporation of said nucleotide analogue into said primer, said acceptor fluorophore is optically detectable;
(b) detecting said acceptor fluorophore, thereby to detect incorporation of said nucleotide into said primer;
(c) repeating steps (a) and (b) at least once; and
(d) compiling a sequence of said template based upon an order of incorporated nucleotides.
38 . The method of claim 37 , wherein said template is attached to a substrate such that it is individually optically resolvable.
39 . The method of claim 25 or 37 , wherein said single-stranded nucleic acid binding protein is attached to a substrate, and wherein said single-stranded nucleic acid binding protein binds said template, thereby attaching said template to said substrate.
40 . The method of claim 37 , further comprising the step of removing unincorporated nucleotide.
41 . The method of claim 37 , further comprising the step of rendering said acceptor fluorophore undetectable subsequent to said detecting step.
42 . A method for sequencing a nucleic acid template, the method comprising the steps of:
(a) exposing a nucleic acid template to a labeled nucleotide and a polymerase under conditions that allow incorporation of said nucleotide into a primer attached to said template, said template being attached to a substrate-bound single-stranded nucleic acid binding protein such that said template is individually optically resolvable;
(b) detecting incorporation of said nucleotide into said primer;
(c) repeating steps (a) and (b) at least once; and
(d) compiling a sequence of said template based upon an order of incorporated nucleotides.
43 . The method of claim 42 , wherein said single-stranded nucleic acid binding protein increases fidelity of said polymerase upon binding of said protein to said template.
44 . The method of claim 42 , wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.
45 . The method of claim 44 , wherein said first and second labels are fluorophores.