POLYMERASES AND METHODS OF USE THEREOF
The invention generally relates to polymerases for efficient and controlled sequencing-by-synthesis reactions. In certain embodiments, the invention provides a polymerase enzyme including at least one mutation that enhances ability of the polymerase as compared to a wild-type polymerase to incorporate a nucleotide into a nascent strand of DNA or cDNA including at least one modified nucleotide.
1 . A polymerase enzyme comprising at least one mutation that enhances ability of the polymerase as compared to a wild-type polymerase to incorporate a nucleotide into a nascent strand of DNA or cDNA comprising at least one modified nucleotide.
2 . The polymerase according to claim 1 , wherein the polymerase is a DNA polymerase.
3 . The polymerase according to claim 1 , wherein the modified nucleotide comprises a modification in the base portion of the nucleotide.
4 . The polymerase according to claim 3 , wherein the modified nucleotide comprises a residue of a cleavable linker attaching a detectable labeled to a nitrogenous base portion of the nucleotide.
5 . The polymerase according to claim 4 , wherein the detectable label is a fluorescent label.
6 . The polymerase according to claim 5 , wherein the fluorescent label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.
7 . The polymerase according to claim 1 , wherein the polymerase is a Taq polymerase comprising mutations that enhance ability of the polymerase to incorporate a nucleotide into a nascent strand of DNA or cDNA comprising at least one modified nucleotide compared to a wild-type Taq polymerase.
8 . The polymerase according to claim 7 , wherein the polymerase comprises the following mutations in the Taq polymerase: H784Q and T664A.
9 . The polymerase according to claim 7 , wherein the polymerase comprises the following mutations in the Taq polymerase: F598I, I614F, V618I, L619M, I638V, T640A, A643G, M646V, A661T, T664V, I665V, L670M, A691V, F700Y, I753V, T756S, A757G, H784Q.
10 . The polymerase according to claim 7 , wherein the polymerase comprises the following mutations in the Taq polymerase: V618I, L619M, V631A, I638V, T640K, M646V, M658L, A661I, T664A, I665V, L670M, F700Y, A757G, H784Q.
11 . The polymerase according to claim 7 , wherein the polymerase comprises the following mutations in the Taq polymerase: I614F, L619M, L622F, T640E, M678K, T684A, M751T, V753I, T756S, A757G, L760I.
12 . A Taq polymerase comprising the following mutations: V618I, L619M, V631A, I638V, T640K, M646V, M658L, A661I, T664A, I665V, L670M, F700Y, A757G, H784Q.
13 . A Taq polymerase comprising the following mutations: I614F, L619M, L622F, T640E, M678K, T684A, M751T, V753I, T756S, A757G, L760I.
14 . A Taq polymerase comprising the following mutations: V618I, L619M, V631A, I638V, T640K, M646V, M658L, A661I, T664A, I665V, L670M, F700Y, A757G, H784Q.
15 . A method of sequencing a nucleic acid, the method comprising:
directly or indirectly anchoring a nucleic acid duplex to a surface, the duplex comprising a primer portion and a template portion having at least one modified nucleotide at its 3′ terminus;
exposing the duplex to at least one detectably labeled nucleotide in the presence of a modified polymerase capable of catalyzing addition of the nucleotide to the duplex;
detecting incorporation of the nucleotide into the primer portion; and
repeating the exposing and detecting steps at least once.
16 . The method according to claim 15 , further comprising: determining a sequence of the template based upon the order of incorporation of the labeled nucleotides.
17 . The method according to claim 15 , further comprising: removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.
18 . The method according to claim 15 , further comprising: neutralizing the label on the labeled nucleotide after the detecting step.
19 . The method according to claim 15 , wherein the template is individually optically resolvable.
20 . The method according to claim 15 , wherein the template is attached to the surface.
21 . The method according to claim 15 , wherein the nucleotide is a non-native nucleotide.
22 . A method for increasing the accuracy of sequencing a nucleic acid, the method comprising:
contacting a nucleic acid duplex comprising a primer nucleic acid hybridized to a template nucleic acid with a polymerase enzyme in the presence of a first labeled nucleotide under conditions that permit the polymerase to add nucleotides to said primer in a template-dependent manner, wherein the polymerase has reduced or eliminated exonuclease activity or has reduced or eliminated binding affinity of the 3′-5′ exonuclease domain;
detecting a signal from the incorporated labeled nucleotide; and
sequentially repeating said contacting and detecting steps at least once, wherein sequential detection of incorporated labeled nucleotide determines the sequence of the nucleic acid.
23 . The method according to claim 22 , wherein said duplex is attached to a surface.
24 . The method according to claim 23 , wherein said surface comprises a plurality of duplex immobilized at different positions on the substrate.
25 . The method according to claim 24 , wherein at least some of said duplex are individually optically resolvable.
26 . The method according to claim 22 , wherein the label is a fluorescent label.
27 . The method according to claim 22 , wherein the polymerase is Klenow fragment of E. coli Pol 1 or Stoffel fragment of Taq polymerase.
28 . The method according to claim 22 , wherein the polymerase comprises at least one mutation that results in the lack of exonuclease activity or the reduced binding affinity of the 3′-5′ exonuclease domain.
29 . The method according to claim 22 , wherein the polymerase comprises at least one mutation that results in absence of the 5′-3′ exonuclease activity of the polymerase.
30 . The method according to claim 22 , wherein the polymerase comprises at least one mutation that results in absence of the 3′-5′ exonuclease activity of the polymerase.
31 . The method according to claim 22 , wherein the polymerase comprises at least one mutation that results in reduced nucleic acid binding affinity of the 3′-5′ exonuclease domain of the polymerase.
32 . The method according to claim 31 , wherein the polymerase is a klenow fragment comprising the following mutations: Leu361, Glu419, Lys422, Tyr423, Arg455, Phe473, and His660.
33 . The method according to claim 32 , wherein Ala is substituted at any of the positions.
34 . The method according to claim 22 , wherein the polymerase comprises at least one mutation that affects metal ion binding.
35 . The method according to claim 34 , wherein the polymerase is a Klenow fragment comprising the following mutations: Asp355, Glu357, Asp424, and Asp501.