U.S. patent application number 14/956231 was filed with the patent office on 2016-04-28 for polymerases.
This patent application is currently assigned to Illumina Cambridge Limited. The applicant listed for this patent is Illumina Cambridge Limited. Invention is credited to Shankar Balasubramanian, Tobias William Barr Ost, Roberto Rigatti, Raquel Maria Sanches-Kuiper, Geoffrey Paul Smith.
Application Number | 20160115461 14/956231 |
Document ID | / |
Family ID | 36645775 |
Filed Date | 2016-04-28 |
United States Patent
Application |
20160115461 |
Kind Code |
A1 |
Smith; Geoffrey Paul ; et
al. |
April 28, 2016 |
Polymerases
Abstract
Modified DNA polymerases have an affinity for DNA such that the
polymerase has an ability to incorporate one or more nucleotides
into a plurality of separate DNA templates in each reaction cycle.
The polymerases are capable of forming an increased number of
productive polymerase-DNA complexes in each reaction cycle. The
modified polymerases may be used in a number of DNA sequencing
applications, especially in the context of clustered arrays.
Inventors: |
Smith; Geoffrey Paul; (Nr
Saffron Walden, GB) ; Rigatti; Roberto; (Nr Saffron
Walden, GB) ; Ost; Tobias William Barr; (Nr Saffron
Walden, GB) ; Balasubramanian; Shankar; (Nr Saffron
Walden, GB) ; Sanches-Kuiper; Raquel Maria; (Nr
Saffron Walden, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Illumina Cambridge Limited |
Nr Saffron Walden |
|
GB |
|
|
Assignee: |
Illumina Cambridge Limited
Nr Saffron Walden
GB
|
Family ID: |
36645775 |
Appl. No.: |
14/956231 |
Filed: |
December 1, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14137434 |
Dec 20, 2013 |
9273352 |
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14956231 |
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11431939 |
May 10, 2006 |
8623628 |
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14137434 |
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60757997 |
Jan 11, 2006 |
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Current U.S.
Class: |
435/6.11 ;
435/194; 435/6.1; 435/6.12 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12N 9/1252 20130101; C12Y 207/07007 20130101 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2005 |
GB |
0509508.8 |
Claims
1-58. (canceled)
59. A polymerase comprising the amino acid sequence
Ile-Gly-Asp-Arg-Ala-Ile-Pro at the positions functionally
equivalent to residues 710-716 of SEQ ID NO:22, having a
substitution mutation at the position functionally equivalent to
Arg713 to a nonpolar amino acid, whereby the polymerase has a
reduced affinity for DNA.
60. The polymerase of claim 59, wherein the polymerase is capable
of incorporating a nucleotide or nucleotides into a plurality of
separate DNA templates in each reaction cycle as compared to a
control polymerase, wherein the control polymerase is the unaltered
polymerase and is capable of incorporating a nucleotide or
nucleotides into a single DNA template in each reaction cycle.
61. The polymerase of claim 59, wherein the polymerase is capable
of forming an increased number of productive polymerase-DNA
complexes in each reaction cycle as compared to a control
polymerase, wherein the control polymerase is the unaltered
polymerase.
62. The polymerase of claim 59, wherein the affinity of the
polymerase for nucleotides and the fidelity of the polymerase is
substantially unaffected by the substitution mutation.
63. The polymerase according to claim 59, wherein the substitution
mutation converts the position functionally equivalent to Arg713 to
glycine (G) or methionine (M).
64. The polymerase according to claim 59, wherein the substitution
mutation converts the position functionally equivalent to Arg713 to
alanine (A).
65. A kit for performing a nucleotide incorporation reaction
comprising: a polymerase as defined in claim 59, 63, or 64, and a
nucleotide solution.
66. The kit of claim 65, wherein the nucleotide solution comprises
labelled nucleotides.
67. The kit of claim 65, wherein the nucleotides comprise synthetic
nucleotides.
68. The kit of claim 65, wherein the nucleotides comprise modified
nucleotides.
69. The kit of claim 68, wherein the modified nucleotides have been
modified at the 3' sugar hydroxyl such that the substituent is
larger in size than the naturally occurring 3' hydroxyl group.
70. The kit according to claim 69, wherein the modified nucleotides
are a modified nucleotide or nucleoside molecule comprising a
purine or pyrimidine base and a ribose or deoxyribose sugar moiety
having a removable 3'-OH blocking group covalently attached
thereto, such that the 3' carbon atom has attached a group of the
structure --O--Z wherein Z is any of --C(R)2-O--R'',
--C(R').sub.2--N(R'').sub.2, --C(R).sub.2--N(H)R'',
--C(R').sub.2--S--R'' and --C(R').sub.2--F, wherein each R'' is or
is part of a removable protecting group; each R' is independently a
hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl,
alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy,
aryloxy, heteroaryloxy or amido group, or a detectable label
attached through a linking group; or (R').sub.2 represents an
alkylidene group of formula=C(R''').sub.2 wherein each R''' may be
the same or different and is selected from the group comprising
hydrogen and halogen atoms and alkyl groups; and wherein said
molecule may be reacted to yield an intermediate in which each R''
is exchanged for H or, where Z is --C(R').sub.2--F, the F is
exchanged for OH, SH or NH.sub.2, preferably OH, which intermediate
dissociates under aqueous conditions to afford a molecule with a
free 3'OH; with the proviso that where Z is --C(R').sub.2--S--R'',
both R' groups are not H.
71. The kit according to claim 70, wherein R' of the modified
nucleotide or nucleoside is an alkyl or substituted alkyl.
72. The kit according to claim 71, wherein --Z of the modified
nucleotide or nucleoside is of formula --C(R').sub.2-N.sub.3.
73. The kit according to claim 72, wherein Z is an azidomethyl
group.
74. The kit according to claim 69, wherein the modified nucleotides
are fluorescently labelled to allow their detection.
75. The kit according to claim 69, wherein the modified nucleotides
comprise a nucleotide or nucleoside having a base attached to a
detectable label via a cleavable linker, wherein the cleavable
linker contains a moiety selected from the group consisting of:
##STR00002## wherein X is selected from the group comprising O, S,
NH and NQ wherein Q is a C.sub.1-10 substituted or unsubstituted
alkyl group, Y is selected from the group comprising O, S, NH and
N(allyl), T is hydrogen or a C.sub.1-10 substituted or
unsubstituted alkyl group and * indicates where the moiety is
connected to the remainder of the nucleotide or nucleoside.
76. The kit according to claim 75, wherein the detectable label
comprises a fluorescent label.
77. The kit of claim 65 further comprising one or more DNA template
molecules and/or primers.
Description
RELATED APPLICATIONS
[0001] The present application claims priority from U.S.
Provisional Application 60/757,997, filed Jan. 11, 2006, and Great
Britain Provisional Application No. 0509508.8, filed May 10, 2005.
Applicants claim the benefits of priority under 35 U.S.C. .sctn.119
as to the United States and Great Britain applications, and the
entire disclosures of each of these applications are incorporated
herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to polymerase enzymes and more
particularly to modified DNA polymerases having an affinity for DNA
such that the polymerase has an ability to incorporate a nucleotide
or nucleotides into a plurality of separate DNA templates in each
reaction cycle and is capable of forming an increased number of
productive polymerase-DNA complexes in each reaction cycle. Also
included in the scope of the present invention are methods of using
the modified polymerases for DNA sequencing, especially in the
context of clustered arrays.
BACKGROUND
[0003] Several publications and patent docments are referenced in
this application in order to more fully describe the state of the
art to which this invention pertains. The disclosure of each of
these publications and documents is incorporated by reference
herein.
[0004] The three-dimensional crystal structure of certain DNA
polymerases has revealed three separate subdomains, named palm,
fingers and thumb (Joyce, C. M. and Steiz, T. A. (1994) Function
and structure relationships in DNA polymerases, Annu. Rev.
Biochem., 63, 777-822), each having key roles during DNA
polymerisation.
[0005] The C terminal thumb subdomain of DNA polymerases has been
implicated in DNA binding and processivity (Doublie et al. 1998.
Nature 391, 251; Truniger et al. 2004. Nucleic Acids Research 32,
371). Residues in this region of DNA polymerases interact with the
primer:template duplex.
[0006] Disruption of the structure of this region either by the
introduction of site-directed mutations or truncation by the
deletion of a small number of amino acids, has provided evidence
for variants with reduced DNA affinity and processivity without
gross changes in other physical properties such as dNTP affinity
and nucleotide insertion fidelity (Truniger et al. 2004. Nucleic
Acids Research 32, 371; Minnick et al. 1996. J. Biol. Chem., 271,
24954; Polesky et al. 1990. J. Biol. Chem., 265, 14579).
[0007] Polymerases may be separated into two structurally distinct
families called family A and family B.
[0008] The C-terminal subdomain of family B polymerases has been
poorly studied, but is believed to be involved in DNA binding based
primarily on the inspection of the x-ray crystal structure of the
closed form (DNA-bound) of polymerase RB69. Mutagenesis studies
have been conducted within this thumb domain for two examples of
the family B class, namely Phi29 and T4. However, these studies
were limited to amino acid deletions of large portions of the
domain. The same type of deletion has been carried out for Klenow
(a family A polymerase). The performance of the variants in these
studies was evaluated in terms of their ability to bind and
incorporate dNTPs, the effect the deletion had on fidelity, their
affinity for DNA and also their interaction with accessory
proteins.
[0009] No studies of the thumb domain of the polymerase from a
thermophilic archaeon have previously been carried out.
[0010] The subject matter of the present invention was presented in
prior filed U.K. Provisional Application No. 0509508.8 filed May
10, 2005, priority of which is believed to be available under 35
U.S.C. .sctn.119, and the disclosure of which is incorporated
herein in its entirety. In said application, the structural aspects
of the polymerases and the related materials of the present
invention were disclosed as they are herein, and were accompanied
by information providing further background and characterization of
function, which was also in accordance with the understanding of
the inventors at the time. Since filing said application, further
study of the enzymes in question has taken place and and additional
data illustrating and advancing the understanding of their function
and application, has resulted, which is now felt to be desirably
presented herein.
[0011] Accordingly, it is toward the advancement of the
understanding and application of the present invention that the
present application is directed.
SUMMARY OF THE INVENTION
[0012] The present invention is based upon the realisation that the
tight binding of a polymerase to the DNA template is not always an
advantageous property. This is particularly the case in the context
of sequencing reactions in which only a single nucleotide
incorporation event is required in each reaction cycle. Thus, for a
polymerase that binds tightly to DNA, the ability of the polymerase
to take part in incorporation of nucleotides on multiple DNA
strands is restricted compared to a variant polymerase that has a
lower affinity for DNA.
[0013] The present inventors have devised a method for sequencing
DNA that uses nucleotide analogues bearing modifications at the 3'
sugar hydroxyl group which block incorporation of further
nucleotides (see WO03/048387, for example, and the citations
described therein). The use of nucleotides bearing a 3' block
allows successive nucleotides to be incorporated into a
polynucleotide chain in a controlled manner. After each nucleotide
addition the presence of the 3' block prevents incorporation of a
further nucleotide into the chain. Once the nature of the
incorporated nucleotide has been determined, the block may be
removed, leaving a free 3' hydroxyl group for addition of the next
nucleotide.
[0014] In addition, in the context of reactions such as sequencing
reactions involving modified nucleotides (as discussed above and in
more detail herein below), tight binding of a polymerase may in
fact present certain disadvantages in terms of reaction completion.
For example, if an inactive polymerase molecule having a tight DNA
binding affinity forms a stable complex with a template DNA
molecule, no extension is possible from that particular template
DNA molecule.
[0015] With this realisation, the present invention provides
altered polymerases which have a weaker interaction with template
DNA. Thus, the polymerase of the invention has an improved ability
to move from one template DNA molecule to another during a reaction
cycle. This ability to form an increased number of productive
polymerase-DNA complexes has the benefit that levels or reaction
completion in reactions involving addition of a single nucleotide
in each reaction cycle are much improved.
[0016] Unmodified polymerases tend to bind DNA with high affinity
such that the equation:
Pol + DNA k a k d [ Pol : DNA ] ##EQU00001##
is heavily shifted to favour the [Pol:DNA] complex.
[0017] In contrast, in the present invention, the altered
polymerases bind to DNA less well, meaning that the equilibrium
position is shifted towards the left hand side.
[0018] Therefore, the invention provides an altered polymerase
having reduced affinity for DNA such that the polymerase has an
ability to incorporate a nucleotide or nucleotides into a plurality
of separate DNA templates in each reaction cycle.
[0019] By "DNA template" is meant any DNA molecule which may be
bound by the polymerase and utilised as a template for nucleic acid
synthesis.
[0020] "Nucleotide" is defined herein to include both nucleotides
and nucleosides. Nucleosides, as for nucleotides, comprise a purine
or pyrimidine base linked glycosidically to ribose or deoxyribose,
but they lack the phosphate residues which would make them a
nucleotide. Synthetic and naturally occurring nucleotides are
included within the definition. Labelled nucleotides are included
within the definition. The advantageous properties of the
polymerases are due to their reduced affinity for the DNA template
in combination with a retained affinity and fidelity for the
nucleotides which they incorporate.
[0021] In one preferred aspect, an altered polymerase is provided
having a reduced affinity for DNA such that the polymerase has an
ability to incorporate at least one synthetic nucleotide into a
plurality of DNA templates in each reaction cycle. Prior to the
present invention, the problem of modifying a polymerase adapted to
incorporate non-natural nucleotides, to reduce its DNA affinity
whilst retaining its advantageous properties has neither been
realised nor addressed.
[0022] In one embodiment, nucleotides comprise dideoxy nucleotide
triphosphates (ddNTPs) such as those used in Sanger sequencing
reactions. These nucleotides may be labelled, e.g., with any of a
mass label, radiolabel or a fluorescent label.
[0023] In a further embodiment, the nucleotides comprise
nucleotides which have been modified at the 3' sugar hydroxyl such
that the substituent is larger in size than the naturally occurring
3' hydroxyl group, compared to a control polymerase.
[0024] In a preferred embodiment, the nucleotides comprise those
having a purine or pyrimidine base and a ribose or deoxyribose
sugar moiety having a removable 3'-OH blocking group covalently
attached thereto, such that the 3' carbon atom has attached a group
of the structure
--O--Z
[0025] wherein Z is any of --C(R').sub.2--O--R'',
--C(R').sub.2--N(R'').sub.2, --C(R').sub.2--N(H)R'',
--C(R').sub.2--S--R'' and --C(R').sub.2--F,
[0026] wherein each R'' is or is part of a removable protecting
group;
[0027] each R' is independently a hydrogen atom, an alkyl,
substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido
group, or a detectable label attached through a linking group; or
(R').sub.2 represents an alkylidene group of formula=C(R''').sub.2
wherein each R''' may be the same or different and is selected from
the group comprising hydrogen and halogen atoms and alkyl groups;
and
[0028] wherein said molecule may be reacted to yield an
intermediate in which each R'' is exchanged for H or, where Z is
--C(R').sub.2--F, the F is exchanged for OH, SH or NH.sub.2,
preferably OH, which intermediate dissociates under aqueous
conditions to afford a molecule with a free 3'OH;
[0029] with the proviso that where Z is --C(R').sub.2--S--R'', both
R' groups are not H.
[0030] The nucleosides or nucleotides which are incorporated by the
polymerases of the present invention according to one embodiment,
comprise a purine or pyrimidine base and a ribose or deoxyribose
sugar moiety which has a blocking group covalently attached
thereto, preferably at the 3'O position, which renders the
molecules useful in techniques requiring blocking of the 3'-OH
group to prevent incorporation of additional nucleotides, such as
for example in sequencing reactions, polynucleotide synthesis,
nucleic acid amplification, nucleic acid hybridisation assays,
single nucleotide polymorphism studies, and other such
techniques.
[0031] Once the blocking group has been removed, it is possible to
incorporate another nucleotide to the free 3'-OH group.
[0032] Preferred modified nucleotides are exemplified in
International Patent Application publication number WO 2004/018497
in the name of Solexa Limited, which reference is incorporated
herein in its entirety.
[0033] In a preferred embodiment the R' group of the modified
nucleotide or nucleoside is an alkyl or substituted alkyl. In a
further embodiment the --Z group of the modified nucleotide or
nucleoside is of formula --C(R').sub.2--N.sub.3. In a most
preferred embodiment the modified nucleotide or nucleoside includes
a Z group which is an azido methyl group.
[0034] The preferred polymerases of the invention, as discussed in
detail below, are particularly preferred for incorporation of
nucleotide analogues wherein Z is an azido methyl group.
[0035] The modified nucleotide can be linked via the base to a
detectable label by a desirable linker, which label may be a
fluorophore, for example. The detectable label may instead, if
desirable, be incorporated into the blocking groups of formula "Z".
The linker can be acid labile, photolabile or contain a disulfide
linkage. Other linkages, in particular phosphine-cleavable
azide-containing linkers, may be employed in the invention as
described in greater detail in WO 2004/018497, the contents of
which are incorporated herein in their entirety.
[0036] Preferred labels and linkages include those disclosed in WO
03/048387, which is incorporated herein in its entirety.
[0037] In one embodiment the modified nucleotide or nucleoside has
a base attached to a detectable label via a cleavable linker,
characterised in that the cleavable linker contains a moiety
selected from the group consisting of:
##STR00001##
(wherein X is selected from the group comprising O, S, NH and NQ
wherein Q is a C.sub.1-10 substituted or unsubstituted alkyl group,
Y is selected from the group comprising O, S, NH and N(allyl), T is
hydrogen or a C.sub.1-10 substituted or unsubstituted alkyl group
and * indicates where the moiety is connected to the remainder of
the nucleotide or nucleoside).
[0038] In one embodiment the detectable label comprises a
fluorescent label. Suitable fluorophores are well known in the art.
In a preferred embodiment each different nucleotide type will carry
a different fluorescent label. This facilitates the identification
and incorporation of a particular nucleotide. Thus, for example
modified Adenine, Guanine, Cytosine and Thymine would all have
attached a separate fluorophore to allow them to be discriminated
from one another readily. Surprisingly, it has been found that the
altered polymerases are capable of incorporating modified
nucleotide analogues carrying a number of different fluorescent
labels. Moreover, the polymerases are capable of incorporating all
four bases. These properties provide substantial advantages with
regard to the use of the polymerases of the present invention in
nucleic acid sequencing protocols.
[0039] As aforesaid, preferred nucleotide analogues include those
containing O-azido methyl functionality at the 3' position. It will
be appreciated that for other nucleotide analogues the preferred
amino acid sequence of the polymerase in the C terminal thumb
sub-domain region, which contributes significantly to DNA binding,
for optimum incorporation may vary. For any given nucleotide
analogue, optimum sequence preferences in the C terminal thumb sub
domain region (such as at residues Lys 790, 800, 844, 874, 878 and
Arg 806 in RB69 and at residues Arg 743, Arg 713 and Lys 705 in
9.degree. N polymerase, as discussed in greater detail below) may
be determined by experiment, for example by construction of a
library or discrete number of mutants followed by testing of
individual variants in an incorporation assay system.
[0040] As aforementioned, the altered polymerases of the invention
are capable of improved incorporation of all nucleotides, including
a wide range of modified nucleotides having large 3' substituent
groups of differing sizes and of varied chemical nature. The
advantageous properties of the polymerases are due to their reduced
affinity for the DNA template leading to increased dissociation of
the polymerase from the DNA without adverse effects on affinity and
fidelity for the nucleotides which they incorporate.
[0041] By virtue of the decreased DNA binding affinity of the
polymerase of the invention, it is able to incorporate one or more
nucleotides into several different DNA molecules in a single
reaction cycle. Thus, the overall efficiency of reaction is
improved, leading to greater levels of completion.
[0042] By "a reaction cycle" is meant a suitable reaction period to
allow the incorporation of nucleotides into the template. Exemplary
conditions for a single reaction cycle are one 30 minute,
45.degree. C. incubation period.
[0043] Many polymerisation reactions occur in the presence of an
excess of DNA compared to polymerase. The polymerase of the present
invention allows such a polymerisation reaction to proceed more
effectively since the polymerase can catalyse numerous rounds of
incorporation of a nucleotide or nucleotides on separate template
DNA molecules. An unaltered polymerase on the other hand,
particularly one which binds DNA much more tightly, will not have
this ability since it is more likely to only participate in
nucleotide incorporation on a single template in each reaction
cycle. The polymerase according to the present invention allows
high levels of reaction completion under conditions where the
concentration of polymerase is limiting with respect to the
concentration of DNA. In particular, the polymerase presents
improved ability to incorporate one or more nucleotides into
separate DNA molecules under conditions wherein the DNA:polymerase
ratio is at least about 2:1, 3:1 or 5:1. However, at high
concentrations of polymerase, the improvement may be masked.
[0044] Thus, an altered polymerase is provided having an affinity
for DNA such that the polymerase is capable of forming an increased
number of productive polymerase-DNA complexes in each reaction
cycle.
[0045] The improved properties of the polymerases of the invention
may be compared to a suitable control. "Control polymerase" is
defined herein as the polymerase against which the activity of the
altered polymerase is compared. The control polymerase is of the
same type as the altered polymerase but does not carry the
alteration(s) which reduce affinity of the polymerase for DNA.
Thus, in a particular embodiment, the control polymerase is a
9.degree. N polymerase and the modified polymerase is the same
9.degree. N polymerase except for the presence of one or more
modifications which reduce the affinity of the 9.degree. N
polymerase for DNA.
[0046] In one embodiment, the control polymerase is a wild type
polymerase which is altered to provide an altered polymerase which
can be directly compared with the unaltered polymerase.
[0047] In one embodiment, the control polymerase comprises
substitution mutations at positions which are functionally
equivalent to Leu408 and Tyr409 and Pro410 in the 9.degree. N DNA
polymerase amino acid sequence. Thus, in this embodiment the
control polymerase has a substitution mutation at position 408 from
leucine to a different amino acid, at position 409 from tyrosine to
a different amino acid and at position 410 from proline to a
different amino acid or at positions which are functionally
equivalent if the polymerase is not a 9.degree. N DNA polymerase.
In a preferred embodiment, the control polymerase is a 9.degree. N
DNA polymerase comprising the said substitution mutations.
[0048] In another embodiment, the control polymerase comprises
substitution mutations which are functionally equivalent to
Leu408Tyr and Tyr409Ala and Pro410Val in the 9.degree. N DNA
polymerase amino acid sequence. Thus, in this embodiment the
control polymerase has a substitution mutation at position 408 from
leucine to tyrosine, at position 409 from tyrosine to alanine and
at position 410 from proline to valine or at positions which are
functionally equivalent if the polymerase is not a 9.degree. N DNA
polymerase. In a preferred embodiment, the control polymerase is a
9.degree. N DNA polymerase comprising the above substitution
mutations.
[0049] The control polymerase may further comprise a substitution
mutation at the position functionally equivalent to Cys223 in the
9.degree. N DNA polymerase amino acid sequence. Thus, in this
embodiment the control polymerase has a substitution mutation at
position 223 from cysteine to a different amino acid, or at a
position which is functionally equivalent if the polymerase is not
a 9.degree. N DNA polymerase. In a preferred embodiment, the
control polymerase is a 9.degree. N DNA polymerase comprising the
said substitution mutation. In another embodiment, the control
polymerase comprises the substitution mutation functionally
equivalent to Cys223Ser in the 9.degree. N DNA polymerase amino
acid sequence. Thus, in this embodiment the control polymerase has
a substitution mutation at position 223 from cysteine to serine, or
at a position which is functionally equivalent if the polymerase is
not a 9.degree. N DNA polymerase. In a preferred embodiment, the
control polymerase is a 9.degree. N DNA polymerase comprising the
said substitution mutation.
[0050] Preferably, the control polymerase is a 9.degree. N DNA
polymerase comprising a combination of the above mentioned
mutations.
[0051] The altered polymerase will generally have a reduced
affinity for DNA. This may be defined in terms of dissociation
constant. Thus, wild type polymerases tend to have dissociation
constants in the nano-picomolar range. For the purposes of the
present invention, an altered polymerase having an affinity for DNA
which is reduced compared to the control unaltered polymerase is
suitable. Preferably, due to the alteration(s), the polymerase has
at least a, or approximately a 2-fold, 3-fold, 4-fold or 5-fold
etc., increase in its dissociation constant when compared to the
control unaltered polymerase.
[0052] By "functionally equivalent" is meant the amino acid
substitution that is considered to occur at the amino acid position
in another polymerase that has the same functional role in the
enzyme. As an example, the mutation at position 412 from Tyrosine
to Valine (Y412V) in the Vent DNA polymerase would be functionally
equivalent to a substitution at position 409 from Tyrosine to
Valine (Y409V) in the 9.degree. N polymerase. The bulk of this
amino acid residue is thought to act as a "steric gate" to block
access of the 2'-hydroxyl of the nucleotide sugar to the binding
site. Also, residue 488 in Vent polymerase is deemed equivalent to
amino acid 485 in 9.degree. N polymerase, such that the Alanine to
Leucine mutation at 488 in Vent (A488L) is deemed equivalent to the
A485L mutation in 9.degree. N polymerase.
[0053] Generally, functionally equivalent substitution mutations in
two or more different polymerases occur at homologous amino acid
positions in the amino acid sequences of the polymerases. Hence,
use herein of the term "functionally equivalent" also encompasses
mutations that are "positionally equivalent" or "homologous" to a
given mutation, regardless of whether or not the particular
function of the mutated amino acid is known. It is possible to
identify positionally equivalent or homologous amino acid residues
in the amino acid sequences of two or more different polymerases on
the basis of sequence alignment and/or molecular modelling.
[0054] The altered polymerase will generally be an "isolated" or
"purified" polypeptide. By "isolated polypeptide" is meant a
polypeptide that is essentially free from contaminating cellular
components, such as carbohydrates, lipids, nucleic acids or other
proteinaceous impurities which may be associated with the
polypeptide in nature. Typically, a preparation of the isolated
polymerase contains the polymerase in a highly purified form, i.e.
at least about 80% pure, preferably at least about 90% pure, more
preferably at least about 95% pure, more preferably at least about
98% pure and most preferably at least about 99% pure. Purity of a
preparation of the enzyme may be assessed, for example, by
appearance of a single band on a standard SDS-polyacrylamide
electrophoresis gel.
[0055] The altered polymerase may be a "recombinant"
polypeptide.
[0056] The altered polymerase according to the invention may be any
DNA polymerase. More particularly, the altered polymerase may be a
family B type DNA polymerase, or a mutant or variant thereof.
Family B DNA polymerases include numerous archael DNA polymerase,
human DNA polymerase .alpha. and T4, RB69 and .phi.29 phage DNA
polymerases. These polymerases are less well studied than the
family A polymerases, which include polymerases such as Taq, and T7
DNA polymerase. In one embodiment the polymerase is selected from
any family B archael DNA polymerase, human DNA polymerase .alpha.
or T4, RB69 and .phi.29 phage DNA polymerases.
[0057] The archael DNA polymerases are in many cases from
hyperthermophilic archea, which means that the polymerases are
often thermostable. Accordingly, in a further preferred embodiment
the polymerase is a thermophilic archaeon polymerase, including,
e.g., Vent, Deep Vent, 9.degree. N and Pfu polymerase. Vent and
Deep Vent are commercial names used for family B DNA polymerases
isolated from the hyperthermophilic archaeon Thermococcus litoralis
and Pyrococcus furiosus respectively. 9.degree. N polymerase was
also identified from Thermococcus sp. Pfu polymerase was isolated
from Pyrococcus furiosus. As mentioned above, prior to the present
invention the thumb domain from a thermophilic polymerase had not
been studied. A preferred polymerase of the present invention is
9.degree. N polymerase, including mutants and variants thereof.
9.degree. N polymerase has no requirement for accessory proteins.
This can be contrasted with previously studied polymerases in which
deletions in the thumb domain were shown to adversely affect the
interaction with accessory proteins whilst not altering other
properties of the polymerase. In contrast, as is shown in the
Experimental Section below, a deletion of a large number of
residues of 9.degree. N has a significant adverse effect on the
important properties of 9.degree. N such that catalytic activity is
severely compromised.
[0058] It is to be understood that the invention is not intended to
be limited to mutants or variants of the family B polymerases. The
altered polymerase may also be a family A polymerase, or a mutant
or variant thereof, for example a mutant or variant Taq or T7 DNA
polymerase enzyme, or a polymerase not belonging to either family A
or family B, such as for example reverse transcriptases.
[0059] A number of different types of alterations are contemplated
by the invention, wherein such alterations produce a polymerase
displaying the desired properties as a result of reduced affinity
for DNA. Particularly preferred are substitution mutations in the
primary amino acid sequence of the polymerase, although addition
and deletion mutations may also produce useful polymerases.
Suitable alteration techniques, such as site directed mutagenesis,
e.g., are well known in the art.
[0060] Thus, by "altered polymerase" it is meant that the
polymerase has at least one amino acid change compared to the
control polymerase enzyme. In general this change will comprise the
substitution of at least one amino acid for another. In preferred
embodiments, these changes are non-conservative changes, although
conservative changes to maintain the overall charge distribution of
the protein are also envisaged in the present invention. Moreover,
it is within the contemplation of the present invention that the
modification in the polymerase sequence may be a deletion or
addition of one or more amino acids from or to the protein,
provided that the resultant polymerase has reduced DNA affinity and
an ability to incorporate a nucleotide or nucleotides into a
plurality of separate DNA templates in each reaction cycle compared
to a control polymerase.
[0061] In one embodiment, the alteration to form the polymerase of
the invention comprises at least one mutation, and preferably at
least one substitution mutation, at a residue in the polymerase
which destabilises the interaction of the polymerase with DNA.
Thus, the resultant polymerase interacts in a less stable manner
with DNA. As aforementioned, a decrease in affinity of the
polymerase for DNA allows it to incorporate one or more nucleotides
into several different DNA molecules in a single reaction cycle.
Thus, the overall efficiency of a reaction is improved, leading to
greater levels of reaction completion.
[0062] In a further embodiment, the alteration comprises at least
one mutation, and preferably at least one substitution mutation, at
a residue in the polymerase which binds to DNA. Suitable target
residues for mutation can be selected according to available
crystal structures for suitable polymerases, particularly when
crystallised in the closed state (bound to DNA). By reducing the
number of binding contacts with the DNA, an overall reduction in
DNA binding affinity may be achieved. Thus, the resultant
polymerase displays improved characteristics in the context of
nucleotide incorporation reactions in which tight binding to DNA is
disadvantageous.
[0063] In similar fashion, the polymerase may also carry an
alteration which comprises at least one mutation, and preferably at
least one substitution mutation, at a residue in the DNA binding
domain of the polymerase. Such a mutation is predicted to decrease
the DNA binding affinity of the altered polymerase such that it is
able to more readily bind to and dissociate from separate template
DNA molecules during a reaction.
[0064] In one embodiment, the polymerase includes an alteration
which comprises at least one mutation, and preferably at least one
substitution mutation, at a basic amino acid residue in the
polymerase. Indeed, many positively charged amino acid residues in
polymerases are known to interact with the overall negatively
charged DNA double helix, in particular with specific phosphate
groups of nucleotides in the DNA.
[0065] As aforementioned, a particular type of alteration resulting
in a polymerase according to the invention comprises at least one
substitution mutation. As is shown in the experimental section
below, deletion of residues from the polymerase amino acid sequence
generate a polymerase which, whilst having a reduced affinity for
DNA, does not have overall advantageous properties since catalytic
ability is impaired. In one particularly preferred embodiment, the
polymerase comprises two substitution mutations, but may contain
four, five, six or seven, etc. mutations provided that the
resultant polymerase has the desired properties.
[0066] Preferably, the affinity of the polymerase for nucleotides
is substantially unaffected by the alteration. As is shown in the
experimental section (in particular example 6), it is possible to
mutate a polymerase such that its affinity for DNA is reduced,
whilst the affinity of the polymerase for a nucleotide, which may
be a dNTP or ddNTP or a modified version thereof for example (see
the definition of nucleotide supra) is not adversely affected. By
"substantially unaffected" in this context is meant that the
affinity for the nucleotide remains of the same order as for the
unaltered polymerase. Preferably, the affinity for nucleotides is
unaffected by the alteration.
[0067] Preferably, the fidelity of the polymerase is substantially
unaffected by the alteration. As is shown in the experimental
section (in particular example 6), it is possible to mutate a
polymerase such that its affinity for DNA is reduced, whilst the
fidelity of the polymerase is substantially unaffected by the
alteration. By "substantially unaffected" in this context is meant
that the misincorporation frequency for each nucleotide remains of
the same order as for the unaltered polymerase. Preferably, the
fidelity of the polymerase is unaffected by the alteration.
[0068] In terms of specific and preferred structural mutants, these
may be based upon the most preferred polymerase, namely 9.degree. N
DNA polymerase. As discussed in example 1 below, an energy
minimised overlaid alignment (contracted by Cresset) of the crystal
structures of the open form of 9.degree. N-7 DNA polymerase
(PDB=lqht), the open structure of a closely related DNA polymerase
RB69 (PDB=lih7) and the closed form of RB69 (PDB=lig9) was used as
a structural model for the identification of key residues involved
in DNA binding. Accordingly, an altered polymerase is provided
which comprises or incorporates one, two or three amino acid
substitution mutations to a different amino acid at the position or
positions functionally equivalent to Lys705, Arg713 and/or Arg743
in the 9.degree. N DNA polymerase amino acid sequence. Preferably,
the polymerase is a 9.degree. N DNA polymerase comprising these
mutations. All combinations and permutations of one, two or three
mutations are contemplated within the scope of the invention.
[0069] Mutations may also be made at other specific residues based
upon alignment of the "open" 9.degree. N DNA polymerase structure
(i.e. not bound to DNA) with the known crystal structure of the
RB69 polymerase complexed with DNA. Thus, an altered polymerase is
provided which comprises or incorporates one or two amino acid
substitution mutations to a different amino acid at the position or
positions functionally equivalent to Arg606 and/or His679 in the
9.degree. N DNA polymerase amino acid sequence. Preferably, the
polymerase is a 9.degree. N DNA polymerase comprising these
mutations. All combinations and permutations of different mutations
are contemplated within the scope of the invention. Thus, these
mutations may be made in combination with the other mutations
discussed supra.
[0070] In one preferred embodiment, the polymerase comprises at
least a substitution mutation to a different amino acid at the
position functionally equivalent to either Arg713 or Arg743 in the
9.degree. N DNA polymerase amino acid sequence. These two positions
represent particularly preferred sites for mutation, as discussed
in more detail in the experimental section below. Both residues may
be mutated in the same polymerase to a different amino acid.
[0071] In terms of the nature of the different amino acid, the
substitution mutation or mutations preferably convert the
substituted amino acid to a non-basic amino acid (i.e. not lysine
or arginine). Any non-basic amino acid may be chosen. Preferred
substitution mutation or mutations convert the substituted amino
acid to an amino acid selected from: [0072] (i) acidic amino acids,
[0073] (ii) aromatic amino acids, particularly tyrosine (Y) or
phenylalanine (F); and [0074] (iii)non-polar amino acids,
particularly, alanine (A), glycine (G) or methionine (M).
[0075] In one embodiment, the substitution mutation or mutations
convert the substituted amino acid to alanine. In a more specific
embodiment, an altered polymerase is provided comprising the
substitution mutation or mutations which are functionally
equivalent to Lys705Ala and/or Arg713Ala and/or Arg743Ala in the
9.degree. N DNA polymerase amino acid sequence. Thus, in this
embodiment the polymerase has a substitution mutation at position
705 from lysine to alanine and/or at position 713 from arginine to
alanine and/or at position 743 from arginine to alanine or at
positions which are functionally equivalent if the polymerase is
not a 9.degree. N DNA polymerase. In a preferred embodiment, the
polymerase is a 9.degree. N DNA polymerase comprising the said
substitution mutations.
[0076] In one embodiment, the altered polymerase comprises the
amino acid substitution functionally equivalent to Arg713Ala; in a
further embodiment, the altered polymerase comprises the amino acid
substitution functionally equivalent to Arg743Ala. Preferably, the
altered polymerase is a 9.degree. N DNA polymerase.
[0077] Specific structural mutants may also be based upon other
types of polymerase, such as the RB69 polymerase for which the
"open" and "closed" structures are known. Accordingly, an altered
polymerase is provided which comprises or incorporates one, two,
three, four, five or six amino acid substitution mutations to a
different amino acid at the position or positions functionally
equivalent to Lys790, Lys800, Arg806, Lys844, Lys874 and/or Lys878
in the RB69 DNA polymerase amino acid sequence. Preferably, the
polymerase is a 9.degree. N DNA polymerase comprising these
analogous or functionally equivalent mutations. All combinations
and permutations of one, two, three, four, five or six mutations
are contemplated within the scope of the invention.
[0078] In terms of the nature of the different amino acid, the
substitution mutation or mutations preferably convert the
substituted amino acid to a non-basic amino acid (i.e. not lysine
or arginine). Any non-basic amino acid may be chosen.
[0079] Preferred substitution mutation or mutations convert the
substituted amino acid to an amino acid selected from: [0080] (i)
acidic amino acids, [0081] (ii) aromatic amino acids, particularly
tyrosine (Y) or phenylalanine (F); and [0082] (iii)non-polar amino
acids, particularly, alanine (A), glycine (G) or methionine
(M).
[0083] In one embodiment, the substitution mutation or mutations
convert the substituted amino acid to alanine.
[0084] It should be noted that the present invention is not limited
to polymerases which have only been altered in the above mentioned
manner. Polymerases of the invention may include a number of
additional mutations, such as for example the preferred mutant
polymerases disclosed in detail in WO 2005/024010. In particular, a
polymerase comprising substitution mutations at positions which are
functionally equivalent to Leu408 and Tyr409 and Pro410 in the
9.degree. N DNA polymerase amino acid sequence is contemplated. In
a preferred embodiment, the polymerase is a 9.degree. N DNA
polymerase comprising the said substitution mutations.
[0085] In a specific embodiment, the polymerase comprises the
substitution mutations which are functionally equivalent to at
least one or two but preferably all of Leu408Tyr and Tyr409Ala and
Pro410Val in the 9.degree. N DNA polymerase amino acid sequence. In
a preferred embodiment, the polymerase is a 9.degree. N DNA
polymerase comprising all the said substitution mutations.
[0086] The polymerase may further comprise a substitution mutation
at the position functionally equivalent to Cys223 in the 9.degree.
N DNA polymerase amino acid sequence. In a preferred embodiment,
the polymerase is a 9.degree. N DNA polymerase comprising the said
substitution mutation. In one embodiment, the polymerase comprises
the substitution mutation functionally equivalent to Cys223Ser in
the 9.degree. N DNA polymerase amino acid sequence. In a preferred
embodiment, the polymerase is a 9.degree. N DNA polymerase
comprising the said substitution mutation.
[0087] Preferably, the polymerase is a 9.degree. N DNA polymerase
comprising a combination of the above mentioned mutations.
[0088] The invention also relates to a 9.degree. N polymerase
molecule comprising, consisting essentially of or consisting of the
amino acid sequence shown as any one of SEQ ID NO: 1, 3, 5 or 21.
The invention also encompasses polymerases having amino acid
sequences which differ from those shown as SEQ ID NOs: 1, 3, 5 and
21 only in amino acid changes which do not affect the function of
the polymerase to a material extent. In this case the relevant
function of the polymerase is defined as a reduced affinity for DNA
such that the polymerase has an ability to incorporate a nucleotide
or nucleotides into a plurality of separate DNA templates in each
reaction cycle (compared to a control polymerase) and/or that the
polymerase is capable of forming an increased number of productive
polymerase-DNA complexes in each reaction cycle (compared to a
control polymerase).
[0089] Thus, conservative substitutions at residues which are not
important for this activity of the polymerase variants having
reduced DNA affinity are included within the scope of the
invention. The effect of further mutations on the function of the
enzyme may be readily tested, for example using well known
nucleotide incorporation assays (such as those described in the
examples of WO 2005/024010 and in examples 3 and 4 below).
[0090] The altered polymerase of the invention may also be defined
directly with reference to its reduced affinity for DNA, which
together with a substantially unaltered fidelity and affinity for
nucleotides produce the advantages associated with the polymerases
of the invention. Thus, an altered polymerase is provided which has
a dissociation constant (K.sub.D) for DNA of at least, or in the
region of between, approximately 2-fold greater, 3-fold greater,
4-fold greater or 5-fold greater than the unaltered control
polymerase.
[0091] In one embodiment, an altered polymerase is provided which
will dissociate from DNA in the presence of a salt solution,
preferably a NaCl solution, having a concentration of less than or
equal to about 500 mM, preferably less than 500 mM. The salt
solution may be of a suitable concentration such that the reduced
affinity polymerase of the invention can be distinguished from an
unaltered polymerase which binds DNA more tightly. Suitable salt
solution concentrations (preferably NaCl) are in the region of
approximately 150 mM, 200 mM, 250 mM, 300 mM or 350 mM preferably
200 mM. Any suitable double stranded DNA molecule may be utilised
to determine whether the alteration has the desired effect in terms
of reducing DNA affinity. Preferably, the DNA molecule from which
the polymerase dissociates comprises the sequence set forth as SEQ
ID No.: 18. Preferably, at least approximately 40%, 50%, 60%, 70%,
etc., of the polymerase will dissociate from the DNA at the
relevant NaCl concentration in the wash solution.
[0092] Dissociation experiments may be carried out by any known
means, such as, for example, by utilising the washing assay
detailed in example 5 of the Experimental Section below (see also
FIGS. 6 and 7).
[0093] As aforementioned, the reduction in DNA affinity is
(preferably) achieved without a notable or significant decrease in
the affinity of the polymerase for nucleotides. Surprisingly, the
altered polymerase of the invention may also display comparable
activity, for example, in terms of Vmax, to the unmodified
polymerase even though the DNA binding affinity has been decreased.
This surprising property displayed by the polymerases of the
present invention is shown in the kinetic analysis of certain
enzymes of the invention in particular in example 6 of the
Experimental Section below and with reference to FIG. 8.
[0094] The altered polymerase of the invention may also be defined
directly with reference to its improved ability to be purified from
host cells in which the polymerase is expressed. Thus, thanks to
the reduced affinity of the altered polymerase for DNA (which
together with a substantially unaltered affinity for nucleotides
and fidelity produce the advantages associated with the polymerases
of the invention) the polymerase can more readily be purified. Less
endogenous DNA from the host cell is co-purified during
purification of the enzyme. Thus, a more pure product results since
less endogenous DNA remains bound to the polymerase following the
purification process. An additional advantage of the reduced
affinity for DNA of the altered polymerases is that less severe
purification procedures need to be utilised in order to provide a
substantially pure polymerase preparation. Accordingly, less
polymerase will be adversely affected by the purification process
itself leading to a polymerase preparation with higher levels of
overall activity. In addition, more uniform purification should be
possible leading to less variability between batches of polymerase.
Representative data regarding the improvement in carry over of
endogenous DNA during the purification procedure is provided in
Example 7 of the experimental section below.
[0095] Preferably, less than about 60 ng/ml, 50 ng/ml, 40 ng/ml, 30
ng/ml, 20 ng/ml, 10 ng/ml and more preferably less than about 5
ng/ml of host DNA is carried over following purification of the
polymerase. Standard purification protocols may be utilised, such
as for example see Colley et al., J. Biol. Chem. 264:17619-17622
(1989); Guide to Protein Purification, in Methods in Enzymology,
vol. 182 (Deutscher, ed., 1990).
[0096] Thus, the invention provides an altered polymerase having an
affinity for DNA such that;
[0097] (i) the polymerase has a dissociation constant for DNA of at
least about, or approximately, 2-fold, 3-fold, 4-fold or 5-fold
greater than the unaltered/control polymerase and/or
[0098] (ii) at least 50%, 60%, 70% or 80% of the polymerase
dissociates from DNA to which the polymerase is bound when a sodium
chloride solution having a concentration of between about 200 nM
and 500 nM, preferably between about 200 nM and 300 nM is applied
thereto, and/or
[0099] (iii) less than about 60, 50, 45, 40, 35, 30, 25, 20, 15,
10, 5, 3, 1 or 0.5 ng/ml of endogenous DNA remains bound to the
polymerase following a purification process from the cell in which
the polymerase is expressed; the alteration not significantly
adversely affecting nucleotide binding ability or fidelity such
that the polymerase is capable of;
[0100] (a) forming an increased number of productive polymerase-DNA
complexes over a reaction cycle (giving improved levels of reaction
completion), and/or
[0101] (b) catalysing an improved (increased/elevated) overall
level of nucleotide incorporation;
especially under conditions where the concentration of polymerase
is limiting with respect to the concentration of DNA.
[0102] The invention further relates to nucleic acid molecules
encoding the altered polymerase enzymes of the invention.
[0103] For any given altered polymerase which is a mutant version
of a polymerase for which the amino acid sequence and preferably
also the wild type nucleotide sequence encoding the polymerase is
known, it is possible to obtain a nucleotide sequence encoding the
mutant according to the basic principles of molecular biology. For
example, given that the wild type nucleotide sequence encoding
9.degree. N polymerase is known, it is possible to deduce a
nucleotide sequence encoding any given mutant version of 9.degree.
N having one or more amino acid substitutions using the standard
genetic code. Similarly, nucleotide sequences can readily be
derived for mutant versions other polymerases from both family A
and family B polymerases such as, for example, Vent.TM., Pfu, Tsp
JDF-3, Taq, etc. Nucleic acid molecules having the required
nucleotide sequence may then be constructed using standard
molecular biology techniques known in the art.
[0104] In one particular embodiment the invention relates to
nucleic acid molecules encoding mutant versions of the 9.degree. N
polymerase.
[0105] Therefore, the invention provides a nucleic acid molecule
which encodes an altered 9.degree. N polymerase, the nucleic acid
molecule comprising, consisting essentially of or consisting of the
nucleotide sequence of any of SEQ ID NO: 2, 4, 6, 19 or 20.
[0106] In accordance with the present invention, a defined nucleic
acid includes not only the identical nucleic acid but also any
minor base variations including, in particular, substitutions in
cases which result in a synonymous codon (a different codon
specifying the same amino acid residue) due to the degenerate code
in conservative amino acid substitutions. The term "nucleic acid
sequence" also includes the complementary sequence to any single
stranded sequence given regarding base variations.
[0107] The nucleic acid molecules described herein may also,
advantageously, be included in a suitable expression vector to
express the polymerase proteins encoded therefrom in a suitable
host. Thus, there is provided an expression vector comprising,
consisting essentially of or consisting of the nucleotide sequence
of any of SEQ ID NO: 2, 4, 6, 19 or 20. Incorporation of cloned DNA
into a suitable expression vector for subsequent transformation of
said cell and subsequent selection of the transformed cells is well
known to those skilled in the art as provided in Sambrook et al.
(1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbour
Laboratory.
[0108] Such an expression vector includes a vector having a nucleic
acid according to the invention operably linked to regulatory
sequences, such as promoter regions, that are capable of effecting
expression of said DNA fragments. The term "operably linked" refers
to a juxtaposition wherein the components described are in a
relationship permitting them to function in their intended manner.
Such vectors may be transformed into a suitable host cell to
provide for the expression of a protein according to the
invention.
[0109] The nucleic acid molecule may encode a mature protein or a
protein having a prosequence, including that encoding a leader
sequence on the preprotein which is then cleaved by the host cell
to form a mature protein.
[0110] The vectors may be, for example, plasmid, virus or phage
vectors provided with an origin of replication, and optionally a
promoter for the expression of said nucleotide and optionally a
regulator of the promoter. The vectors may contain one or more
selectable markers, such as, for example, an antibiotic resistance
gene.
[0111] Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and to direct an appropriate level
of transcription initiation and also translation initiation
sequences for ribosome binding. For example, a bacterial expression
vector may include a promoter such as the lac promoter and for
translation initiation the Shine-Delgarno sequence and the start
codon AUG. Similarly, a eukaryotic expression vector may include a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors may
be obtained commercially or be assembled from the sequences
described by methods well known in the art.
[0112] Transcription of DNA encoding the polymerase of the
invention by higher eukaryotes may be optimised by including an
enhancer sequence in the vector. Enhancers are cis-acting elements
of DNA that act on a promoter to increase the level of
transcription. Vectors will also generally include origins of
replication in addition to the selectable markers.
Preferred Uses of the Altered Polymerases
[0113] In a further aspect the invention relates to use of an
altered polymerase having reduced affinity for DNA according to the
invention for the incorporation of a nucleotide into a
polynucleotide. As mentioned above, the nature of the nucleotide is
not limiting since the altered polymerases of the invention retain
affinity for the relevant nucleotides.
[0114] As aforementioned, the invention is based upon the
realization that, the tight binding of a polymerase to the DNA
template is not always an advantageous property. This is
particularly the case in the context of sequencing reactions in
which only a single nucleotide incorporation event is required in
each reaction cycle for each template DNA molecule. In many of
these sequencing reactions a labelled nucleotide is utilised.
[0115] Thus, the invention provides for use of a polymerase which
has been altered such that it displays a reduced affinity for DNA
and an ability to incorporate a labelled nucleotide into a
plurality of separate DNA templates in each reaction cycle for
incorporation of a labelled nucleotide into a polynucleotide, the
label being utilised to determine the nature of the nucleotide
added.
[0116] In one embodiment, the nucleotide comprises a ddNTP. Thus,
the polymerase of the invention may be utilised in a conventional
Sanger sequencing reaction, the details of which are well known in
the art.
[0117] In a preferred embodiment, the nucleotide is a modified
nucleotide which has been modified at the 3' sugar hydroxyl such
that the substituent is larger in size than the naturally occurring
3' hydroxyl group.
[0118] The polymerases of the invention may be used in any area of
technology where it is required/desirable to be able to incorporate
nucleotides, for example modified nucleotides having a substituent
at the 3' sugar hydroxyl position which is larger in size than the
naturally occurring hydroxyl group, into a polynucleotide chain.
They may be used in any area of technology where any of the
desirable properties of the enzyme, for example improved rate of
incorporation of nucleotides even under conditions where the DNA is
present in excess and increased levels of reaction completion under
these conditions, are required. This may be a practical, technical
or economic advantage.
[0119] Although the altered polymerases exhibit desirable
properties in relation to incorporation of modified nucleotides
having a large 3' substituent due to their decreased affinity for
DNA, the utility of the enzymes is not confined to incorporation of
such nucleotide analogues. The desirable properties of the altered
polymerase due to its reduced affinity for DNA may provide
advantages in relation to incorporation of any other nucleotide,
including unmodified nucleotides, relative to enzymes known in the
art. In essence, the altered polymerases of the invention may be
used to incorporate any type of nucleotide that they have the
ability to incorporate.
[0120] The polymerases of the present invention are useful in a
variety of techniques requiring incorporation of a nucleotide into
a polynucleotide, which include sequencing reactions,
polynucleotide synthesis, nucleic acid amplification, nucleic acid
hybridisation assays, single nucleotide polymorphism studies, and
other such techniques. Use in sequencing reactions represents a
most preferred embodiment. All such uses and methods utilizing the
modified polymerases of the invention are included within the scope
of the present invention.
[0121] The invention also relates to a method for incorporating
nucleotides into DNA comprising allowing the following components
to interact:
[0122] (i) A polymerase according to the invention;
[0123] (ii) a DNA template; and
[0124] (iii) a nucleotide solution.
[0125] As discussed above, the polymerase of the invention has
particular applicability in reactions where incorporation of only a
single or relatively few nucleotides are required in each reaction
cycle. Often in these reactions one or more of the nucleotides will
be labelled. Accordingly, the invention provides a method for
incorporating labelled nucleotides into DNA comprising allowing the
following components to interact: [0126] (i) A polymerase which has
been altered such that it displays a reduced affinity for DNA and
an ability to incorporate a labelled nucleotide into a plurality of
separate DNA templates in each reaction cycle, [0127] (ii) a DNA
template; and [0128] (iii) a nucleotide solution.
[0129] In one specific embodiment, the invention provides a method
for incorporating nucleotides which have been modified at the 3'
sugar hydroxyl such that the substituent is larger in size than the
naturally occurring 3' hydroxyl group into DNA comprising allowing
the following components to interact: [0130] A polymerase according
to the present invention (as described above) [0131] a DNA
template; and [0132] a nucleotide solution containing the
nucleotides which have been modified at the 3' sugar hydroxyl such
that the substituent is larger in size than the naturally occurring
3' hydroxyl group.
[0133] Particularly preferred are uses and methods carried out on a
clustered array. Clustered arrays of nucleic acid molecules may be
produced using techniques generally known in the art. By way of
example, WO 98/44151 and WO 00/18957 (both of which are
incorporated by reference herein) both describe methods of nucleic
acid amplification which allow amplification products to be
immobilised on a solid support in order to form arrays comprised of
clusters or "colonies" of immobilised nucleic acid molecules.
Reference is also made to WO 2005/078130 including the citations
referred to therein, the contents of all of which are hereby
incorporated by reference. Incorporation on clusters, in particular
sequencing on clustered arrays, provides specific advantages
because the polymerase is able to incorporate nucleotides into
multiple DNA templates located in close proximity, thus providing a
highly efficient reaction.
[0134] The above components are allowed to interact under
conditions which permit the formation of a phosphodiester linkage
between the 5' phosphate group of a nucleotide and a free 3'
hydroxyl group on the DNA template, whereby the nucleotide is
incorporated into a polynucleotide. Preferred nucleotides,
including modified nucleotides, are described in detail above.
[0135] The incorporation reactions may occur in free solution or
the DNA templates may be fixed to a solid support.
[0136] The rate of incorporation of the nucleotide exhibited by a
mutant enzyme may be similar to the rate of incorporation of
nucleotides exhibited by the unaltered enzyme. Due to the improved
activity of the modified enzyme, thanks to its reduced affinity for
DNA, the same rate of incorporation combined with the ability to
incorporate nucleotides into a plurality of templates in a single
reaction cycle improves the overall rates of completion. However,
it is not necessary for the rate of incorporation of nucleotides to
be precisely the same to that of the unaltered enzyme for a mutant
enzyme to be of practical use. The rate of incorporation may be
less than, equal to or greater than the rate of incorporation of
nucleotides by the unaltered enzyme, provided the overall reaction
efficiency in terms of reaction completion is improved.
[0137] In one particular embodiment of the invention, the altered
polymerases of the invention may be used to incorporate modified
nucleotides into a polynucleotide chain in the context of a
sequencing-by-synthesis protocol. In this particular aspect of the
method the nucleotides may have been modified at the 3' sugar
hydroxyl such that the substituent is larger in size than the
naturally occurring 3' hydroxyl group. These nucleotides are
detected in order to determine the sequence of a DNA template.
[0138] Thus, in a still further aspect, the invention provides a
method of sequencing DNA comprising allowing the following
components to interact: [0139] A polymerase according to the
present invention (as described above) [0140] a DNA template; and
[0141] a nucleotide solution containing the nucleotides which have
been modified at the 3' sugar hydroxyl such that the substituent is
larger in size than the naturally occurring 3' hydroxyl group
[0142] followed by detection of the incorporated modified
nucleotides thus allowing sequencing of the DNA template.
[0143] The DNA template for a sequencing reaction will typically
comprise a double-stranded region having a free 3' hydroxyl group
which serves as a primer or initiation point for the addition of
further nucleotides in the sequencing reaction. The region of the
DNA template to be sequenced will overhang this free 3' hydroxyl
group on the complementary strand. The primer bearing the free 3'
hydroxyl group may be added as a separate component (e.g. a short
oligonucleotide) which hybridises to a region of the template to be
sequenced. Alternatively, the primer and the template strand to be
sequenced may each form part of a partially self-complementary
nucleic acid strand capable of forming an intramolecular duplex,
such as for example a hairpin loop structure. Nucleotides are added
successively to the free 3' hydroxyl group, resulting in synthesis
of a polynucleotide chain in the 5' to 3' direction. After each
nucleotide addition the nature of the base which has been added
will be determined, thus providing sequence information for the DNA
template.
[0144] Such DNA sequencing may be possible if the modified
nucleotides can act as chain terminators. Once the modified
nucleotide has been incorporated into the growing polynucleotide
chain complementary to the region of the template being sequenced
there is no free 3'-OH group available to direct further sequence
extension and therefore the polymerase can not add further
nucleotides. Once the nature of the base incorporated into the
growing chain has been determined, the 3' block may be removed to
allow addition of the next successive nucleotide. By ordering the
products derived using these modified nucleotides it is possible to
deduce the DNA sequence of the DNA template. Such reactions can be
done in a single experiment if each of the modified nucleotides has
attached a different label, known to correspond to the particular
base, to facilitate discrimination between the bases added at each
incorporation step. Alternatively, a separate reaction may be
carried out containing each of the modified nucleotides
separately.
[0145] In a preferred embodiment the modified nucleotides carry a
label to facilitate their detection. Preferably this is a
fluorescent label. Each nucleotide type may carry a different
fluorescent label. However the detectable label need not be a
fluorescent label. Any label can be used which allows the detection
of the incorporation of the nucleotide into the DNA sequence.
[0146] One method for detecting the fluorescently labelled
nucleotides, suitable for use in the methods of the invention,
comprises using laser light of a wavelength specific for the
labelled nucleotides, or the use of other suitable sources of
illumination.
[0147] In one embodiment, the fluorescence from the label on the
nucleotide may be detected by a CCD camera.
[0148] If the DNA templates are immobilised on a surface they may
preferably be immobilised on a surface to form a high density
array, which is preferably a clustered or "colonial" array as
discussed supra. In one embodiment, and in accordance with the
technology developed by the applicants for the present invention,
the high density array comprises a single molecule array, wherein
there is a single DNA molecule at each discrete site that is
detectable on the array. Single-molecule arrays comprised of
nucleic acid molecules that are individually resolvable by optical
means and the use of such arrays in sequencing are described, for
example, in WO 00/06770, the contents of which are incorporated
herein by reference. Single molecule arrays comprised of
individually resolvable nucleic acid molecules including a hairpin
loop structure are described in WO 01/57248, the contents of which
are also incorporated herein by reference. The polymerases of the
invention are suitable for use in conjunction with single molecule
arrays prepared according to the disclosures of WO 00/06770 of WO
01/57248. However, it is to be understood that the scope of the
invention is not intended to be limited to the use of the
polymerases in connection with single molecule arrays.
[0149] Single molecule array-based sequencing methods may work by
adding fluorescently labelled modified nucleotides and an altered
polymerase to the single molecule array. Complementary nucleotides
base-pair to the first base of each nucleotide fragment and are
then added to the primer in a reaction catalysed by the improved
polymerase enzyme. Remaining free nucleotides are removed.
[0150] Then, laser light of a specific wavelength for each modified
nucleotide excites the appropriate label on the incorporated
modified nucleotides, leading to the fluorescence of the label.
This fluorescence may be detected by a suitable CCD camera that can
scan the entire array to identify the incorporated modified
nucleotides on each fragment. Thus millions of sites may
potentially be detected in parallel. Fluorescence may then be
removed.
[0151] The identity of the incorporated modified nucleotide reveals
the identity of the base in the sample sequence to which it is
paired. The cycle of incorporation, detection and identification
may then be repeated approximately 25 times to determine the first
25 bases in each oligonucleotide fragment attached to the array,
which is detectable.
[0152] Thus, by simultaneously sequencing all molecules on the
array, which are detectable, the first 25 bases for the hundreds of
millions of oligonucleotide fragments attached in single copy to
the array may be determined. Obviously the invention is not limited
to sequencing 25 bases. Many more or less bases may be sequenced
depending on the level of detail of sequence information required
and the complexity of the array.
[0153] Using a suitable bioinformatics program the generated
sequences may be aligned and compared to specific reference
sequences. This allows determination of any number of known and
unknown genetic variations such as single nucleotide polymorphisms
(SNPs) for example.
[0154] The utility of the altered polymerases of the invention is
not limited to sequencing applications using single-molecule
arrays. The polymerases may be used in conjunction with any type of
array-based (and particularly any high density array-based)
sequencing technology requiring the use of a polymerase to
incorporate nucleotides into a polynucleotide chain, and in
particular any array-based sequencing technology which relies on
the incorporation of modified nucleotides having large 3'
substituents (larger than natural hydroxyl group), such as 3'
blocking groups.
[0155] The polymerases of the invention may be used for nucleic
acid sequencing on essentially any type of array formed by
immobilisation of nucleic acid molecules on a solid support. In
addition to single molecule arrays suitable arrays may include, for
example, multi-polynucleotide or clustered arrays in which distinct
regions on the array comprise multiple copies of one individual
polynucleotide molecule or even multiple copies of a small number
of different polynucleotide molecules (e.g. multiple copies of two
complementary nucleic acid strands).
[0156] In particular, the polymerases of the invention may be
utilised in the nucleic acid sequencing method described in WO
98/44152, the contents of which are incorporated herein by
reference. This International application describes a method of
parallel sequencing of multiple templates located at distinct
locations on a solid support. The method relies on incorporation of
labelled nucleotides into a polynucleotide chain.
[0157] The polymerases of the invention may be used in the method
described in International Application WO 00/18957, the contents of
which are incorporated herein by reference. This application
describes a method of solid-phase nucleic acid amplification and
sequencing in which a large number of distinct nucleic acid
molecules are arrayed and amplified simultaneously at high density
via formation of nucleic acid colonies and the nucleic acid
colonies are subsequently sequenced. The altered polymerases of the
invention may be utilised in the sequencing step of this
method.
[0158] Multi-polynucleotide or clustered arrays of nucleic acid
molecules may be produced using techniques generally known in the
art. By way of example, WO 98/44151 and WO 00/18957 both describe
methods of nucleic acid amplification which allow amplification
products to be immobilised on a solid support in order to form
arrays comprised of clusters or "colonies" of immobilised nucleic
acid molecules. The contents of WO 98/44151 and WO 00/18957
relating to the preparation of clustered arrays and use of such
arrays as templates for nucleic acid sequencing are incorporated
herein by reference. The nucleic acid molecules present on the
clustered arrays prepared according to these methods are suitable
templates for sequencing using the polymerases of the invention.
However, the invention is not intended to be limited to use of the
polymerases in sequencing reactions carried out on clustered arrays
prepared according to these specific methods.
[0159] The polymerases of the invention may further be used in
methods of fluorescent in situ sequencing, such as that described
by Mitra et al. Analytical Biochemistry 320, 55-65, 2003.
[0160] The present invention also contemplates kits which include
the polymerase of the invention, possibly packaged together with
suitable instructions for use. The polymerase will be provided in a
form suitable for use, for example provided in a suitable buffer or
may be in a form which can be reconstituted for use (e.g. in a
lyophilized form).
[0161] Thus, a kit is provided for use in a nucleotide
incorporation reaction or assay comprising a polymerase of the
invention as described herein and a solution of nucleotides, the
nucleotides being such that the polymerase can incorporate them
into a growing DNA strand. Preferred nucleotides include suitably
labelled nucleotides which can thus be used in sequencing reactions
for example. Labels may include fluorescent labels, radiolabels
and/or mass labels as are known in the art.
[0162] In one preferred embodiment, the nucleotide solution
comprises, consists essentially of or consists of synthetic (i.e.
non-natural) nucleotides such as ddNTPS for example. The kit may
thus be utilised in a Sanger sequencing reaction for example.
[0163] In a further embodiment, the nucleotide solution comprises,
consists essentially of or consists of modified nucleotides.
Preferred modified nucleotides are defined above with respect to
the polymerases of the invention and this description applies
mutatis mutandis here.
[0164] The kit may, in a further embodiment, also incorporate
suitable primer and/or DNA template molecules which allow a
nucleotide incorporation reaction to be carried out.
[0165] In a still further aspect, the invention provides a method
for producing a polymerase according to the invention
comprising:
[0166] (i) selecting residues for mutagensis in the polymerase;
[0167] (ii) producing a mutant polymerase in accordance with the
selection made in (i);
[0168] (iii) determining the affinity of the mutant polymerase for
DNA; and
[0169] (iv) if the affinity for DNA is reduced, testing the
polymerase for an ability to form an increased number of productive
polymerase-DNA complexes in each reaction cycle.
[0170] Preferably affinity for nucleotides is unaffected, but may
be considered satisfactory if it remains of the same order as for
the unmodified polymerase.
[0171] In one embodiment, the method further comprises ensuring
that the fidelity of the polymerase remains of the same order
following mutagenesis.
[0172] Preferably fidelity is unaffected, but may be considered
acceptable if it remains of the same order as for the modified
polymerase.
[0173] Reaction cycle is as defined above.
[0174] In a preferred embodiment, the test of the polymerase
includes the use of synthetic nucleotides to determine whether an
increased number of productive polymerase-DNA complexes are being
formed. Suitable nucleotide incorporation assays in which the
polymerase may be tested are known in the art (e.g. see
WO2005/024010) and are described in more detail in the experimental
section below.
[0175] In one embodiment, residues are selected on the basis of the
9.degree. N primary amino acid sequence. In one embodiment, the
selection is made by predicting which amino acids will contact the
DNA. Alternatively, residues may be selected which are predicted to
stabilise the interaction of the polymerase with DNA and/or which
are found in the DNA binding domain of the polymerase and/or which
are basic. Predictions may be based on crystal structures of a
suitable polymerase, as discussed supra and in the experimental
section (example 1).
[0176] Methods of mutagenesis, in particular site-directed
mutagenesis, are well characterised in the art and kits are
commercially available. Accordingly, these techniques are not
discussed in detail. Any suitable technique may be utilized in the
method of the invention.
[0177] The reduction in affinity for DNA may be measured by any
suitable method. Preferably, the affinity is reduced at least, or
approximately, 1.5-fold, 2-fold, 3-fold, 4-fold or 5-fold etc.
compared to the original unaltered polymerase. This affinity may be
measured with reference to the dissociation constant for
example.
[0178] In a preferred embodiment, the polymerase is a family B
polymerase, preferably derived from a thermophilic archaeon and
most preferably is 9.degree. N polymerase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0179] The invention will be further understood with reference to
the following experimental section and figures in which:
[0180] FIGS. 1A and 1B show overexpression of mutant enzymes of the
invention.
[0181] FIG. 2 shows results of a NUNC tube assay using crude
preparations of the mutant enzymes.
[0182] FIGS. 3A and 3B show results of a single base incorporation
assay utilising the mutant enzymes.
[0183] FIG. 4 is a further presentation of the activity of the
mutant enzymes.
[0184] FIG. 5 presents results of timecourses for a single base
incorporation assay at [DNA]>[pol] (ratio 5:1) for the control
polymerase (YAV) and for each of the three mutant enzymes (K705A,
R713A and R743A).
[0185] FIG. 6 shows results of the washing assay, with the
fluorescence image of the NUNC wells shown.
[0186] FIG. 7 also presents results of the washing assay, showing
the affinity of the respective polymerases for a DNA template (the
data for K705A has been omitted for clarity).
[0187] FIG. 8 represents Michaelis plots showing the kinetic
characterization of the polymerase enzymes, which are shown
overlaid.
[0188] FIG. 9 presents the nucleotide and amino acid sequences
encoded by the codon-modified gene of clone 9. FIG. 9 discloses SEQ
ID NOS 23 and 22, in order of appearance.
[0189] FIG. 10 presents the results of SDS-PAGE experiments
comparing the expression of 3 clones (1, 2 and 4) of Pol 52
(codon-modified gene of clone 9 when expressed in pET11-a
expression vector) with Pol 19 (clone 9 gene expressed from the
pNEB917 expression vector) and Pol 43 (clone 9 gene expressed from
the pET11-a expression vector) in the crude lysates of uninduced
(gel I) and induced (Gel I,II) cultures.
[0190] Abbreviations: MW-Molecular Weight; PM-protein marker; cl
9-clone 9.
DETAILED DESCRIPTION OF THE INVENTION
Experimental Section
Example 1
Preparation of Altered Polymerases
Rationale
[0191] Site-directed mutations were introduced in the C-terminal
region of 9.degree. N-7 YAV C223S polymerase in an attempt to
reduce the affinity of the enzyme for DNA (wild-type 9.degree. N-7
polymerase has a very high affinity for DNA, Kd=50 pM; Southworth
et al. 1996. PNAS. 93, 5281).
[0192] An energy minimised overlaid alignment (contracted by
Cresset) of the crystal structures of the open form of 9.degree.
N-7 DNA polymerase (PDB=lqht), the open structure of a closely
related DNA polymerase RB69 (PDB=lih7) and the closed form of RB69
(PDB=lig9) was used as a structural model for the identification of
key residues involved in DNA binding. The crystal structure of the
closed form of RB69 polymerase (Franklin et al. 2001. Cell 105,
657) identified a number of residues that formed H-bond or
electrostatic interactions with the complexed DNA, either directly
to the nucleotide bases or the phosphate backbone. A high
proportion of these residues were basic (Lys790, 800, 844, 874, 878
and Arg806), consistent with their likely interaction with acidic
phosphate groups. Inspection of the closed RB69 structure showed
that the majority of these residues adopted orientations toward the
bound duplex. No analogous structure for the closed form of
9.degree. N-7 pol exists and so we used our structural alignment to
identify basic residues in the open form of 9.degree. N-7 pol which
adopted analogous conformations to the basic residues (of those
above) from the RB69 open structure. Of the 6 basic residues from
RB69, 3 were found to have a corresponding basic residue in
9.degree. N-7, these were: Arg743 (RB69 Lys878), Arg713 (Lys800)
and Lys705 (Lys844). It was decided to engineer 4 mutant enzymes,
the alanine variants of the residues shown (R743A, R713A and K705A)
and a 71 amino acid deletion (71), which removed an .alpha.-helix
from the thumb subdomain (residues disordered in the 9.degree. N-7
pol structure) within which the three residues above were
located.
Mutagenesis and Cloning
[0193] Mutations were introduced into pSV19 (plasmid encoding
9.degree. N-7 YAV C223S exo-polymerase) via a PCR method using
Stratagene Quikchange XL kit and the protocol thereof (also see WO
2005/024010)
[0194] Mutagenic primers used:
TABLE-US-00001 R743A. (SEQ ID NO: 9) fwd
5'-CCCGGCGGTGGAGGCGATTCTAAAAGCC-3' (SEQ ID NO: 10) rev
3'-GGGCCGCCACCTCCGCTAAGATTTTCGG-5' R713A (SEQ ID NO: 11) fwd
5'-GAAGGATAGGCGACGCGGCGATTCCAGCTG-3' (SEQ ID NO: 12) rev
3'-CTTCCTATCCGCTGCGCCGCTAAGGTCGAC-5' K705A (SEQ ID NO: 13) fwd
5'-GCTACATCGTCCTAGCGGGCTCTGGAAGG-3' (SEQ ID NO: 14) rev
3'-CGATGTAGCAGGATCGCCCGAGACCTTCC-5' 71 (C-terminus 704) (SEQ ID NO:
15) fwd 5'-GCTACATCGTCCTATGAGGCTCTGGAAGG-3' (SEQ ID NO: 16) rev
3'-CGATGTAGCAGGATACTCCGAGACCTTCC-5'
[0195] Potential clones were selected and PCR fragments of the gene
sequenced to confirm the presence of the mutation. Positive clones
were produced for all mutants.
Overexpression and Growth:
[0196] Transformed into expression strain Novagen RosettaBlue DE3
pLysS
[0197] Growth and induction carried out as described in
Experimental section of WO 2005/024010.
[0198] Harvest and lysis carried out as described in Experimental
section of WO 2005/024010.
[0199] Purification carried out as described in Experimental
section of WO 2005/024010.
Results:
[0200] Successful overexpression of mutant enzymes was achieved.
All mutant enzymes were overexpressed. SDS-PAGE gels were run to
check overexpression of the constructs (-=uninduced; +=IPTG
induced). The resulting gels are shown in FIG. 1.
Example 2
NUNC Tube Assay Using Crude Protein Preparation
[0201] Small 5 ml cultures of the mutant enzymes (along with a
culture of YAV C223S exo- for direct comparison) were taken through
a quick purification as outlined in WO 2005/024010 up until the
heat treatment step. At this point, the samples were considered to
be sufficiently pure to test their activity.
[0202] The buffers for each of the crude preparations were
exchanged into enzymology buffer (50 mM Tris pH 8.0, 6 mM MgSO4, 1
mM EDTA, 0.05% Tween20) using an S300 gel filtration spin-column.
The samples were not normalised for concentration. The test
employed was a simple incorporation of ffTTP into surface-coupled
A-template hairpin. 2 pmoles of 5'-amino oligo 815
(5'-CGATCACGATCACGATCACGATCACGATCACGATCACGCTGATGTGCATGCTGTTG
TTTTTTTACAACAGCATGCACATCAGCG-3') (SEQ ID NO: 17) was coupled to a
NUNC-nucleolink strip according to the manufacturers protocol.
[0203] Once washed, each well was incubated with a 20 .quadrature.l
aliquot of a crude enzyme preparation (identity of enzyme listed
below) and 5 DM ffT-N3-647. The strip was then incubated at
45.degree. C. for 30 minutes. The experiment was performed in
duplicate. Upon completion of the 30 minute incubation, wells were
washed with 3.times.100 .quadrature.l of high salt wash buffer (10
mM Tris pH 8.0, 1M NaCl, 10 mM EDTA) and then 3.times.100
.quadrature.l of MilliQ water. Strips were scanned on a typhoon
fluorescence imager CY5 filter, PMT=450 V).
[0204] The results are presented in FIG. 2, in which the wells are
as follows:
1=20 .mu.l enzymology buffer only+1 .mu.l 100 .mu.M ffT-N3-647 2=20
.mu.l crude YAV C223S exo-+1 .mu.l 100 .mu.M ffT-N3-647 3=20 .mu.l
crude YAV C223S R743A exo-(clone 12)+1 .mu.l 100 .mu.M ffT-N3-647
4=20 .mu.l crude YAV C223S K705A exo-(clone 15)+1 .mu.l 100 .mu.M
ffT-N3-647 5=20 .mu.l crude YAV C223S R743A exo-(clone 16)+1 .mu.l
100 .mu.M ffT-N3-647 6=20 .mu.l crude YAV C223S R713A exo-(clone
24)+1 .mu.l 100 .mu.M ffT-N3-647 7=20 .mu.l crude YAV C223S 71
exo-(clone 38)+1 .mu.l 100 .mu.M ffT-N3-647 8=20 .mu.l crude YAV
C223S R713A exo-(clone 39)+1 .mu.l 100 .mu.M ffT-N3-647
Results
[0205] Enzymology was observed in all wells except the background
wells (MilliQ only) and well 1 (no enzyme control). The
fluorescence density is proportional to the amount of ffTTP
incorporation--the darker the well, the greater the level of
incorporation. Performance of the mutant enzymes will be discussed
relative to YAV (clone 9)(YAV C223S exo-). Deletion of the tip of
the thumb subdomain (71 mutant) results in an enzyme that is
severely catalytically compromised, and only incorporates to 35% of
the level seen for clone 9. Mutant K705A was equivalent to clone 9.
The two arginine mutants R743A and R713A showed elevated levels of
incorporation, showing .about.45% improvements over clone 9.
Conclusion
[0206] Mutant enzymes K705A, R713A and R743A display improved
levels of incorporation and decreased affinity of the enzyme for
DNA. Removal of all three of these basic residues, in combination
with deletion of additional residues, abolishes activity (71
mutant). It may be that substitution of all three residues would
not lead to a decrease in activity, in the absence of further
mutations/deletions.
Example 3
Single Base Incorporation Assay
[0207] The activity of the crude enzyme preparations (normalised
concentrations) was measured using the single base incorporation
assay as described in WO 2005/024010. 10 minute incubations were
run with either 30 or 3 .mu.g/ml crude enzyme preparation in the
presence of 2 .mu.M ffT-N3-cy3 and 20 nM 10 A hairpin DNA
(.sup.32P-labelled), aliquots of the reaction mixture were
withdrawn at 0, 30, 60, 180 and 600 s and run on a 12% acrylamide
gel.
Results
[0208] The gel images are shown in FIG. 3.
[0209] The band intensities were quantified using Imagequant and
the fluorescence intensity plotted versus incubation time to
generate the time-courses shown in FIG. 4.
[0210] These data give an estimate of the performance of the mutant
enzymes for the first base incorporation of ffTTP relative to YAV.
Due to the concentration normalisation, the activities are directly
comparable. The 71 mutant is essentially inactive (kobs is 21% of
that observed for YAV), R743A and K705A have comparable activities
to YAV, but R713A shows a significant enhancement in both kobs
(2.times. that observed for YAV) and the level of cycle
completion.
Example 4
Single Base Incorporation Assay for Purified Polymerases Under
Conditions where [DNA] is Greater than [Pol]
[0211] The activity of the purified enzyme preparations of Clone 9
polymerase (YAV C223S exo-) and the thumb sub-domain mutants K705A,
R713A and R743A was measured using the single base incorporation
assay as described in WO 2005/024010. The experiment was carried
out such that the respective concentrations of DNA and polymerase
were at a ratio of approximately 5:1. Thus, the ability of the
enzyme to incorporate nucleotides into multiple DNA template
molecules in a single reaction cycle was investigated. 30 minute
incubations were run with 4 nM purified enzyme in the presence of
20 nM 10 A hairpin DNA (.sup.32P-labelled) and 2 .mu.M ffT-N3-cy3,
aliquots of the reaction mixture were withdrawn at 0, 15, 30, 60,
180, 480, 900 and 1800 s intervals and run on a 12% acrylamide
gel.
Results
[0212] The band intensities were quantified using Imagequant and
the fluorescence intensity, converted into percentage completion
(based on the relative intensities of the starting material and
final product bands on the gel) plotted versus incubation time to
generate the timecourses shown in FIG. 5.
[0213] Timecourse plots for clone 9 and K705A are biphasic in
nature, displaying an initial exponential "burst" phase (black
line) followed by a linear dependence of product conversion with
time (grey line). The amplitude of the burst phase is greater for
K705A than for clone 9 (.about.28% and 19% respectively) and the
gradient of the linear phase is steeper (hence faster) for K705A
than clone 9. The significance of this observation is discussed
below.
[0214] In contrast to this, both R713A and R743A mutant enzymes do
not show this biphasic nature, instead, only the fast exponential
phase is observed. In both cases, the amplitude of the exponential
phase is .about.90% indicating a higher degree of product
conversion within this exponential phase than either clone 9 or
K705A. The burst phase equates to the rate of incorporation of
ffTTP of the population of DNA molecules associated with a
polymerase prior to reaction initiation i.e. maximum rate at which
the ternary pol:DNA:ffTTP complex can turnover. Any subsequent
phase is attributed to a slower dissociation/re-association process
required for the polymerase to sequester new substrate molecules
(DNA and ffTTP). The biphasic nature observed for clone 9 and K705A
suggests that the slow post-burst phase is caused by the difficulty
of the enzyme to dissociate and re-associate with DNA, most likely
due to their low Kd (DNA).
[0215] The mutation of basic residues that may contact duplex DNA
when bound by the polymerase (namely R713 and R743) to remove this
functionality results in mutant enzymes which only display burst
kinetics (R713A and R743A). We interperet this in one of two ways,
i) as having improved the enzymes ability to dissociate and
re-associate with DNA by decreasing the affinity for DNA (increased
Kd(DNA)) and/or ii) the decrease in affinity for DNA in these
mutants results in a larger "active enzyme" fraction in the
polymerase preparation. It has been shown that impure DNA
polymerase (contaminated with E. coli genomic DNA carried through
from lysis) inhibits the enzyme by reducing the active enzyme
fraction of the preparation.
[0216] The crude fitting of the timecourses suggests that the
observed rate constants for the burst phase seen for clone 9 and
K705A are comparable (kobs.about.0.06 s-1) whereas this rate
constant is smaller for R713A (kobs.about.0.01 s-1) and R743A
(kobs.about.0.004 s-1). Under these experimental conditions, the
burst is faster for clone 9 and K705A than for R713A or R743A, but
the latter two enzymes reach completion in a shorter period of time
due to the absence of the slow, linear dissociation/re-association
phase inherent to clone 9 and K705A.
Example 5
Washing Assay
[0217] Employing a washing assay qualitatively assesses the
affinity of purified enzyme preparations for DNA. 4 (1.times.8)
NUNC nucleolink strips were functionalized with 2 pmoles of
5'-amino A-template hairpin, oligo 815 (5'
H2N-CGATCACGATCACGATCACGATCACGATCACGATCACGCTGATGTGCATGCTGTTGTTTTTTTACAACA-
GC ATGCACATCAGCG-3') (SEQ ID NO: 18) according to the
manufacturer's protocol.
[0218] Once washed, each well was incubated with a 20 .mu.l aliquot
of 500 nM enzyme (clone 9, K705A, R713A or R743A mutants) at
45.degree. C. for 30 minutes. Post incubation, each well was washed
with 3.times.100 ml of 10 mM Tris pH 8.0, 10 mM EDTA including
varying concentrations of NaCl (0, 0.05, 0.1, 0.3, 0.4, 0.75, 1.0,
2.0 M) and then 3.times.100 ml MilliQ water. Wells were
subsequently pre-equilibrated with enzymology buffer prior to a
further incubation of 20 .mu.l of 2 .mu.M ffT-N3-647 at 45.degree.
C. for 30 minutes.sub.-- Wells were washed with 3.times.100 ml high
salt wash buffer (10 mM Tris pH 8.0, 1M NaCl, 10 mM EDTA) and then
3.times.100 ml MilliQ water. Strips scanned on Typhoon fluorescence
imager (y5 filter, PMT=500 V).
Results
[0219] The fluorescence image of the NUNC wells is shown in FIG.
6.
[0220] Any fluorescence in the wells is due to residual enzyme
bound to the surface-coupled DNA post-wash. Increasing the ionic
strength of the wash buffer between incubation should destabilise
the interaction between the polymerase and the DNA by masking
electrostatic interactions. Enzyme should be more effectively
washed off the DNA at higher ionic strength.
[0221] When a low ionic strength wash is employed between
incubations all enzymes tested displayed a high level of
incorporation, therefore ineffective at dissociating enzyme from
DNA. As the concentration of NaCl in the wash buffer increased, the
behaviour of the enzymes relative to each other changed. Mutant
enzymes R713A and R743A were more effectively removed from the DNA
at [NaCl]<200 mM, whereas K705A and clone 9 showed a similar
response to each other, but required higher [NaCl] to remove them
from the DNA. Even after a wash with 2 M NaCl, a significant (ca.
75%) level of incorporation relative to a 0 M NaCl wash was
observed for clone 9. This is clearly illustrated in the plot shown
in FIG. 7 (the data for K705A has been omitted for clarity).
Interestingly, none of the enzymes tested appeared to be completely
removed from the DNA after experiencing a 2 M NaCl wash.
[0222] From this experiment, it is clear that mutating residues
8713 and 8743 result in enzymes that display lower affinity for DNA
than clone 9, as evidenced by their ability to be washed from DNA
by lower ionic strength washes.
Example 6
Incorporation Kinetics of ffT-N3-Cy3 by Clone 9, R713A and
R743A
[0223] The kinetic characterization of the enzymes was conducted
using NUNC tube assay and involved the measurement of rate
constants for the first order incorporation of ffT N3 cy3 where
[DNA]<<[pol] or [ffNTP], at a variety of [ffTTP]. Below is
described the methodology used for each of the three polymerases
tested.
[0224] Six (1.times.8) NUNC nucleolink strips were functionalized
with 2 pmoles of 5'-amino A template hairpin oligo 815 (5'
H2N-CGATCACGATCACGATCACGATCACGATCACGATCACGCTGATGTGCATGCTGTTGTTTTTTTACAACA-
GC ATGCACATCAGCG-3') (SEQ ID NO: 18), according to the
manufacturer's protocol.
[0225] Each strip was employed for a time-course experiment at a
particular [ffT-N3-cy3]. 20 .mu.l of enzymology buffer (50 mM Tris
pH 8.0, 6 mM MgSO4, 1 mM EDTA, 0.05% Tween20) was incubated in each
NUNC well at 45.degree. C. for 2 minutes.
[0226] Time-courses were initiated by addition of a 20 .mu.l
aliquot of 2.times. enzymology mix (X .mu.M ffT-N3-cy3, 1.1 .mu.M
polymerase in enzymology buffer) pre-equilibrated at 45.degree. C.
for 2 minutes using an 8-channel multipipette in order to start
reactions in individual wells at identical time-points. The action
of adding the 2.times. enzymology mix to the buffer in the well is
sufficient to allow adequate mixing. The reactions were stopped at
desired time-points by the addition of 125 .mu.l of 250 mM EDTA.
After reactions in all 8 wells stopped, strips were washed with
3.times.100 ml high salt wash (10 mM Tris pH 8.0, 1 M NaCl, 10 mM
EDTA) and then 3.times.100 ml MilliQ water and then scanned on a
Typhoon fluorescence imager (Cy3 filter, PMT=500 V). Fluorescence
intensities in each well were quantified using Imagequant. Plotting
the variation in Cy3 fluorescence intensity vs. time generates
time-course graphs. Under our experimental conditions, these
time-course plots evaluate well to a single exponential decay
process (fitted to equation: y=yo+Aexp (x/t)) from which the
reaction half life, t, is determined, the inverse of which is
termed the observed rate constant kobs (kobs=l/t).
[0227] The magnitude of the observed rate constant is dependent on
the concentration of ffT-N3-cy3, so by repeating this experiment at
different ffT-N3-cy3 concentrations a range of kobs values can be
determined for a particular enzyme. The variation of kobs with
ffT-N3-cy3 concentration is hyperbolic and fits well to the
Michealis-Menten equation: Vmax=(kpolx[S])/(Kd+[S]) here S=ffT
N3-cy3, according to standard enzymological analysis. From the
Michaelis plot, key values characteristic of a particular enzyme
catalyzing a particular reaction can be obtained, namely kpol
(defined as the rate constant for the process at infinite substrate
concentration) and Kd (defined as the dissociation constant, the
concentration of substrate at kpol/2). This process was repeated
for clone 9, R713A and R743A mutants.
[0228] Michaelis plots for all of the enzymes are shown overlaid in
FIG. 8.
Results
[0229] The kinetic characteristics of ffT-N3-cy3 incorporation for
the enzymes tested are summarized below.
TABLE-US-00002 Clone 9 R713A R743A k.sub.pol/s.sup.-1 0.061 0.10
0.068 K.sub.d/.quadrature.M 1.72 3.32 1.92
[0230] From this, it appears as though the mutations to the
DNA-binding region of the polymerases have not adversely affected
either the activity of the enzymes (at high substrate
concentrations, kpol approximates to Vmax) or the affinity the
enzymes have for fully functional nucleotide (in this case
ffT-N3-cy3, but the trend is considered to be applicable to all
bases). This is an ideal situation, as the mutations have had the
desired effect of modifying the DNA-binding affinity of the enzymes
without affecting other key catalytic properties.
Example 7
Purification of the Polymerases and Measurement of Levels of Carry
Over DNA
DNA Contamination
[0231] Pico green assay (Molecular Probes kit, cat # P11496).
Solutions Required
[0232] TE buffer: 10 mM Tris.HCl pH 7.5, 1 mM EDTA
40 mL required, 2 mL of 20.times.TE buffer added to 38 mL
H.sub.2O
.lamda. DNA
[0233] Solution 1 (2 .mu.g/mL .lamda.DNA) dilute 15 .mu.L of
.lamda. DNA with 735 .lamda.L of 1.times.TE buffer.
[0234] Solution 2 (50 ng/mL A) dilute 25 .mu.L of .lamda. DNA with
975 .mu.l of 1.times.TE buffer.
Standard Curve
[0235] In 2 mL eppendorfs the following samples were made:
TABLE-US-00003 Sample .lamda. DNA @ .lamda. DNA @ glycerol .lamda.
DNA 2 mg mL 50 ng mL storage buffer TE (ng) (.mu.L) (.mu.L) (.mu.L)
(.mu.L) 100 160 400 1040 25 40 400 1160 10 16 400 1184 2.5 160 400
1040 1 64 400 1136 0.25 16 400 1184 0.025 1.6 400 1198.4 0 400
1200
3.times.500 .mu.L from each sample was put into 3 eppendorfs.
Enzyme Samples
[0236] In 5 mL bijou bottles the following samples were made:
TABLE-US-00004 glycerol Amount storage buffer TE sample (.mu.L)
(.mu.L) (.mu.L) 1 enzyme 400 1800 stock 2 sample 1 1100 200 900 3
sample 2 1100 200 900 4 sample 3 1100 200 900
2.times.500 .mu.L from each sample was put into 2 eppendorfs.
[0237] A picogreen solution was prepared; 85 .mu.L of picogreen
stock added to 17 mL of 1.times.TE buffer.
[0238] 500 .mu.L of this solution was added to each of the standard
curve and enzyme samples, and was mixed well by pipetting and then
all samples were transferred to 1.5 mL fluorimeter cuvettes.
Using the Fluorimeter
[0239] The advanced reads program of the Cary Eclipse file was
utilised. The .lamda. excitation was set to 480 nm and the A
emission was set to 520 nm, and 1000 volts were used.
Analysis
[0240] Data for the standard curve was entered into Graph pad Prism
a standard curve of the formula y=ax+c was fitted. The
concentration values, x, was then determined.
Results
TABLE-US-00005 [0241] Concentration of DNA associated Polymerase
sample with purified polymerase Clone 9 batch 5 62.9 ng .+-. 1.9 ng
Clone 9 batch 6 63.7 ng .+-. 2.1 ng Clone 9 R743A 0.04 ng .+-. 6.4
ng Clone 9 R713A 8.2 ng .+-. 4.2 ng
[0242] From this experiment, it is clear that the alterations in
the polymerases enhance purification of the enzyme since less
endogenous DNA is carried over during purification. As mentioned
above, carry over of endogenous DNA can adversely influence
activity of the enzyme and so the mutations are clearly
advantageous.
Example 8
Preparation of a Modified Optimised Codon Usage Nucleic Acid
Sequence which Encodes the Clone 9 Polymerase
[0243] The amino acid sequence shown in SEQ ID NO 1 was translated
into a nucleic acid sequence using the optimal nucleic acid
sequence at each codon to encode for the required/desired
amino-acid.
[0244] The deduced nucleic acid sequence is shown in SEQ ID NO.
19.
[0245] In a similar scenario, the nucleic acid sequence presented
as SEQ ID NO:20 was deduced based upon the amino-acid sequence of
the polymerase presented as SEQ ID NO: 21. The polymerase having
the amino acid sequence presented as SEQ ID NO: 21 comprises the
R743A mutation and also carries a substitution mutation to Serine
at both residues 141 and 143. Nucleic acid molecules and proteins
comprising the respective nucleotide and amino acid sequences form
a part of the invention.
Cloning of a codon-modified gene of clone 9 into the expression
vector pET11-a using NdeI-Nhe I sites (to preserve the internal Bam
H I site).
Synthesis of a Codon-Optimised Gene of Clone 9
[0246] The nucleic acid sequence of SEQ ID NO 19 was synthesized
and supplied in pPCR-Script by GENEART.
The DNA and protein sequences were confirmed (results not shown).
Cloning of pSV57 (Codon-Modified Gene of Clone 9 in the pPCRScript
Vector) into pET11-a (Hereinafter Named pSV 52) Preparation of the
pET11-a Vector
[0247] The pET11-a vector (Novagen catalog No. 69436-3) was
digested with Nde I and Nhe I, dephosphorylated, and any undigested
vector ligated using standard techniques.
[0248] The digested vector was purified on a 0.8% agarose gel and
using the MinElute.RTM. Gel extraction kit protocol from
Qiagen.RTM..
[0249] The purified digested pET11-a vector was quantified using a
polyacrylamide TB 4-20% gel.
Preparation of the Insert (Codon-Modified Gene of Clone 9)
[0250] The codon-modified gene of clone 9 synthesized by GENEART in
the pPCRSCript vector (hereinafter pSV 57) was digested with Nde I
and Nhe.
[0251] The digested insert was purified on a 0.8% agarose gel and
using the MinElute.RTM. Gel extraction kit protocol from
Qiagen.RTM..
[0252] The purified digested insert was quantified using a
polyacrylamide TB 4-20% gel.
Ligation
[0253] The pET11-a vector and the insert were ligated (ratio 1:3)
at the Nde I and Nhe I restriction sites using the Quick ligation
kit (NEB, M2200S).
Transformation
[0254] 2 .mu.l of the ligation mixture was used to transform
XL10-gold ultracompetent cells (Stratagene catalog No 200315). PCR
screening of the colonies containing the insert.
[0255] Transformants were picked and DNA minipreps of 3 positive
clones of XL10-gold transformed with the ligation product were
prepared. The three purified plasmids (hereinafter pSV52, clones 1,
2 and 4 were sequenced at the cloning sites and all three clones
were found to have the correct sequence at the cloning sites.
[0256] The minipreps were also used to transform the expression E.
coli host BL21-CodonPlus (DE3)-RIL (Stratagene catalog No. 230245)
as described below.
Southern Blotting
[0257] pVent (pNEB917 derived vector), pSV43 (clone 9 in pETlla),
pSV54 (codon-optimised clone in pET11-a) and pSV57 (codon-modified
gene in pPCR-Script supplied by GENEART) were restricted and
Southern blotted to check for cross hybridisation between the genes
(results not shown).
Expression Studies of Pol 52
[0258] Transformation of pSV52 (clones 1, 2 and 4) into the
expression host E. coli BL21-CodonPlus (DE3) RIL (Stratagene
catalog No 230245).
[0259] 21-25 ng of purified pSV52 .mu.lasmid DNA (clones 1, 2 and
4) was used to transform competent cells of the expression host E.
coli BL21-CodonPlus (DE3) RIL (hereinafter RIL) using the
manufacturer's instructions.
[0260] 50 .mu.l of each transformation was plated onto fresh
Luria-Bertani (LB) agar medium containing 100 .mu.g/ml of
carbenicillin and 34 .mu.g/ml of chloramphenicol (LBCC agar medium)
and incubated overnight at 37.degree. C.
[0261] The following glycerol stocks were also plated onto LBCC
agar plates to be used as controls for the expression studies and
incubated overnight at 37.degree. C.
SOL10204:RIL-pSV19 (clone 9 in pNEB 917 vector) SOL10354:RIL-pSV43
(clone 9 in pET11-a vector)
Production of Cell Pellets Expressing Pol 52 and the Positive
Controls of Clone 9
[0262] Single transformed E. coli colonies were used to inoculate
starter cultures of 3 ml LBCC media in culture tubes and incubated
overnight at 37.degree. C. with shaking (225 rpm).
[0263] The starter cultures were diluted 1/100 into 50 ml LBCC
media in sterile vented Erlenmeyer flasks and incubated at
37.degree. C. with vigorous shaking (300 rpm) for approximately 4
hours until OD.sub.600nm was approximately 1.0.
[0264] 10 ml of the uninduced cultures was removed and the cells
harvested (as described below).
[0265] IPTG was added to a final concentration of 1 mM and the
cultures induced for 2 hours at 37.degree. C. with vigorous shaking
(300 rpm).
[0266] 10 ml of the induced cultures was removed and the cells
harvested as follows:
[0267] Induced and uninduced cells were harvested by centrifugation
at 5000.times.g for 30 min at 4.degree. C.
[0268] The cell pellets were washed and resuspended in 1/10.sup.th
of the culture volume of 1.times. Phosphate Buffered Saline (PBS)
and centrifuged as above.
[0269] The supernatants were decanted and the pellets stored at
-20.degree. C. until required for the cell lysis and purification
steps.
Cell Lysis and Crude Purification of Pol 52 and Clone 9
[0270] The cell pellets were thawed and resuspended in 1/50.sup.th
of culture volume of 1.times. Wash buffer (50 mM Tris-HCl pH 7.9,
50 mM glucose, 1 mM EDTA) containing 4 mg/ml lysozyme freshly added
to the 1.times. buffer and incubated at room temperature for 15
min.
[0271] An equal volume of 1.times. Lysis buffer (10 mM Tris-HCl pH
7.9, 50 mM KCl, 1 mM EDTA, 0.5% (w/v) Tween 20) containing
0.5%(w/v) Tergitol NP-40 and 1.times. "complete EDTA-free"
proteinase inhibitor cocktail (both added freshly to the 1.times.
Lysis buffer) was added to the cells which were gently mixed and
incubated at room temperature for 30 min.
[0272] The cells were heated at 80.degree. C. for 1 hr in a water
bath then centrifuged at 38,800.times.g for 30 min at 4.degree. C.
to remove cell debris and denatured protein.
Preparation of Samples Normalised for Volume and SDS-PAGE
Analysis
[0273] The expression of Pol 52 and clone 9 DNA polymerases was
assessed by analysis of the crude lysates of the uninduced and
induced control samples on SDS-PAGE followed by Coomassie blue
staining.
[0274] Supernatants were carefully removed and the samples
normalised to volume by the addition of 50:50 (v/v) 1.times. Wash
buffer and 1.times. Lysis buffer to a final volume of 370
.mu.l.
Preparation of Samples for Gel I
[0275] 10 .mu.l of the normalised crude lysates (from uninduced and
induced samples) were mixed with 10 .mu.l of loading buffer
containing 143 mM DTT.
Preparation of Samples for Gel II
[0276] Normalised crude lysates from the induced samples only were
dilute 1/10 in distilled water to a final volume of 10 .mu.l and
mixed with 10 .mu.l of loading buffer containing 143 mM DTT.
[0277] All samples were heated at 70.degree. C. for 10 minutes.
SDS-Page
[0278] A NuPage.RTM. 4-12% Bis-Tris gel (Invitrogen catalog No
NP0321BOX) was prepared according to the manufacturer's
instructions.
[0279] 10 .mu.l of SeeBlue.RTM. Plus2 pre-stained proteins standard
(Invitrogen catalog No LC5925) and .mu.l of each sample were loaded
and the gels run at a constant 200V for 50 minutes.
[0280] The gels were stained with Coomassie blue (SimplyBlue.RTM.
Safe stain, Invitrogen, catalog No. LC 6060).
Results
[0281] The results of the SDS-PAGE are shown in FIG. 10.
The estimated expression level in this experiment is 20 mg/L of
culture.
[0282] Similar levels of expression of the codon-modified gene of
clone 9 in E. coli host BL21-CodonPlus (DE3)-RIL (Po152) were
obtained using the expression vector pET11-a when compared to the
un-modified gene of clone 9 in the same cells using either the
expression vector pNEB917 (P0119) or pET11 (Pol 43).
[0283] No significant differences were observed in the levels of
expression of the 3 different clones of Pol 52.
REFERENCES
[0284] Crystal structure of a bacteriophage T7 DNA replication
complex at 2.2 .ANG. resolution. [0285] Doublie et al. 1998. Nature
391, 251. [0286] Function of the C-terminus of Phi29 DNA polymerase
in DNA and terminal protein binding. [0287] Truniger et al. 2004.
Nucleic Acids Research 32, 371. [0288] A thumb subdomain mutant of
the large fragment of Escherichia coli DNA polymerase I with
reduced DNA binding affinity, processivity and frameshift fidelity.
[0289] Minnick et al. 1996. J. Biol. Chem., 271. 24954. [0290]
Identification of residues critical for the polymerase activity of
the Klenow fragment of DNA polymerase I from Escherichia coli.
[0291] Polesky et al. 1990. J. Biol. Chem., 265, 14579. [0292]
Cloning of thermostable DNA polymerases from hyperthermophilic
marine archaea with emphasis on Thermococcus sp. 9.degree. N-7 and
mutations affecting 3'-5' exonuclease activity. [0293] Southworth
et al. 1996. PNAS. 93, 5281 [0294] Structure of the replicating
complex of a pol alpha family DNA polymerase. Franklin et al. 2001.
Cell 105, 657. [0295] Crystal structure of a pol alpha family DNA
polymerase from the hyperthermophilic archaeon Thermococcus sp.
9.degree. N-7. [0296] Rodriguez et al. 2000. J. Mol. Biol., 299,
471.
[0297] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
Sequence CWU 1
1
241775PRTArtificial sequenceMutant polymerase 1Met Ile Leu Asp Thr
Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe
Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr
Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40
45 Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Thr Val Val Lys
50 55 60 Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg
Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Asn His Pro Gln Asp
Val Pro Ala Ile 85 90 95 Arg Asp Arg Ile Arg Ala His Pro Ala Val
Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr
Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu
Leu Thr Met Leu Ala Phe Ala Ile Ala Thr 130 135 140 Leu Tyr His Glu
Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser
Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170
175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190 Arg Phe Leu Arg Val Val Arg Glu Lys Asp Pro Asp Val Leu
Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys
Lys Arg Ser Glu 210 215 220 Glu Leu Gly Ile Lys Phe Thr Leu Gly Arg
Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg
Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr
Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu
Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys
Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly 290 295
300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu
Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser
Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala
Tyr Lys Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg
Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val
Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr
Leu Asp Phe Arg Ser Tyr Ala Val Ser Ile Ile Ile Thr His 405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420
425 430 Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly
Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln
Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Val Asp Pro Leu Glu
Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Leu Ile Lys Ile
Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Lys Ala
Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp
Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 515 520 525 Glu Glu
Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 530 535 540
His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545
550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Pro Lys Leu Pro Gly Leu
Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe
Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys
Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser
Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile
Leu Lys His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val
Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro
Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp 660 665
670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala
675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile
Val Leu 690 695 700 Ala Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro
Ala Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Arg Tyr Asp Ala
Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg
Ile Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr
Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp 755 760 765 Leu Lys Val
Lys Gly Lys Lys 770 775 22328DNAArtificial sequenceMutant
polymerase 2atgattctcg ataccgacta catcaccgag aacgggaagc ccgtgataag
ggtcttcaag 60aaggagaacg gcgagtttaa aatcgagtac gacagaacct tcgagcccta
cttctacgcc 120cttctgaagg acgattctgc gatagaggac gtcaagaagg
taaccgcaaa gaggcacgga 180acggttgtca aggtgaagcg cgccgagaag
gtgcagaaga agttcctcgg caggccgata 240gaggtctgga agctctactt
caaccatcct caggacgtcc cggcgattcg agacaggata 300cgtgcccacc
ccgctgtcgt tgacatctac gagtacgaca tacccttcgc caagcgctac
360ctcatcgaca agggcctgat tccgatggag ggcgacgagg agcttacgat
gctcgccttc 420gcgatcgcaa ccctctatca cgagggcgag gagttcggaa
ccgggccgat tctcatgata 480agctacgccg acgggagcga ggcgagggtg
ataacctgga agaagattga ccttccgtac 540gttgacgtcg tctcgaccga
gaaggagatg attaagcgct tcctccgcgt cgtcagggag 600aaggaccccg
acgtgctcat cacctacaac ggcgacaact tcgacttcgc ctacctgaag
660aagcgctctg aggaactcgg aataaagttc acactcggca gggacgggag
cgagccgaag 720atacagcgaa tgggcgaccg ctttgccgtt gaggtgaagg
gcaggattca cttcgacctc 780taccccgtca taaggcgcac gataaacctc
ccgacctaca cccttgaggc cgtttacgag 840gccgtctttg gaaagcccaa
ggagaaggtt tacgcagagg agatagcgca ggcctgggag 900agcggggagg
gccttgaaag ggttgcaaga tactcgatgg aggacgctaa ggtgacctac
960gagctgggaa gggagttctt cccgatggag gcccagcttt cgaggcttat
aggccagagc 1020ctctgggacg tctcgcgctc gagcaccgga aatttggtgg
agtggttcct cctgcggaag 1080gcctacaaga ggaacgagct cgccccaaac
aagcccgacg agagggagct cgcgagacgg 1140cgcgggggct acgctggcgg
gtacgttaag gaaccagagc ggggattgtg ggacaacatt 1200gtgtatctag
acttccgctc gtatgcggtt tcaatcatca taacccacaa cgtctcgccg
1260gataccctca accgcgaggg ctgtaaagag tacgacgtcg cccctgaggt
tggacacaag 1320ttctgcaagg acttccccgg cttcatacca agcctcctgg
gagatttgct cgaggagagg 1380cagaagataa agcggaagat gaaggcaacg
gttgacccgc tggagaagaa actcctcgat 1440tacaggcaga ggctgatcaa
aatcctcgcc aacagcttct acggctacta cggctacgcc 1500aaggcccggt
ggtactgcaa ggagtgcgcc gagagcgtta cggcctgggg aagggagtat
1560atagaaatgg ttatccggga actcgaagaa aaattcggtt ttaaagttct
ctatgccgat 1620acagacggtc tccatgctac cattcccgga gcagacgctg
aaacagtcaa gaaaaaagca 1680aaggagttct taaaatacat taatccaaaa
ctgcccggcc tgctcgaact tgagtacgag 1740ggcttctacg tgaggggctt
cttcgtcacg aagaagaagt acgctgtgat agacgaggag 1800ggcaagataa
ccacgagggg tcttgagatt gtgaggcgcg actggagcga gatagcgaag
1860gagacccagg ccagggtctt agaggcgata ctcaagcacg gtgacgtcga
ggaggccgtt 1920aggatagtca aggaagtgac ggaaaagctg agcaagtatg
aggtcccgcc cgagaagctg 1980gtaatccacg agcagataac gcgcgatttg
agggattaca aagccaccgg cccgcacgtt 2040gccgttgcga agaggctcgc
ggcgcgtgga gtgaaaatcc ggcccggcac ggtgataagc 2100tacatcgtcc
tagcgggctc tggaaggata ggcgacaggg cgattccagc tgatgagttc
2160gacccgacga agcaccgcta cgatgcggaa tactacatcg agaaccaggt
tctcccggcg 2220gtggagagga ttctaaaagc cttcggctat cggaaggagg
atttgcgcta ccagaagacg 2280aagcaggtcg gcttgggcgc gtggctgaag
gtgaagggga agaagtga 23283775PRTArtificial sequenceMutant polymerase
3Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1
5 10 15 Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp
Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp
Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Val Thr Ala Lys Arg His
Gly Thr Val Val Lys 50 55 60 Val Lys Arg Ala Glu Lys Val Gln Lys
Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe
Asn His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Arg Ile Arg
Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro
Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met
Glu Gly Asp Glu Glu Leu Thr Met Leu Ala Phe Ala Ile Ala Thr 130 135
140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160 Ser Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp
Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu
Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val Val Arg Glu Lys
Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp
Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220 Glu Leu Gly Ile Lys
Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln
Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260
265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys
Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser
Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp
Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro
Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp
Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe
Leu Leu Arg Lys Ala Tyr Lys Arg Asn Glu Leu Ala 355 360 365 Pro Asn
Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385
390 395 400 Val Tyr Leu Asp Phe Arg Ser Tyr Ala Val Ser Ile Ile Ile
Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys
Lys Glu Tyr Asp 420 425 430 Val Ala Pro Glu Val Gly His Lys Phe Cys
Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu
Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr
Val Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln
Arg Leu Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 Tyr
Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505
510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu
515 520 525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp
Gly Leu 530 535 540 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val
Lys Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Pro
Lys Leu Pro Gly Leu Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr
Val Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile
Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val
Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg
Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val 625 630
635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val
Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp
Leu Arg Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala
Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr
Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly
Asp Ala Ala Ile Pro Ala Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys
His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu
Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp 755
760 765 Leu Lys Val Lys Gly Lys Lys 770 775 42328DNAArtificial
sequenceMutant polymerase 4atgattctcg ataccgacta catcaccgag
aacgggaagc ccgtgataag ggtcttcaag 60aaggagaacg gcgagtttaa aatcgagtac
gacagaacct tcgagcccta cttctacgcc 120cttctgaagg acgattctgc
gatagaggac gtcaagaagg taaccgcaaa gaggcacgga 180acggttgtca
aggtgaagcg cgccgagaag gtgcagaaga agttcctcgg caggccgata
240gaggtctgga agctctactt caaccatcct caggacgtcc cggcgattcg
agacaggata 300cgtgcccacc ccgctgtcgt tgacatctac gagtacgaca
tacccttcgc caagcgctac 360ctcatcgaca agggcctgat tccgatggag
ggcgacgagg agcttacgat gctcgccttc 420gcgatcgcaa ccctctatca
cgagggcgag gagttcggaa ccgggccgat tctcatgata 480agctacgccg
acgggagcga ggcgagggtg ataacctgga agaagattga ccttccgtac
540gttgacgtcg tctcgaccga gaaggagatg attaagcgct tcctccgcgt
cgtcagggag 600aaggaccccg acgtgctcat cacctacaac ggcgacaact
tcgacttcgc ctacctgaag 660aagcgctctg aggaactcgg aataaagttc
acactcggca gggacgggag cgagccgaag 720atacagcgaa tgggcgaccg
ctttgccgtt gaggtgaagg gcaggattca cttcgacctc 780taccccgtca
taaggcgcac gataaacctc ccgacctaca cccttgaggc cgtttacgag
840gccgtctttg gaaagcccaa ggagaaggtt tacgcagagg agatagcgca
ggcctgggag 900agcggggagg gccttgaaag ggttgcaaga tactcgatgg
aggacgctaa ggtgacctac 960gagctgggaa gggagttctt cccgatggag
gcccagcttt cgaggcttat aggccagagc 1020ctctgggacg tctcgcgctc
gagcaccgga aatttggtgg agtggttcct cctgcggaag 1080gcctacaaga
ggaacgagct cgccccaaac aagcccgacg agagggagct cgcgagacgg
1140cgcgggggct acgctggcgg gtacgttaag gaaccagagc ggggattgtg
ggacaacatt 1200gtgtatctag acttccgctc gtatgcggtt tcaatcatca
taacccacaa cgtctcgccg 1260gataccctca accgcgaggg ctgtaaagag
tacgacgtcg cccctgaggt tggacacaag 1320ttctgcaagg acttccccgg
cttcatacca agcctcctgg gagatttgct cgaggagagg 1380cagaagataa
agcggaagat gaaggcaacg gttgacccgc tggagaagaa actcctcgat
1440tacaggcaga ggctgatcaa aatcctcgcc aacagcttct acggctacta
cggctacgcc 1500aaggcccggt ggtactgcaa ggagtgcgcc gagagcgtta
cggcctgggg aagggagtat 1560atagaaatgg ttatccggga actcgaagaa
aaattcggtt ttaaagttct ctatgccgat 1620acagacggtc tccatgctac
cattcccgga gcagacgctg aaacagtcaa gaaaaaagca 1680aaggagttct
taaaatacat taatccaaaa ctgcccggcc tgctcgaact tgagtacgag
1740ggcttctacg tgaggggctt cttcgtcacg aagaagaagt acgctgtgat
agacgaggag 1800ggcaagataa ccacgagggg tcttgagatt gtgaggcgcg
actggagcga gatagcgaag 1860gagacccagg ccagggtctt agaggcgata
ctcaagcacg gtgacgtcga ggaggccgtt 1920aggatagtca aggaagtgac
ggaaaagctg agcaagtatg aggtcccgcc cgagaagctg 1980gtaatccacg
agcagataac gcgcgatttg agggattaca aagccaccgg cccgcacgtt
2040gccgttgcga agaggctcgc ggcgcgtgga gtgaaaatcc ggcccggcac
ggtgataagc 2100tacatcgtcc taaagggctc tggaaggata ggcgacgcgg
cgattccagc tgatgagttc 2160gacccgacga agcaccgcta cgatgcggaa
tactacatcg agaaccaggt tctcccggcg 2220gtggagagga ttctaaaagc
cttcggctat cggaaggagg atttgcgcta ccagaagacg 2280aagcaggtcg
gcttgggcgc gtggctgaag gtgaagggga agaagtga 23285775PRTArtificial
sequenceMutant polymerase 5Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu
Asn Gly Lys Pro Val Ile 1
5 10 15 Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp
Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp
Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Val Thr Ala Lys Arg His
Gly Thr Val Val Lys 50 55 60 Val Lys Arg Ala Glu Lys Val Gln Lys
Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe
Asn His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Arg Ile Arg
Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro
Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met
Glu Gly Asp Glu Glu Leu Thr Met Leu Ala Phe Ala Ile Ala Thr 130 135
140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160 Ser Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp
Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu
Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val Val Arg Glu Lys
Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp
Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220 Glu Leu Gly Ile Lys
Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln
Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260
265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys
Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser
Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp
Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro
Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp
Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe
Leu Leu Arg Lys Ala Tyr Lys Arg Asn Glu Leu Ala 355 360 365 Pro Asn
Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385
390 395 400 Val Tyr Leu Asp Phe Arg Ser Tyr Ala Val Ser Ile Ile Ile
Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys
Lys Glu Tyr Asp 420 425 430 Val Ala Pro Glu Val Gly His Lys Phe Cys
Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu
Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr
Val Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln
Arg Leu Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 Tyr
Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505
510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu
515 520 525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp
Gly Leu 530 535 540 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val
Lys Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Pro
Lys Leu Pro Gly Leu Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr
Val Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile
Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val
Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg
Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val 625 630
635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val
Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp
Leu Arg Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala
Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr
Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly
Asp Arg Ala Ile Pro Ala Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys
His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu
Pro Ala Val Glu Ala Ile Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp 755
760 765 Leu Lys Val Lys Gly Lys Lys 770 775 62328DNAArtificial
sequenceMutant polymerase 6atgattctcg ataccgacta catcaccgag
aacgggaagc ccgtgataag ggtcttcaag 60aaggagaacg gcgagtttaa aatcgagtac
gacagaacct tcgagcccta cttctacgcc 120cttctgaagg acgattctgc
gatagaggac gtcaagaagg taaccgcaaa gaggcacgga 180acggttgtca
aggtgaagcg cgccgagaag gtgcagaaga agttcctcgg caggccgata
240gaggtctgga agctctactt caaccatcct caggacgtcc cggcgattcg
agacaggata 300cgtgcccacc ccgctgtcgt tgacatctac gagtacgaca
tacccttcgc caagcgctac 360ctcatcgaca agggcctgat tccgatggag
ggcgacgagg agcttacgat gctcgccttc 420gcgatcgcaa ccctctatca
cgagggcgag gagttcggaa ccgggccgat tctcatgata 480agctacgccg
acgggagcga ggcgagggtg ataacctgga agaagattga ccttccgtac
540gttgacgtcg tctcgaccga gaaggagatg attaagcgct tcctccgcgt
cgtcagggag 600aaggaccccg acgtgctcat cacctacaac ggcgacaact
tcgacttcgc ctacctgaag 660aagcgctctg aggaactcgg aataaagttc
acactcggca gggacgggag cgagccgaag 720atacagcgaa tgggcgaccg
ctttgccgtt gaggtgaagg gcaggattca cttcgacctc 780taccccgtca
taaggcgcac gataaacctc ccgacctaca cccttgaggc cgtttacgag
840gccgtctttg gaaagcccaa ggagaaggtt tacgcagagg agatagcgca
ggcctgggag 900agcggggagg gccttgaaag ggttgcaaga tactcgatgg
aggacgctaa ggtgacctac 960gagctgggaa gggagttctt cccgatggag
gcccagcttt cgaggcttat aggccagagc 1020ctctgggacg tctcgcgctc
gagcaccgga aatttggtgg agtggttcct cctgcggaag 1080gcctacaaga
ggaacgagct cgccccaaac aagcccgacg agagggagct cgcgagacgg
1140cgcgggggct acgctggcgg gtacgttaag gaaccagagc ggggattgtg
ggacaacatt 1200gtgtatctag acttccgctc gtatgcggtt tcaatcatca
taacccacaa cgtctcgccg 1260gataccctca accgcgaggg ctgtaaagag
tacgacgtcg cccctgaggt tggacacaag 1320ttctgcaagg acttccccgg
cttcatacca agcctcctgg gagatttgct cgaggagagg 1380cagaagataa
agcggaagat gaaggcaacg gttgacccgc tggagaagaa actcctcgat
1440tacaggcaga ggctgatcaa aatcctcgcc aacagcttct acggctacta
cggctacgcc 1500aaggcccggt ggtactgcaa ggagtgcgcc gagagcgtta
cggcctgggg aagggagtat 1560atagaaatgg ttatccggga actcgaagaa
aaattcggtt ttaaagttct ctatgccgat 1620acagacggtc tccatgctac
cattcccgga gcagacgctg aaacagtcaa gaaaaaagca 1680aaggagttct
taaaatacat taatccaaaa ctgcccggcc tgctcgaact tgagtacgag
1740ggcttctacg tgaggggctt cttcgtcacg aagaagaagt acgctgtgat
agacgaggag 1800ggcaagataa ccacgagggg tcttgagatt gtgaggcgcg
actggagcga gatagcgaag 1860gagacccagg ccagggtctt agaggcgata
ctcaagcacg gtgacgtcga ggaggccgtt 1920aggatagtca aggaagtgac
ggaaaagctg agcaagtatg aggtcccgcc cgagaagctg 1980gtaatccacg
agcagataac gcgcgatttg agggattaca aagccaccgg cccgcacgtt
2040gccgttgcga agaggctcgc ggcgcgtgga gtgaaaatcc ggcccggcac
ggtgataagc 2100tacatcgtcc taaagggctc tggaaggata ggcgacaggg
cgattccagc tgatgagttc 2160gacccgacga agcaccgcta cgatgcggaa
tactacatcg agaaccaggt tctcccggcg 2220gtggaggcga ttctaaaagc
cttcggctat cggaaggagg atttgcgcta ccagaagacg 2280aagcaggtcg
gcttgggcgc gtggctgaag gtgaagggga agaagtga 23287704PRTArtificial
sequenceMutant polymerase 7Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu
Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe Lys Lys Glu Asn Gly
Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe
Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Asp Val Lys
Lys Val Thr Ala Lys Arg His Gly Thr Val Val Lys 50 55 60 Val Lys
Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Asn His Pro Gln Asp Val Pro Ala Ile 85
90 95 Arg Asp Arg Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu
Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly
Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Thr Met Leu Ala
Phe Ala Ile Ala Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly
Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Gly Ser
Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr
Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe
Leu Arg Val Val Arg Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210
215 220 Glu Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro
Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val
Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg
Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu
Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu
Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val
Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu
Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330
335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Lys Arg Asn Glu
Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg
Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg
Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser
Tyr Ala Val Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp
Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Val Ala Pro
Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile
Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455
460 Arg Lys Met Lys Ala Thr Val Asp Pro Leu Glu Lys Lys Leu Leu Asp
465 470 475 480 Tyr Arg Gln Arg Leu Ile Lys Ile Leu Ala Asn Ser Phe
Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys
Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile
Glu Met Val Ile Arg Glu Leu 515 520 525 Glu Glu Lys Phe Gly Phe Lys
Val Leu Tyr Ala Asp Thr Asp Gly Leu 530 535 540 His Ala Thr Ile Pro
Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Lys Glu
Phe Leu Lys Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 580
585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly
Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu
Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp
Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu
Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile
His Glu Gln Ile Thr Arg Asp Leu Arg Asp 660 665 670 Tyr Lys Ala Thr
Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly
Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700
82328DNAArtificial sequenceMutant polymerase 8atgattctcg ataccgacta
catcaccgag aacgggaagc ccgtgataag ggtcttcaag 60aaggagaacg gcgagtttaa
aatcgagtac gacagaacct tcgagcccta cttctacgcc 120cttctgaagg
acgattctgc gatagaggac gtcaagaagg taaccgcaaa gaggcacgga
180acggttgtca aggtgaagcg cgccgagaag gtgcagaaga agttcctcgg
caggccgata 240gaggtctgga agctctactt caaccatcct caggacgtcc
cggcgattcg agacaggata 300cgtgcccacc ccgctgtcgt tgacatctac
gagtacgaca tacccttcgc caagcgctac 360ctcatcgaca agggcctgat
tccgatggag ggcgacgagg agcttacgat gctcgccttc 420gcgatcgcaa
ccctctatca cgagggcgag gagttcggaa ccgggccgat tctcatgata
480agctacgccg acgggagcga ggcgagggtg ataacctgga agaagattga
ccttccgtac 540gttgacgtcg tctcgaccga gaaggagatg attaagcgct
tcctccgcgt cgtcagggag 600aaggaccccg acgtgctcat cacctacaac
ggcgacaact tcgacttcgc ctacctgaag 660aagcgctctg aggaactcgg
aataaagttc acactcggca gggacgggag cgagccgaag 720atacagcgaa
tgggcgaccg ctttgccgtt gaggtgaagg gcaggattca cttcgacctc
780taccccgtca taaggcgcac gataaacctc ccgacctaca cccttgaggc
cgtttacgag 840gccgtctttg gaaagcccaa ggagaaggtt tacgcagagg
agatagcgca ggcctgggag 900agcggggagg gccttgaaag ggttgcaaga
tactcgatgg aggacgctaa ggtgacctac 960gagctgggaa gggagttctt
cccgatggag gcccagcttt cgaggcttat aggccagagc 1020ctctgggacg
tctcgcgctc gagcaccgga aatttggtgg agtggttcct cctgcggaag
1080gcctacaaga ggaacgagct cgccccaaac aagcccgacg agagggagct
cgcgagacgg 1140cgcgggggct acgctggcgg gtacgttaag gaaccagagc
ggggattgtg ggacaacatt 1200gtgtatctag acttccgctc gtatgcggtt
tcaatcatca taacccacaa cgtctcgccg 1260gataccctca accgcgaggg
ctgtaaagag tacgacgtcg cccctgaggt tggacacaag 1320ttctgcaagg
acttccccgg cttcatacca agcctcctgg gagatttgct cgaggagagg
1380cagaagataa agcggaagat gaaggcaacg gttgacccgc tggagaagaa
actcctcgat 1440tacaggcaga ggctgatcaa aatcctcgcc aacagcttct
acggctacta cggctacgcc 1500aaggcccggt ggtactgcaa ggagtgcgcc
gagagcgtta cggcctgggg aagggagtat 1560atagaaatgg ttatccggga
actcgaagaa aaattcggtt ttaaagttct ctatgccgat 1620acagacggtc
tccatgctac cattcccgga gcagacgctg aaacagtcaa gaaaaaagca
1680aaggagttct taaaatacat taatccaaaa ctgcccggcc tgctcgaact
tgagtacgag 1740ggcttctacg tgaggggctt cttcgtcacg aagaagaagt
acgctgtgat agacgaggag 1800ggcaagataa ccacgagggg tcttgagatt
gtgaggcgcg actggagcga gatagcgaag 1860gagacccagg ccagggtctt
agaggcgata ctcaagcacg gtgacgtcga ggaggccgtt 1920aggatagtca
aggaagtgac ggaaaagctg agcaagtatg aggtcccgcc cgagaagctg
1980gtaatccacg agcagataac gcgcgatttg agggattaca aagccaccgg
cccgcacgtt 2040gccgttgcga agaggctcgc ggcgcgtgga gtgaaaatcc
ggcccggcac ggtgataagc 2100tacatcgtcc tgacgggctc tggaaggata
ggcgacaggg cgattccagc tgatgagttc 2160gacccgacga agcaccgcta
cgatgcggaa tactacatcg agaaccaggt tctcccggcg 2220gtggagagga
ttctaaaagc cttcggctat cggaaggagg atttgcgcta ccagaagacg
2280aagcaggtcg gcttgggcgc gtggctgaag gtgaagggga agaagtga
2328928DNAArtificial sequenceFwd primer 9cccggcggtg gaggcgattc
taaaagcc 281028DNAArtificial sequenceRev primer 10gggccgccac
ctccgctaag attttcgg 281130DNAArtificial sequenceFwd primer
11gaaggatagg cgacgcggcg attccagctg 301230DNAArtificial sequenceRev
primer 12cttcctatcc gctgcgccgc taaggtcgac 301329DNAArtificial
sequenceFwd primer 13gctacatcgt cctagcgggc tctggaagg
291429DNAArtificial sequenceRev primer 14cgatgtagca ggatcgcccg
agaccttcc 291529DNAArtificial sequenceFwd primer 15gctacatcgt
cctatgaggc tctggaagg
291629DNAArtificial sequenceRev primer 16cgatgtagca ggatactccg
agaccttcc 291784DNAArtificial sequenceTemplate DNA 17cgatcacgat
cacgatcacg atcacgatca cgatcacgct gatgtgcatg ctgttgtttt 60tttacaacag
catgcacatc agcg 841884DNAArtificial sequenceNH2 coupled template
18cgatcacgat cacgatcacg atcacgatca cgatcacgct gatgtgcatg ctgttgtttt
60tttacaacag catgcacatc agcg 84192328DNAArtificial sequenceCodon
optimised polymerase 19atgatcttag ataccgacta tatcaccgag aacggtaaac
cggtgataag ggtgttcaaa 60aaggaaaatg gcgaattcaa gatcgagtat gatagaacct
tcgaaccgta cttctacgcc 120ttgttgaagg acgatagtgc catcgaagat
gtgaaaaaag ttaccgccaa acgtcacggc 180accgtggtaa aggttaaacg
cgccgaaaag gttcagaaga agttcctagg ccgtccgatc 240gaggtgtgga
aattgtactt taaccatccg caggatgtcc cggcgattag agatcgtatt
300cgtgcccacc cggcggtagt ggatatctat gagtacgata tcccgttcgc
aaaaagatac 360ttgattgata aaggactaat cccgatggaa ggcgatgaag
aattaaccat gttagcgttc 420tccatctcca ccctgtacca cgaaggcgaa
gagttcggca ccggtccgat tctgatgatc 480tcctacgcag acggtagcga
agcacgtgtg ataacctgga agaaaataga cctaccttac 540gtggacgtcg
taagtaccga gaaggagatg atcaaaagat tcctgagggt ggtccgtgag
600aaggatccgg acgtactgat tacctataac ggcgataact tcgacttcgc
ctacttgaaa 660aagagatctg aggaattagg catcaaattc accctgggcc
gtgatggcag tgagccgaaa 720atccaacgta tgggcgaccg cttcgccgtc
gaggtgaaag gccgtataca tttcgacttg 780tatccggtga ttaggcgtac
cattaatttg ccgacctaca ccttggaagc ggtgtacgag 840gcggtcttcg
gcaagccgaa ggaaaaggtg tacgccgaag agatcgcgca ggcgtgggag
900agcggtgagg gtctagaacg tgttgcaaga tatagcatgg aggacgccaa
agttacctac 960gaattgggcc gcgagttttt tccgatggag gcccagttat
ctcgtttaat tggccagtcc 1020ctgtgggatg ttagccgcag ttctactggt
aatttggtag aatggttctt actgcgcaaa 1080gcgtataaac gtaacgagtt
agcgccaaat aagccggacg aacgtgaact ggcccgtcgt 1140cgtggtggct
atgccggcgg ttacgtgaag gaaccggagc gtggcctatg ggataacatt
1200gtgtaccttg actttagaag ctatgcggtt agcatcatca tcacccataa
tgttagtccg 1260gacacattga atcgtgaagg atgcaaagaa tatgacgtcg
ccccagaggt gggccacaaa 1320ttttgtaaag atttcccagg attcatccca
agtttgttgg gtgatctgct ggaagaacgc 1380cagaaaatca aacgtaagat
gaaggcgacc gtcgatccac tggagaaaaa gctattggac 1440taccgtcagc
gcctgatcaa gattttggcg aattctttct atggatacta cggctacgcc
1500aaagcccgtt ggtattgtaa agagtgcgcc gagtctgtca ctgcctgggg
tcgtgaatat 1560atcgaaatgg tgatccgcga gctggaagag aaatttggat
tcaaagtctt gtacgccgat 1620accgatggtc tgcacgcgac cattccgggt
gccgatgccg agaccgtgaa gaaaaaggcg 1680aaagagtttt tgaaatatat
caatccgaag ttgccgggat tattagaatt ggaatacgaa 1740ggtttctatg
ttcgcggctt tttcgtgacc aagaaaaaat acgccgtgat cgacgaggaa
1800ggaaaaatta ccacccgtgg tctagagatt gttcgtcgtg actggtccga
aatcgccaaa 1860gaaacccagg cccgtgtact ggaagcgatt ttgaagcatg
gcgatgtgga ggaggcggtt 1920cgtatcgtca aagaagtgac cgaaaagctg
agcaagtatg aagtgccgcc ggagaaattg 1980gtcatacacg aacaaatcac
acgtgacctg cgcgattata aggcgaccgg tccgcacgtt 2040gccgtggcga
agcgtttggc ggcccgtggt gttaagattc gtccaggaac cgtgattagt
2100tacatagtgt tgaagggcag tggtcgtatt ggtgaccgtg ccatcccggc
ggatgagttt 2160gacccgacca agcatcgtta tgacgccgaa tattatatcg
agaatcaggt gctaccagcg 2220gttgaacgta ttttgaaggc attcggctat
cgtaaagaag acctgcgcta ccagaaaacc 2280aagcaggttg gtctgggtgc
ctggttgaaa gtgaaaggca aaaaataa 2328202328DNAArtificial
sequenceCodon optimised thumb mutant polymerase 20atgatcttag
ataccgacta tatcaccgag aacggtaaac cggtgataag ggtgttcaaa 60aaggaaaatg
gcgaattcaa gatcgagtat gatagaacct tcgaaccgta cttctacgcc
120ttgttgaagg acgatagtgc catcgaagat gtgaaaaaag ttaccgccaa
acgtcacggc 180accgtggtaa aggttaaacg cgccgaaaag gttcagaaga
agttcctagg ccgtccgatc 240gaggtgtgga aattgtactt taaccatccg
caggatgtcc cggcgattag agatcgtatt 300cgtgcccacc cggcggtagt
ggatatctat gagtacgata tcccgttcgc aaaaagatac 360ttgattgata
aaggactaat cccgatggaa ggcgatgaag aattaaccat gttagcgttc
420tccatctcca ccctgtacca cgaaggcgaa gagttcggca ccggtccgat
tctgatgatc 480tcctacgcag acggtagcga agcacgtgtg ataacctgga
agaaaataga cctaccttac 540gtggacgtcg taagtaccga gaaggagatg
atcaaaagat tcctgagggt ggtccgtgag 600aaggatccgg acgtactgat
tacctataac ggcgataact tcgacttcgc ctacttgaaa 660aagagatctg
aggaattagg catcaaattc accctgggcc gtgatggcag tgagccgaaa
720atccaacgta tgggcgaccg cttcgccgtc gaggtgaaag gccgtataca
tttcgacttg 780tatccggtga ttaggcgtac cattaatttg ccgacctaca
ccttggaagc ggtgtacgag 840gcggtcttcg gcaagccgaa ggaaaaggtg
tacgccgaag agatcgcgca ggcgtgggag 900agcggtgagg gtctagaacg
tgttgcaaga tatagcatgg aggacgccaa agttacctac 960gaattgggcc
gcgagttttt tccgatggag gcccagttat ctcgtttaat tggccagtcc
1020ctgtgggatg ttagccgcag ttctactggt aatttggtag aatggttctt
actgcgcaaa 1080gcgtataaac gtaacgagtt agcgccaaat aagccggacg
aacgtgaact ggcccgtcgt 1140cgtggtggct atgccggcgg ttacgtgaag
gaaccggagc gtggcctatg ggataacatt 1200gtgtaccttg actttagaag
ctatgcggtt agcatcatca tcacccataa tgttagtccg 1260gacacattga
atcgtgaagg atgcaaagaa tatgacgtcg ccccagaggt gggccacaaa
1320ttttgtaaag atttcccagg attcatccca agtttgttgg gtgatctgct
ggaagaacgc 1380cagaaaatca aacgtaagat gaaggcgacc gtcgatccac
tggagaaaaa gctattggac 1440taccgtcagc gcctgatcaa gattttggcg
aattctttct atggatacta cggctacgcc 1500aaagcccgtt ggtattgtaa
agagtgcgcc gagtctgtca ctgcctgggg tcgtgaatat 1560atcgaaatgg
tgatccgcga gctggaagag aaatttggat tcaaagtctt gtacgccgat
1620accgatggtc tgcacgcgac cattccgggt gccgatgccg agaccgtgaa
gaaaaaggcg 1680aaagagtttt tgaaatatat caatccgaag ttgccgggat
tattagaatt ggaatacgaa 1740ggtttctatg ttcgcggctt tttcgtgacc
aagaaaaaat acgccgtgat cgacgaggaa 1800ggaaaaatta ccacccgtgg
tctagagatt gttcgtcgtg actggtccga aatcgccaaa 1860gaaacccagg
cccgtgtact ggaagcgatt ttgaagcatg gcgatgtgga ggaggcggtt
1920cgtatcgtca aagaagtgac cgaaaagctg agcaagtatg aagtgccgcc
ggagaaattg 1980gtcatacacg aacaaatcac acgtgacctg cgcgattata
aggcgaccgg tccgcacgtt 2040gccgtggcga agcgtttggc ggcccgtggt
gttaagattc gtccaggaac cgtgattagt 2100tacatagtgt tgaagggcag
tggtcgtatt ggtgaccgtg ccatcccggc ggatgagttt 2160gacccgacca
agcatcgtta tgacgccgaa tattatatcg agaatcaggt gctaccagcg
2220gttgaagcta ttttgaaggc attcggctat cgtaaagaag acctgcgcta
ccagaaaacc 2280aagcaggttg gtctgggtgc ctggttgaaa gtgaaaggca aaaaataa
232821775PRTArtificial sequenceCodon optimised thumb mutant
polymerase 21Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys
Pro Val Ile 1 5 10 15 Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys
Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu
Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Val Thr
Ala Lys Arg His Gly Thr Val Val Lys 50 55 60 Val Lys Arg Ala Glu
Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp
Lys Leu Tyr Phe Asn His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg
Asp Arg Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105
110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125 Met Glu Gly Asp Glu Glu Leu Thr Met Leu Ala Phe Ser Ile
Ser Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro
Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Gly Ser Glu Ala Arg
Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val
Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val
Val Arg Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly
Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220 Glu
Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230
235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg
Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn
Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe
Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Gln
Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr
Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg
Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly
Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Lys Arg Asn Glu Leu Ala 355
360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly
Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp
Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Tyr Ala Val
Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn
Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Val Ala Pro Glu Val Gly
His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu
Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys
Met Lys Ala Thr Val Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475
480 Tyr Arg Gln Arg Leu Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr
485 490 495 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala
Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val
Ile Arg Glu Leu 515 520 525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr
Ala Asp Thr Asp Gly Leu 530 535 540 His Ala Thr Ile Pro Gly Ala Asp
Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys
Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 565 570 575 Leu Glu Tyr
Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys
Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600
605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620 Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu
Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser
Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln
Ile Thr Arg Asp Leu Arg Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His
Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile
Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser
Gly Arg Ile Gly Asp Arg Ala Ile Pro Ala Asp Glu Phe 705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725
730 735 Val Leu Pro Ala Val Glu Ala Ile Leu Lys Ala Phe Gly Tyr Arg
Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu
Gly Ala Trp 755 760 765 Leu Lys Val Lys Gly Lys Lys 770 775
22775PRTArtificial sequenceMutant polymerase 22Met Ile Leu Asp Thr
Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile1 5 10 15 Arg Val Phe
Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr
Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40
45 Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Thr Val Val Lys
50 55 60 Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg
Pro Ile65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Asn His Pro Gln Asp
Val Pro Ala Ile 85 90 95 Arg Asp Arg Ile Arg Ala His Pro Ala Val
Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr
Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu
Leu Thr Met Leu Ala Phe Ser Ile Ser Thr 130 135 140 Leu Tyr His Glu
Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile145 150 155 160 Ser
Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170
175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190 Arg Phe Leu Arg Val Val Arg Glu Lys Asp Pro Asp Val Leu
Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys
Lys Arg Ser Glu 210 215 220 Glu Leu Gly Ile Lys Phe Thr Leu Gly Arg
Asp Gly Ser Glu Pro Lys225 230 235 240 Ile Gln Arg Met Gly Asp Arg
Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr
Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu
Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys
Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly 290 295
300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr
Tyr305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln
Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg
Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys
Ala Tyr Lys Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu
Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr
Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile385 390 395 400 Val
Tyr Leu Asp Phe Arg Ser Tyr Ala Val Ser Ile Ile Ile Thr His 405 410
415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430 Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro
Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg
Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Val Asp Pro Leu
Glu Lys Lys Leu Leu Asp465 470 475 480 Tyr Arg Gln Arg Leu Ile Lys
Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Lys
Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala
Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 515 520 525 Glu
Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 530 535
540 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys
Ala545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Pro Lys Leu Pro
Gly Leu Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly
Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu
Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp
Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu
Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val625 630 635 640 Arg
Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650
655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp
660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu
Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser
Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala
Ile Pro Ala Asp Glu Phe705 710 715
720 Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr
Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly
Leu Gly Ala Trp 755 760 765 Leu Lys Val Lys Gly Lys Lys 770
775232374DNAArtificial SequenceMutant polymerase 23gggcgaattg
ggtacccata tgatcttaga taccgactat atcaccgaga acggtaaacc 60ggtgataagg
gtgttcaaaa aggaaaatgg cgaattcaag atcgagtatg atagaacctt
120cgaaccgtac ttctacgcct tgttgaagga cgatagtgcc atcgaagatg
tgaaaaaagt 180taccgccaaa cgtcacggca ccgtggtaaa ggttaaacgc
gccgaaaagg ttcagaagaa 240gttcctaggc cgtccgatcg aggtgtggaa
attgtacttt aaccatccgc aggatgtccc 300ggcgattaga gatcgtattc
gtgcccaccc ggcggtagtg gatatctatg agtacgatat 360cccgttcgca
aaaagatact tgattgataa aggactaatc ccgatggaag gcgatgaaga
420attaaccatg ttagcgttct ccatctccac cctgtaccac gaaggcgaag
agttcggcac 480cggtccgatt ctgatgatct cctacgcaga cggtagcgaa
gcacgtgtga taacctggaa 540gaaaatagac ctaccttacg tggacgtcgt
aagtaccgag aaggagatga tcaaaagatt 600cctgagggtg gtccgtgaga
aggatccgga cgtactgatt acctataacg gcgataactt 660cgacttcgcc
tacttgaaaa agagatctga ggaattaggc atcaaattca ccctgggccg
720tgatggcagt gagccgaaaa tccaacgtat gggcgaccgc ttcgccgtcg
aggtgaaagg 780ccgtatacat ttcgacttgt atccggtgat taggcgtacc
attaatttgc cgacctacac 840cttggaagcg gtgtacgagg cggtcttcgg
caagccgaag gaaaaggtgt acgccgaaga 900gatcgcgcag gcgtgggaga
gcggtgaggg tctagaacgt gttgcaagat atagcatgga 960ggacgccaaa
gttacctacg aattgggccg cgagtttttt ccgatggagg cccagttatc
1020tcgtttaatt ggccagtccc tgtgggatgt tagccgcagt tctactggta
atttggtaga 1080atggttctta ctgcgcaaag cgtataaacg taacgagtta
gcgccaaata agccggacga 1140acgtgaactg gcccgtcgtc gtggtggcta
tgccggcggt tacgtgaagg aaccggagcg 1200tggcctatgg gataacattg
tgtaccttga ctttagaagc tatgcggtta gcatcatcat 1260cacccataat
gttagtccgg acacattgaa tcgtgaagga tgcaaagaat atgacgtcgc
1320cccagaggtg ggccacaaat tttgtaaaga tttcccagga ttcatcccaa
gtttgttggg 1380tgatctgctg gaagaacgcc agaaaatcaa acgtaagatg
aaggcgaccg tcgatccact 1440ggagaaaaag ctattggact accgtcagcg
cctgatcaag attttggcga attctttcta 1500tggatactac ggctacgcca
aagcccgttg gtattgtaaa gagtgcgccg agtctgtcac 1560tgcctggggt
cgtgaatata tcgaaatggt gatccgcgag ctggaagaga aatttggatt
1620caaagtcttg tacgccgata ccgatggtct gcacgcgacc attccgggtg
ccgatgccga 1680gaccgtgaag aaaaaggcga aagagttttt gaaatatatc
aatccgaagt tgccgggatt 1740attagaattg gaatacgaag gtttctatgt
tcgcggcttt ttcgtgacca agaaaaaata 1800cgccgtgatc gacgaggaag
gaaaaattac cacccgtggt ctagagattg ttcgtcgtga 1860ctggtccgaa
atcgccaaag aaacccaggc ccgtgtactg gaagcgattt tgaagcatgg
1920cgatgtggag gaggcggttc gtatcgtcaa agaagtgacc gaaaagctga
gcaagtatga 1980agtgccgccg gagaaattgg tcatacacga acaaatcaca
cgtgacctgc gcgattataa 2040ggcgaccggt ccgcacgttg ccgtggcgaa
gcgtttggcg gcccgtggtg ttaagattcg 2100tccaggaacc gtgattagtt
acatagtgtt gaagggcagt ggtcgtattg gtgaccgtgc 2160catcccggcg
gatgagtttg acccgaccaa gcatcgttat gacgccgaat attatatcga
2220gaatcaggtg ctaccagcgg ttgaacgtat tttgaaggca ttcggctatc
gtaaagaaga 2280cctgcgctac cagaaaacca agcaggttgg tctgggtgcc
tggttgaaag tgaaaggcaa 2340aaaataagct agcggagctc cagcttttgt tccc
2374242374DNAArtificial SequenceMutant polymerase 24gggaacaaaa
gctggagctc cgctagctta ttttttgcct ttcactttca accaggcacc 60cagaccaacc
tgcttggttt tctggtagcg caggtcttct ttacgatagc cgaatgcctt
120caaaatacgt tcaaccgctg gtagcacctg attctcgata taatattcgg
cgtcataacg 180atgcttggtc gggtcaaact catccgccgg gatggcacgg
tcaccaatac gaccactgcc 240cttcaacact atgtaactaa tcacggttcc
tggacgaatc ttaacaccac gggccgccaa 300acgcttcgcc acggcaacgt
gcggaccggt cgccttataa tcgcgcaggt cacgtgtgat 360ttgttcgtgt
atgaccaatt tctccggcgg cacttcatac ttgctcagct tttcggtcac
420ttctttgacg atacgaaccg cctcctccac atcgccatgc ttcaaaatcg
cttccagtac 480acgggcctgg gtttctttgg cgatttcgga ccagtcacga
cgaacaatct ctagaccacg 540ggtggtaatt tttccttcct cgtcgatcac
ggcgtatttt ttcttggtca cgaaaaagcc 600gcgaacatag aaaccttcgt
attccaattc taataatccc ggcaacttcg gattgatata 660tttcaaaaac
tctttcgcct ttttcttcac ggtctcggca tcggcacccg gaatggtcgc
720gtgcagacca tcggtatcgg cgtacaagac tttgaatcca aatttctctt
ccagctcgcg 780gatcaccatt tcgatatatt cacgacccca ggcagtgaca
gactcggcgc actctttaca 840ataccaacgg gctttggcgt agccgtagta
tccatagaaa gaattcgcca aaatcttgat 900caggcgctga cggtagtcca
atagcttttt ctccagtgga tcgacggtcg ccttcatctt 960acgtttgatt
ttctggcgtt cttccagcag atcacccaac aaacttggga tgaatcctgg
1020gaaatcttta caaaatttgt ggcccacctc tggggcgacg tcatattctt
tgcatccttc 1080acgattcaat gtgtccggac taacattatg ggtgatgatg
atgctaaccg catagcttct 1140aaagtcaagg tacacaatgt tatcccatag
gccacgctcc ggttccttca cgtaaccgcc 1200ggcatagcca ccacgacgac
gggccagttc acgttcgtcc ggcttatttg gcgctaactc 1260gttacgttta
tacgctttgc gcagtaagaa ccattctacc aaattaccag tagaactgcg
1320gctaacatcc cacagggact ggccaattaa acgagataac tgggcctcca
tcggaaaaaa 1380ctcgcggccc aattcgtagg taactttggc gtcctccatg
ctatatcttg caacacgttc 1440tagaccctca ccgctctccc acgcctgcgc
gatctcttcg gcgtacacct tttccttcgg 1500cttgccgaag accgcctcgt
acaccgcttc caaggtgtag gtcggcaaat taatggtacg 1560cctaatcacc
ggatacaagt cgaaatgtat acggcctttc acctcgacgg cgaagcggtc
1620gcccatacgt tggattttcg gctcactgcc atcacggccc agggtgaatt
tgatgcctaa 1680ttcctcagat ctctttttca agtaggcgaa gtcgaagtta
tcgccgttat aggtaatcag 1740tacgtccgga tccttctcac ggaccaccct
caggaatctt ttgatcatct ccttctcggt 1800acttacgacg tccacgtaag
gtaggtctat tttcttccag gttatcacac gtgcttcgct 1860accgtctgcg
taggagatca tcagaatcgg accggtgccg aactcttcgc cttcgtggta
1920cagggtggag atggagaacg ctaacatggt taattcttca tcgccttcca
tcgggattag 1980tcctttatca atcaagtatc tttttgcgaa cgggatatcg
tactcataga tatccactac 2040cgccgggtgg gcacgaatac gatctctaat
cgccgggaca tcctgcggat ggttaaagta 2100caatttccac acctcgatcg
gacggcctag gaacttcttc tgaacctttt cggcgcgttt 2160aacctttacc
acggtgccgt gacgtttggc ggtaactttt ttcacatctt cgatggcact
2220atcgtccttc aacaaggcgt agaagtacgg ttcgaaggtt ctatcatact
cgatcttgaa 2280ttcgccattt tcctttttga acacccttat caccggttta
ccgttctcgg tgatatagtc 2340ggtatctaag atcatatggg tacccaattc gccc
2374
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