U.S. patent application number 12/446746 was filed with the patent office on 2009-12-10 for polymerase.
This patent application is currently assigned to Medical Research Council. Invention is credited to Claudia Baar, Marc D'Abbadie, Philipp Holliger.
Application Number | 20090305345 12/446746 |
Document ID | / |
Family ID | 38925721 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090305345 |
Kind Code |
A1 |
Holliger; Philipp ; et
al. |
December 10, 2009 |
POLYMERASE
Abstract
The present invention relates to an engineered polymerase
characterized in that the polymerase exhibits an enhanced ability
to process nucleic acid in the presence of environmental and
biological inhibitors compared to wild type DNA polymerase.
Inventors: |
Holliger; Philipp;
(Cambridge, GB) ; D'Abbadie; Marc; (Cambridge,
GB) ; Baar; Claudia; (Cambridge, GB) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 SOUTH WACKER DRIVE, 6300 SEARS TOWER
CHICAGO
IL
60606-6357
US
|
Assignee: |
Medical Research Council
London
GB
|
Family ID: |
38925721 |
Appl. No.: |
12/446746 |
Filed: |
October 23, 2007 |
PCT Filed: |
October 23, 2007 |
PCT NO: |
PCT/GB07/04031 |
371 Date: |
August 10, 2009 |
Current U.S.
Class: |
435/69.1 ;
435/193; 536/23.2 |
Current CPC
Class: |
C12N 9/1252 20130101;
C12N 9/1241 20130101 |
Class at
Publication: |
435/69.1 ;
435/193; 536/23.2 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 9/10 20060101 C12N009/10; C07H 21/00 20060101
C07H021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 2006 |
GB |
0621094.2 |
Nov 30, 2006 |
GB |
0623977.6 |
Claims
1. An engineered polymerase characterised in that it exhibits an
enhanced ability to process nucleic acid (i) in the presence of
humic acid; (ii) in the presence of one or more phenolic compounds
or derivatives thereof; or (iii) in the presence of soil, compared
to wild type polymerase.
2-5. (canceled)
6. The engineered polymerase according to claim 1 which exhibits an
enhanced ability to process nucleic acid in the presence of (i)
humic acid; phenolic acid or derivatives thereof; or phytophenolic
acid or derivatives thereof, at a concentration of between 5 and
20%.
7-8. (canceled)
9. The engineered polymerase according to claim 1 wherein the
engineered polymerase is derived from wild type polymerase by
substitution, deletion or insertion of one or more amino acids.
10. The engineered polymerase according to claim 1 wherein said
ability to process nucleic acid is enhanced at least four fold,
eight fold, or sixteen fold, when compared to the wild type
polymerase.
11-12. (canceled)
13. The engineered polymerase according to claim 1 wherein the
ability to process nucleic acid within a polymerase chain reaction
is enhanced.
14. The engineered polymerase according to claim 1 comprising an
engineered polymerase that is generated from a library derived by
recombining related wild type polymerase genes.
15. The engineered polymerase according to claim 1 wherein said
wild type polymerase is selected from a group consisting of Taq,
T8, TTh and Ttl.
16. The engineered polymerase according to claim 1 wherein said
polymerase is generated from a library of nucleic acids derived by
error prone polymerase chain reaction mutagenesis and/or
recombination of related wild type polymerase genes.
17. The engineered polymerase according to claim 1 wherein the
polymerase is a DNA polymerase.
18. A method for producing the engineered polymerase of claim 1,
which comprises: (a) preparing a nucleic acid molecule encoding a
polymerase; (b) introducing a mutation into the nucleic acid
molecule encoding that polymerase according to step (a) so that one
or more nucleotides in one or more regions are not identical to the
polymerase from which it is derived; (c) selecting a modified
polymerase expressed by the mutated nucleic acid molecule by the
ability of said modified polymerase to process nucleic acid in the
presence of (i) humic acid; (ii) one or more phenolic compounds or
derivatives thereof; or (iii) soil; and (d) isolating and purifying
that polymerase.
19-21. (canceled)
22. A method for the generation of an engineered polymerase
according to claim 1 which comprises the steps of: (a) providing a
pool of nucleic acids comprising members each encoding an
engineered polymerase; (b) providing (i) humic acid; (ii) one or
more phenolic compounds or derivatives thereof; or (iii) soil; (c)
subdividing the pool of nucleic acids into compartments, such that
each compartment comprises substantially a nucleic acid member of
the pool together with the engineered polymerase encoded by the
nucleic acid member, and (i) humic acid; (ii) one or more phenolic
compounds or derivatives thereof; or (iii) soil; (d) allowing
processing of the nucleic acid member to occur; and (e) detecting
processing of the nucleic acid member by that engineered
polymerase; (f) optionally repeating the series of steps (a) to (f)
one or more times; and (g) isolating and purifying that engineered
polymerase.
23-25. (canceled)
26. The method according to claim 22 wherein humic acid is provided
at a concentration that inhibits wild type polymerase activity.
27. The method according to claim 22 wherein one or more phenolic
compounds are provided at a concentration that inhibits wild type
polymerase activity.
28-32. (canceled)
33. An isolated nucleic acid molecule which encodes an engineered
DNA polymerase polypeptide comprising an amino acid sequence having
at least 80%, 90%, 95%, or 99% identity to any of SEQ ID NOs 2, 4
or 6 and wherein said polypeptide has DNA polymerase activity in
the presence of 5 to 20% humic acid.
34-36. (canceled)
37. An isolated nucleic acid molecule encoding an engineered DNA
polymerase comprising a nucleotide sequence as set forth in any of
SEQ ID NOs 1, 3, 5, 7 or 9, or a nucleotide sequence having at
least 80%, 90%, or 95% sequence identity with any of SEQ ID NOs 1,
3, 5, 7, or 9, wherein said polymerase exhibits an enhanced ability
to process nucleic acid in the presence of humic acid compared to
wild type polymerase.
38-40. (canceled)
41. The engineered polymerase according to claim 1 wherein said
engineered polymerase comprises an amino acid sequence that has at
least 80%, 90%, or 95% identity to amino residues of the wild type
polymerase.
42-43. (canceled)
44. A polypeptide with DNA polymerase activity, characterized in
that the amino acid sequence of that polypeptide comprises the
amino acid sequence of any of SEQ ID NOs 2, 4, 6, 8 or 10.
45-46. (canceled)
47. A recombinant nucleic acid molecule comprising a promoter
sequence operably linked to the nucleic acid molecule according to
claim 33.
48. A cell transformed with the recombinant nucleic acid molecule
according to claim 47.
49-50. (canceled)
51. A kit for amplifying nucleic acid comprising an isolated,
engineered polymerase according to claim 1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to engineered polymerases. In
particular, the invention relates to engineered polymerases that
are resistant to certain environmental and biological inhibitors.
Uses for said engineered polymerases and methods of generating said
engineered polymerases are also described.
BACKGROUND TO THE INVENTION
[0002] Polymerase enzymes, such as DNA polymerase, RNA polymerase,
or reverse transcriptase, can catalyse the formation of
polynucleotides of DNA or RNA using an existing strand of DNA or
RNA as a template. RNA polymerases and DNA polymerases can catalyse
the polymerisation of RNA and DNA respectively using a DNA
template, whereas reverse transcriptase can catalyse the formation
of DNA using an RNA template.
[0003] DNA polymerases, for example are naturally occurring
intracellular enzymes, and are used by a cell to replicate a
nucleic acid strand using a template molecule to manufacture a
complementary nucleic acid strand. DNA polymerases are also widely
used in vitro for various biochemical applications including cDNA
synthesis and DNA sequencing reactions, amplification of nucleic
acids by methods such as the polymerase chain reaction (PCR) and
for RNA transcription-medicated amplification methods.
[0004] The polymerase chain reaction (PCR) is a widely used
technique that allows a specific region of DNA to be amplified
exponentially, provided that at least part of its nucleotide
sequence is already known. This known region of sequence is used to
design synthetic DNA oligonucleotides complementary to each strand
of the DNA double helix. These oligonucleotides serve as primers
for in vitro DNA synthesis, which is catalyzed by DNA
polymerase.
[0005] Unfortunately, the effectiveness of this technique in basic
research or in forensic or clinical applications is limited by some
technical problems. A number of substances are known that are
potent inhibitors of polymerase activity and limit the use of
polymerase chain reaction (PCR) in biological samples where they
are present. Examples include heme (and its degradation products
such as bilirubin) present in blood and faeces. Another potent
inhibitor present in the environment is humic acid.
[0006] Humic acids are a complex mixture of polyphenolic acids
produced by the decomposition of organic matter (e.g. decomposing
terrestrial vegetation). Humic acids are ubiquitous in soil and
water and thus are present in any sample exposed to the
environment. Inhibition of PCR by humic acids is thus especially
relevant for samples of paelontological, archaeological or forensic
interest, which are exposed to soil for extended periods of
time.
[0007] Some attempts have been made to circumvent the problem of
humic acid contamination in PCR samples. One approach to the
problem has been purification or extraction of DNA from samples in
advance of PCR (LaMontagne et al (2002) J Microbiol Methods
49:255-64; Howeler et al J Microbiol Methods 2003 54:37-45).
Unfortunately, humic acid contamination may still be a problem
depending on the extraction method used (LaMontagne et al 2002).
Furthermore, not all contaminants are completely removed during
classical extraction protocols (such as detergent, protease and
phenol-chloroform treatments), and loss of the original sample may
occur. Another problem with extraction procedures includes the use
of expensive materials such as ion-exchange columns, glass bead
extraction, immunomagnetic separation, size-exclusion
chromatography, anion-binding resins or spin columns (Wilson, I G
(1997) Appl. Environ. Microbiol. 63:3741-3751). Moreover, the extra
steps required in each PCR protocol may increase
cross-contamination risks and subsequent false-positive
results.
[0008] Another approach to tackle the inhibitory effect of humic
acid on polymerase activity is to increase the concentration of
polymerase in each reaction mixture (Sutlovic et al Croat Med J
2005 46:556-62). Various additives such as BSA, T4 gp32 or salmon
sperm DNA are also reported to relieve inhibition of polymerase
activity, but these need to be added at substantial concentrations
(typically greater than 0.2 mg/ml) (Tebbe et al Appl Environ
Microbiol. (1993) 59:2657-65).
[0009] With all previous attempts to avoid inhibition of PCR by
humic acid--such as extraction techniques or the addition of
supplements--extra time and expense is associated with the use of
additional reagents or protocols.
[0010] There remains a need in the art for a simple and more
effective way of dealing with the inhibitory effect of humic acid
on DNA polymerases, particularly in PCR.
SUMMARY OF THE INVENTION
[0011] The present invention addresses the problem of inhibition of
DNA polymerase activity by humic acid. Specifically, the present
invention provides a DNA polymerase that is resistant to the
inhibitory effects of humic acid. Importantly, the problem of humic
acid intolerance encountered by DNA polymerase in PCR reactions is
solved not by altering-the-amount or potency of humic acid present
in a sample, as in the prior art, but via changes in the property
of the polymerase itself.
[0012] Thus in a first aspect, the invention provides an engineered
polymerase wherein that polymerase exhibits an enhanced ability to
process nucleic acid in the presence of humic acid compared to wild
type polymerase.
[0013] According to the above aspect of the invention, the term
`engineered polymerase` refers to a polymerase which has a nucleic
acid sequence which is not 100% identical at the nucleic acid level
to the one or more polymerase/s or fragments thereof, from which it
is derived, and which is synthetic. According to the invention, the
engineered polymerase may be derived from wild type DNA polymerase
by the substitution, deletion or insertion of one or more amino
acids. The term `engineered polymerase` also includes within its
scope fragments, derivatives and homologues of an `engineered
polymerase` as herein defined so long as it exhibits the requisite
property of possessing an enhanced ability to process nucleic acid
in the presence of humic acid compared to that of wild type
polymerase.
[0014] A "wild-type" polymerase is a polymerase which has not been
engineered in accordance with the present invention. Preferably, a
wild-type polymerase is the polymerase which is subjected to the
claimed engineering procedure; thus, the wild-type polymerase is
unmodified form of the engineered polymerase.
[0015] "Enhanced ability" is taken to mean an increase in any
function of engineered polymerase that enables it to process
nucleic acid, as compared to that of wild type polymerase. This
includes an increase in the ability of polymerase to catalyze
formation of a bond between the 3' hydroxyl group at the growing
end of a nucleic acid primer and the 5' phosphate group of a
nucleotide triphosphate. Functions of DNA polymerases also include
but are not limited to, incorporation of deoxyribonucleotide
subunits or derivatives thereof, phosphoryl transfer, translocation
along a DNA template, extension of primer substrates, template
recognition and replication or amplification of template DNA.
[0016] An engineered polymerase according to the invention may be a
DNA polymerase. A DNA polymerase will be known to those in the art
and the function and properties of which will be well known. An
engineered DNA polymerase will have similar properties and
characteristics to the engineered polymerase of the invention in
that they will have an enhanced ability to process nucleic acid
compared to the wild type DNA polymerases from which they may have
been derived.
[0017] The engineered DNA polymerase isolated by the present
inventors has an enhanced ability to process nucleic acid at
concentrations of between 0.1% and 50% humic acid. Preferably, the
engineered DNA polymerase has an enhanced ability to process
nucleic acid at concentrations of between 1 and 30% humic acid.
Most preferably, the engineered DNA polymerase has an enhanced
ability to process nucleic acid at concentrations of between 5 and
20% humic acid. Humic acid may be derived from decomposed organic
material such as peat soil. Methods for the derivation of a
solution of humic acid at concentrations of between 5 and 20% are
enclosed herein. Using these methods those skilled in the art would
be able to determine other polymerases that can process nucleic
acids at different concentrations of humic acid that are within the
scope of this invention. The present inventors measure the ability
of engineered DNA polymerase to process nucleic acid by comparing
the activity of engineered DNA polymerase to wild type DNA
polymerase at various concentrations of humic acid. Engineered DNA
polymerases can then be identified that are active under humic acid
concentrations where wild type DNA polymerases are not.
[0018] Engineered DNA polymerases of the present invention are
found to be active at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19 or 20 fold greater concentrations of humic
acid than concentrations under which wild type DNA polymerase is
still active. Accordingly there is provided an engineered DNA
polymerase with an enhanced ability to process nucleic acid in the
presence of humic acid wherein said ability is enhanced 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fold when
compared to wild type DNA polymerase.
[0019] Engineered DNA polymerases of the present invention may be
used in any in vitro reaction wherein the property of humic acid
resistance is regarded as beneficial. DNA polymerases are used in
many in vitro molecular biology applications including mutagenesis,
cDNA libraries, sequencing and polymerase chain reaction (PCR). It
is known that inhibition of wild type DNA polymerase activity by
humic acid can inhibit or impair a polymerase chain reaction (PCR).
Central to this technique is the activity of DNA polymerase, which
is involved in replicating template-DNA at sites marked by primers,
by incorporating deoxyribonucleotide subunits to synthesise a new
DNA strand. Biological samples of paelontological, archaeological
or forensic interest containing template DNA, may be exposed to
soil for extended periods of time and may contain humic acid. The
engineered DNA polymerases of the present invention are
particularly suitable for use in PCR reactions performed on such
samples. Therefore, preferably, the engineered DNA polymerase of
the present invention has an enhanced ability to process nucleic
acid within a polymerase chain reaction.
[0020] Nucleic acid molecules encoding engineered DNA polymerases
of the present invention can readily be obtained in a variety of
ways including, without limitation, chemical synthesis, cDNA or
genomic library screening, expression library screening, and/or PCR
amplification of cDNA.
[0021] The present inventors also provide methods for the
introduction of mutations into nucleic acid and for generation of
libraries thereof. Those skilled in the art will be aware of
several techniques to generate diversity within a gene or within
nucleic acid. Nucleic acid molecules encoding variants may be
produced using site directed mutagenesis, error prone PCR
amplification, or other appropriate methods, where the primer(s)
have the desired point mutations (see Sambrook et al., supra, and
Ausubel et al., supra, for descriptions of mutagenesis techniques).
Chemical synthesis using methods described by Engels et al., supra,
may also be used to prepare such variants. Other methods known to
the skilled artisan may be used as well.
[0022] The present inventors describe generation of a library of
chimeric polymerase gene variants that can be derived by a gene
shuffling technique such as "staggered extension process" (StEP)
(Zhao et al Biotechnol (1998) 16:258-261). This technique allows
two or more genes of interest from different species to be randomly
recombined to produce chimeras, the sequence of which contains
parts of the original input parent genes.
[0023] Accordingly, there is provided in the present invention, an
engineered DNA polymerase comprising a DNA polymerase that is
generated from a library derived by recombining related wild type
DNA polymerase genes. Advantageously, an engineered DNA polymerase
with humic acid resistance according to the invention is derived
from a pol A-family DNA polymerase. Preferably, the wild type DNA
polymerase is selected from a group consisting of Taq, T8 (a
previously selected 11 fold more thermostable Taq variant;
Ghadessey et al. 2001), TTh (Thermus thermophilus) and Ttl (Thermus
flavus).
[0024] There is also provided in the present invention, an
engineered DNA polymerase generated from a library of nucleic acids
derived by error prone polymerase chain reaction mutagenesis and/or
recombination of related wild type DNA polymerase genes.
[0025] In a second aspect of the invention, there are provided
methods for the generation of engineered DNA polymerases that are
humic acid resistant. Accordingly, there is provided a method for
producing a DNA polymerase of the present invention which
comprises:
(a) preparing a nucleic acid molecule encoding a DNA polymerase;
(b) introducing a mutation into the nucleic acid molecule encoding
that polymerase according to step (a) so that one or more
nucleotides in one or more regions are not identical to the DNA
polymerase from which it is derived; (c) selecting a modified DNA
polymerase expressed by the mutated nucleic acid molecule; and (d)
isolating and purifying that DNA polymerase.
[0026] A highly preferred method of generating engineered DNA
polymerases of the present invention is by directed evolution. The
techniques of directed evolution and compartmentalised self
replication are detailed in GB 97143002 and GB 98063936 and GB
01275643, in the name of the present inventors. These documents are
herein incorporated by reference.
[0027] The inventors modified the methods of compartmentalised self
replication and surprisingly generated DNA polymerases which
exhibited humic acid resistance. Accordingly, in a further aspect
of the invention, there is provided a method for the generation of
an engineered DNA polymerase which comprises the steps of: [0028]
a) providing a pool of nucleic acids comprising members each
encoding an engineered DNA polymerase; [0029] b) providing humic
acid; [0030] c) subdividing the pool of nucleic acids into
compartments, such that each compartment comprises substantially a
nucleic acid member of the pool together with the engineered DNA
polymerase or variant encoded by the nucleic acid member, and humic
acid; [0031] d) allowing processing of the nucleic acid member to
occur; and [0032] e) detecting processing of the nucleic acid
member by that engineered DNA polymerase; and [0033] f) optionally
repeating the series of steps (a) to (f) one or more times.
[0034] Preferably, the processing of said nucleic acid member is
part of a polymerase chain reaction.
[0035] Preferably, humic acid is provided at a concentration that
inhibits wild type DNA polymerase activity. Advantageously, humic
acid is added at a concentration sufficient to provide a selection
pressure, but not so great that all polymerase activity is
inhibited. Using the above method of generating an engineered DNA
polymerase, only those DNA polymerases that are resistant to a
given amount of humic acid will be able to process nucleic acid and
subsequently be detected.
[0036] In another aspect of the above method, the member comprises
a bacterial cell expressing an engineered DNA polymerase according
to the present invention. Preferably the bacterial cell is E.
Coli.
[0037] In the above method of generating an engineered DNA
polymerase, only those DNA polymerases which exhibit at least some
resistance to humic acid will be able to process nucleic acid and
subsequently be detected. Accordingly, the post-amplification copy
number of the nucleic acid member which encodes engineered DNA
polymerase according to the invention, is substantially
proportional to the activity of the DNA polymerase. Preferably,
nucleic acid processing is detected by assaying the copy number of
the nucleic acid member.
[0038] In a preferred embodiment, the compartments consist of the
encapsulated aqueous component of a water-in-oil emulsion. The
water-in-oil emulsion is preferably produced by emulsifying an
aqueous phase with an oil phase in the presence of a surfactant
comprising 4.5% v/v Span 80, 0.4% v/v Tween 80 and 0.1% v/v Triton
X100, or a surfactant comprising Span 80, Tween 80 and Triton X100
in substantially the same proportions. Preferably, the water:oil
phase ratio is 1:2, which leads to adequate droplet size. Such
emulsions have a higher thermal stability than more oil-rich
emulsions.
[0039] In a further aspect of the invention, there is provided an
engineered DNA polymerase characterized in that the amino acid
sequence of that polymerase comprises, preferably consists of, the
amino acid sequence designated herein as SEQ ID NO: 2. There is
also provided an isolated nucleic acid molecule which encodes an
engineered DNA polymerase polypeptide comprising an amino acid
sequence having at least 80% identity to any of SEQ ID NOs 2, 4 or
6 and wherein said polypeptide has DNA polymerase activity in the
presence of 5 to 20% humic acid. Preferably, said polypeptide has
at least 90% identity to amino residues of SEQ ID NO: 2, 4 or 6.
More preferably, said polypeptide has at least 95% identity to
amino residues of SEQ ID NO: 2, 4 or 6. Most preferably, said
polypeptide has at least 99% identity to residues of SEQ ID NO: 2,
4 or 6.
[0040] In a further embodiment of the invention, there is provided
an isolated nucleic acid molecule encoding an engineered DNA
polymerase according to the present invention comprising a
nucleotide sequence as set forth in SEQ ID NO. 1, 3, or 5.
Preferably, the isolated nucleic acid molecule comprises a
nucleotide sequence having at least 90% sequence identity with SEQ
ID NO. 1, 3 or 5
[0041] There is also provided in the present invention, an
engineered DNA polymerase wherein said engineered DNA polymerase
has at least 80% identity to amino residues of the wild type
polymerase. Preferably, said engineered DNA polymerase has at least
90% identity to amino residues of the wild type polymerase. Most
preferably, said engineered DNA polymerase has at least 95%
identity to amino residues of the wild type polymerase. Preferably,
said wild type DNA polymerase is a Pol A family DNA polymerase.
Advantageously said wild type DNA polymerase is selected from the
group comprising Taq, T8, TTh and Ttl DNA polymerases.
[0042] Preferably an engineered DNA polymerase of the present
invention has at least 95% amino acid sequence homology and at
least 95% of the proof-reading capability and thermostability of
wild type DNA polymerase isolated from Thermus aquaticus, Thermus
thermophilus, or Thermus flavus.
[0043] In a further aspect of the invention, there is provided, a
nucleotide sequence encoding the polypeptides described above.
There is also provided, a recombinant nucleic acid molecule
comprising a promoter sequence operably linked to nucleic acid
molecule in which said promoter sequence can be constitutive,
inducible, or tissue-specific in function. There is furthermore
provided a cell transformed with said recombinant nucleic acid
molecule. Host cells may be prokaryotic host cells (such as E.
coli) or eukaryotic host cells (such as yeast, insect, or
vertebrate cells). Preferably, the host cell is a bacterial host
cell. Most preferably the host cell is E. coli.
[0044] Advantageously, the polypeptide described above is used for
producing primer extension products. Preferably, the engineered DNA
polymerase of the present invention is used in a polymerase chain
reaction.
[0045] In a further aspect still, there is provided a kit for
amplifying DNA comprising an isolated, engineered DNA polymerase of
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA and immunology,
which are within the capabilities of a person of ordinary skill in
the art. Such techniques are explained in the literature. See,
e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold
Spring Harbor Laboratory Press; B. Roe, J. Crabtree, and A. Kahn,
1996, DNA Isolation and Sequencing: Essential Techniques, John
Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ
Hybridization: Principles and Practice; Oxford University Press; M.
J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical
Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992,
Methods of Enzymology: DNA Structure Part A: Synthesis and Physical
Analysis of DNA Methods in Enzymology, Academic Press. Each of
these general texts are herein incorporated by reference.
Humic Acid
[0047] Humic acid is a complex substance found in certain deposits
of partially decomposed organic matter, particularly dead plants
(Hertkorn N. et al. 2002). These deposits exist especially in
previously heavily forested areas with moist, swamp-like
conditions. The deposits represent a stage between decaying
vegetation (humus/humates/peat) and eventual potential formation of
coal and oil.
[0048] The term "humic acid" refers to any of various organic acids
obtained from humus wherein humus is partially decomposed organic
matter. Other terms that are used for humic acid include but are
not limited to humin, humic substance, natural organic matter,
fulvic acid, moor, ulmin, gein, ulmic or geic acid. Humic
substances are endowed with acidic functional groups mainly
carboxylic acid, which confer on these molecules the ability to
chelate multivalent cations such as Mg.sup.2+, Ca.sup.2+, and
Fe.sup.2+. Humic acid contains a diverse array of relatively low
molecular weight entities including metals, aliphatic acids,
ethers, esters, alcohols, phenols (carbolic acids), phenolic
compounds, aromatic lignin derived fragments, polysaccharides and
polypeptides (Simpson A J et al. 2002). Additional consulting
references include Flaig, Soil Components pp. 1-219 (Gieseking Ed.,
Springer, Berlin 1975) and Humic Substances II Hays et al. Ed.,
Wiley Interscience, John Wiley, New York (1989), as well as Humus
Chemistry, Genesis Composition Reactions, author F. J. Stevenson,
John Wiley & Sons, New York (1994).
[0049] Environmental samples in which humic acid may be present
include but are not limited to soil, sediment, sludge, decomposing
biological matter, archaeological remains, peat bogs, compost and
water that are terrestrial or subterranean in origin. Engineered
DNA polymerases of the present invention may be particularly useful
for replication or amplification reactions such as PCR wherein the
nucleic acid to be replicated or amplified is comprised within or
has been exposed to such environmental samples. Uses include for
example, analytical, cloning, diagnostic and detection reactions in
the fields of agriculture, horticulture, forestry, forensics,
biological research and in the identification of organism and
sample compositions.
Isolation and Use of Humic Acid in the Present Invention
[0050] Humic acid is typically extracted from humus on the basis of
its solubility in strong alkali and subsequent precipitation in
strong acid (Swift, R. S. in "Methods of soil analysis. Part 3:
Chemical methods", Sparks, D. L. (Ed.), Soil Sci. Soc. Am.,
Madison, 1996, pp. 1018-1020). The remaining solubilized material
is a somewhat refined version of humic acid, referred to as fulvic
acid. Soluble preparations of humic acids are also commercially
available, especially as plant food supplements. Technical grade
humic acid can be obtained for example from Sigma-Alrich Company
Ltd, Gillingham, UK, product number 53680, CAS number 1415-93-6. As
illustrated in Example 1 below, solutions of humic acid may be
prepared and used to test candidate engineered DNA polymerases for
resistance to humic acid. Candidate DNA polymerases may be tested
in any replication or amplification reaction, for example, PCR.
Preferably, candidate DNA polymerases are selected by directed
evolution of DNA polymerases in the presence of humic acid as a
selection pressure. Humic acid may be added to each compartment or
microcapsule during compartmentalised self replication, for
example, or in any other method of directed evolution. Addition of
humic acid to each compartment can be used to select for DNA
polymerases having activity under such conditions.
[0051] Resistance or an enhanced ability to process nucleic acid,
is conveniently expressed in terms of humic acid concentration,
which is found to inhibit the activity of the selected engineered
DNA polymerase, compared to the concentration, which is found to
inhibit the wild type DNA polymerase enzyme. Thus, the engineered
DNA polymerases, selected by our invention may have 2.times.,
4.times., 6.times., 8.times., 10.times., 12.times., 14.times.,
15.times., 16.times., 18.times., 20.times., 22.times., 25.times.,
30.times., or more resistance or enhanced ability to process
nucleic acid, compared to the wild type DNA polymerase enzyme. Most
preferably, the engineered DNA polymerases of the present invention
have 16.times. or more fold enhanced ability to process nucleic
acid when compared this way. The selected engineered DNA
polymerases preferably have 50% or more, 60% or more, 70% or more,
80% or more, 90% or more, or even 100% activity at the
concentration of the inhibitory factor.
Phenolic Compounds
[0052] Humic acid consists of a mixture of complex macromolecules
having polymeric phenolic structures (Merck Index 13.sup.th
Edition).
[0053] The term "phenolic compounds" or polyphenols refers to a
range of substances that possess an aromatic ring bearing one or
more hydroxyl substitutions. Phenolic compounds are products of
secondary metabolism in plants and are widespread throughout the
plant kingdom. Major classes of plant phenolic compounds include
simple phenols, phenolic acids, phenylacetic acids, courmarines,
naphthoquinones, stilbenes/anthraquinones, flavonoids/isoflavonoids
and lignins (for more details see Harbone J B 1980, "Plant
Phenolics in Encyclopedia of Plant Physiology, volume 8, pages
329-395, edited by Bell E A and Charlwood B V, published by
Springer-Verlag, Berlin Heidelberg New York). The detection and
extraction of phenolic compounds from soil or other biological
samples is well known to those skilled in the art (Mahugo Santana
et al Anal Bioanal Chem (2005) 382(1):125-33, Shin et al J
Biotechnol (2005) 119(1):36-43).
[0054] The term "phenolic acids" refers to acidic derivatives of
phenol including but not limited to caffeic acid, vanillin, ferulic
acid, gallic acid, ellagic acid and coumaric acid. Phenolic acids
form a diverse group that includes two main categories: the
hydroxybenzoic acids and the hydroxycinnamic acids (King and Young,
J Am Diet Assoc. (1999) 99(2):213-8). "Phytophenolic acids" refers
to phenolic acids derived from plant material. Phenolic acids may
occur in plants as esters or glycosides conjugated with other
natural compounds such as flavonoids, alcohols, hydroxyfatty acids,
sterols, and glucosides. Hydroxycinnamic acid compounds occur most
frequently as simple esters with hydroxy carboxylic acids or
glucose. Hydroxybenzoic acid compounds are present mainly in the
form of glucosides. Methods for the extraction of phenolic acids
from biological samples are discussed in Luthria and Mukhopadhyay
(2006) J. Agric. Food Chem 54:41-47.
Polymerases
[0055] Polymerase enzymes are able to catalyse the production of
new DNA or RNA from an existing DNA or RNA template--a process
known as polymerisation. There are many different types of
polymerases including DNA polymerases, RNA polymerases and reverse
transcriptases. The methods described in the present application
may be used to generate engineered polymerases including RNA
polymerases, DNA polymerase or reverse transcriptases that are
resistant to humic acid or to phenolic compounds.
RNA Polymerases
[0056] RNA polymerases (RNAP) catalyze the polymerisation of an RNA
strand from a DNA template in the process of transcription. RNAP
can initiate transcription at specific DNA sequences known as
promoters. It then produces an RNA chain which is complementary to
the DNA strand used as a template. The process of adding
nucleotides to the RNA strand is known as elongation. In contrast
to DNA polymerases, RNAP includes a helicase activity therefore no
separate enzyme is needed to unwind DNA. However, RNAPs do work in
association with a number of accessory factors. Such factors may
control a variety of polymerase related processes such as the
timing and specificity of gene expression (Kaiser et al Trends
Biochem Sci. (1996) 21(9):325-6) or transcription-coupled repair
(Lane T F, Cancer Biol Ther. (2004) 3(6):528-33).
[0057] In eukaryotes the transcription of nucleus-encoded genes is
performed by three distinct RNA polymerases termed, I, II and III
(Archambault J et al; Microbiol Rev. (1993) 57(3):703-24). RNA
polymerase I is involved in the synthesis of ribosomal RNA, RNA
polymerase II is involved in the synthesis of mRNA precursors and
snRNA, and RNA polymerase III synthesises tRNA and other small
RNAs. In bacteria the same enzyme catalyses the synthesis of three
types of RNA: mRNA, rRNA and tRNA. The core enzyme has 5 subunits
(two .alpha. subunits, .beta., .beta..sup.1 and .omega.) of which
the .beta. subunit catalyses the synthesis of RNA. A further
discussion of the structure, function and regulation of other RNA
polymerases in eukaryotes and prokaryotes is provided in Mooney et
al Cell, (1999), Vol. 98:687-690 and Cramer P (2002) Curr Opin
Struct Biol 12(1):89-97.
DNA Polymerases
[0058] Engineered DNA polymerases according to the present
invention exhibit at least some resistance to humic acid or
phenolic compounds
[0059] DNA polymerase enzymes are naturally occurring intracellular
enzymes, and are used by a cell to replicate nucleic acid strands.
During the process of replication, a nucleotide sequence of a DNA
strand is copied by complementary base-pairing into a complementary
nucleic acid sequence. Each nucleotide in the DNA strand is
recognised by an unpolymerised complementary nucleotide and
requires that the two strands of the DNA helix be separated, at
least transiently, so that the hydrogen bond donor and acceptor
groups on each base become exposed for base-pairing. The
appropriate incoming single nucleotides are thereby aligned for
their enzyme-catalysed polymerization into a new nucleic acid
chain.
[0060] Enzymes having DNA polymerase activity catalyze the
formation of a bond between the 3' hydroxyl group at the growing
end of a nucleic acid primer sequence and the 5' phosphate group of
a nucleotide triphosphate. These nucleotide triphosphates are
usually selected from deoxyadenosine triphosphate (A),
deoxythymidine triphosphate (T), deoxycytidine triphosphate (C) and
deoxyguanosine triphosphate (G). However, DNA polymerases may
incorporate modified or altered versions of these nucleotides. The
order in which the nucleotides are added is dictated by base
pairing to a DNA template strand; such base pairing is accomplished
through "canonical" hydrogen-bonding (hydrogen-bonding between A
and T nucleotides and G and C nucleotides of opposing DNA strands),
although non-canonical base pairing, such as G:U base pairing, is
known in the art. See e.g., Adams et al., The Biochemistry of the
Nucleic Acids 14-32 (11th ed. 1992). The in-vitro use of enzymes
having DNA polymerase activity has in recent years become more
common in a variety of biochemical applications including cDNA
synthesis and DNA sequencing reactions (see Sambrook et al., (2nd
ed. Cold Spring Harbor Laboratory Press, 1989) hereby incorporated
by reference herein), and amplification of nucleic acids by methods
such as the polymerase chain reaction (PCR) (Mullis et al., U.S.
Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, hereby incorporated
by reference herein) and RNA transcription-mediated amplification
methods (e.g., Kacian et al., PCT Publication No. WO91/01384).
[0061] Methods such as PCR make use of cycles of primer extension
through the use of a DNA polymerase activity, followed by thermal
denaturation of the resulting double-stranded nucleic acid in order
to provide a new template for another round of primer annealing and
extension. Because the high temperatures necessary for strand
denaturation result in the irreversible inactivations of many DNA
polymerases, the discovery and use of DNA polymerases able to
remain active at temperatures above about 37.degree. C. to
42.degree. C. (thermostable DNA polymerase enzymes) provides an
advantage in cost and labor efficiency. Thermostable DNA
polymerases have been discovered in a number of thermophilic
organisms including, but not limited to Thermus aquaticus, Thermus
thermophilus, and species of the Bacillus, Thermococcus,
Sulfolobus, Pyrococcus genera. DNA polymerases can be purified
directly from these thermophilic organisms. However, substantial
increases in the yield of DNA polymerase can be obtained by first
cloning the gene encoding the enzyme in a multicopy expression
vector by recombinant DNA technology methods, inserting the vector
into a host cell strain capable of expressing the enzyme, culturing
the vector-containing host cells, then extracting the DNA
polymerase from a host cell strain which has expressed the
enzyme.
[0062] Preferably, the DNA polymerase of the present invention is a
thermostable polymerase. A "thermostable" DNA polymerase as used
here is a polymerase, which demonstrates significant resistance to
thermal denaturation at elevated temperatures, typically above body
temperature (37.degree. C.). Preferably, such a temperature is in
the range 42.degree. C. to 160.degree. C., more preferably, between
60 to 100.degree. C., most preferably, above 90.degree. C. Compared
to a non-thermostable polymerase, the thermostable polymerase
displays a significantly increased half-life (time of incubation at
elevated temperature that results in 50% loss of activity).
Preferably, the thermostable polymerase retains 30% or more of its
activity after incubation at the elevated temperature, more
preferably, 40%, 50%, 60%, 70% or 80% or more of its activity. Yet
more preferably, the replicase retains 80% activity. Most
preferably, the activity retained is 90%, 95% or more, even 100%.
None-thermostable polymerases would exhibit little or no retention
of activity after similar incubations at the elevated
temperature.
[0063] The bacterial DNA polymerases that have been characterized
to date have certain patterns of similarities and differences which
has led some to divide these enzymes into two groups: those whose
genes contain introns/inteins (Class B DNA polymerases), and those
whose DNA polymerase genes are roughly similar to that of E. coli
DNA polymerase I and do not contain introns (Class A DNA
polymerases).
[0064] Several Class A and Class B thermostable DNA polymerases
derived from thermophilic organisms have been cloned and expressed.
Among the class A enzymes: Lawyer, et al., J. Biol. Chem.
264:6427-6437 (1989) and Gelfund et al, U.S. Pat. No. 5,079,352,
report the cloning and expression of a full length thermostable DNA
polymerase derived from Thermus aquaticus (Taq). Lawyer et al., in
PCR Methods and Applications, 2:275-287 (1993), and Barnes, PCT
Publication No. WO92/06188 (1992), disclose the cloning and
expression of truncated versions of the same DNA polymerase, while
Sullivan, EPO Publication No. 0482714A1 (1992), reports cloning a
mutated version of the Taq DNA polymerase. Asakura et al., J.
Ferment. Bioeng. (Japan), 74:265-269 (1993) have reportedly cloned
and expressed a DNA polymerase from Thermus thermophilus. Gelfund
et al., PCT Publication No. WO92/06202 (1992), have disclosed a
purified thermostable DNA polymerase from Thermosipho africanus. A
thermostable DNA polymerase from Thermus flavus is reported by
Akhmetzjanov and Vakhitov, Nucleic Acids Res., 20:5839 (1992).
Uemori et al., J. Biochem. 113:401-410 (1993) and EPO Publication
No. 0517418A2 (1992) have reported cloning and expressing a DNA
polymerase from the thermophilic bacterium Bacillus caldotenax.
Ishino et al., Japanese Patent Application No. HEI 4[1992]-131400
(publication date Nov. 19, 1993) report cloning a DNA polymerase
from Bacillus stearothermophilus. Among the Class B enzymes: A
recombinant thermostable DNA polymerase from Thermococcus litoralis
is reported by Comb et al., EPO Publication No. 0 455 430 A3
(1991), Comb et al., EPO Publication No. 0547920A2 (1993), and
Perler et al., Proc. Natl. Acad. Sci. (USA), 89:5577-5581 (1992). A
cloned thermostable DNA polymerase from Sulfolobus solofatarius is
disclosed in Pisani et al., Nucleic Acids Res. 20:2711-2716 (1992)
and in PCT Publication WO93/25691 (1993). The thermostable enzyme
of Pyrococcus furiosus is disclosed in Uemori et al., Nucleic Acids
Res., 21:259-265 (1993), while a recombinant DNA polymerase is
derived from Pyrococcus sp. as disclosed in Comb et al., EPO
Publication No. 0547359A1 (1993).
[0065] Many thermostable DNA polymerases possess activities
additional to a DNA polymerase activity; these may include a 5'-3'
exonuclease activity and/or a 3'-5' exonuclease activity. The
activities of 5'-3' and 3'-5' exonucleases are well known to those
of ordinary skill in the art. The 3'-5' exonuclease activity
improves the accuracy of the newly-synthesized strand by removing
incorrect bases that may have been incorporated; DNA polymerases in
which such activity is low or absent, reportedly including Taq DNA
polymerase, (see Lawyer et al., J. Biol Chem. 264:6427-6437), have
elevated error rates in the incorporation of nucleotide residues
into the primer extension strand. In applications such as nucleic
acid amplification procedures in which the replication of DNA is
often geometric in relation to the number of primer extension
cycles, such errors can lead to serious artifactual problems such
as sequence heterogeneity of the nucleic acid amplification product
(amplicon). Thus, a 3'-5' exonuclease activity is a desired
characteristic of a thermostable DNA polymerase used for such
purposes.
[0066] By contrast, the 5'-3' exonuclease activity often present in
DNA polymerase enzymes is often undesired in a particular
application since it may digest nucleic acids, including primers,
that have an unprotected 5' end. Thus, a thermostable DNA
polymerase with an attenuated 5'-3' exonuclease activity, or in
which such activity is absent, is also a desired characteristic of
an enzyme for biochemical applications. Various DNA polymerase
enzymes have been described where a modification has been
introduced in a DNA polymerase, which accomplishes this object. For
example, the Klenow fragment of E. coli DNA polymerase I can be
produced as a proteolytic fragment of the holoenzyme in which the
domain of the protein controlling the 5'-3' exonuclease activity
has been removed. The Klenow fragment still retains the polymerase
activity and the 3'-5' exonuclease activity. Barnes, supra, and
Gelfund et al., U.S. Pat. No. 5,079,352 have produced 5'-3'
exonuclease-deficient recombinant Taq DNA polymerases. Ishino et
al., EPO Publication No. 0517418A2, have produced a 5'-3'
exonuclease-deficient DNA polymerase derived from Bacillus
caldotenax. On the other hand, polymerases lacking the 5'-3'
exonuclease domain often have reduced processivity.
Polymerase Chain Reaction and Other Amplification Techniques
Amplification
[0067] The methods for generation of engineered DNA polymerases in
our invention involve the templated amplification of desired
nucleic acids. "Amplification" refers to the increase in the number
of copies of a particular nucleic acid fragment (or a portion of
this) resulting either from an enzymatic chain reaction (such as a
polymerase chain reaction), or from the replication of all or part
of the vector into which it has been cloned. Preferably, the
amplification according to our invention is an exponential
amplification, as exhibited by for example the polymerase chain
reaction.
[0068] Many target and signal amplification methods have been
described in the literature, for example, general reviews of these
methods in Landegren, U., et al., Science 242:229-237 (1988) and
Lewis, R., Genetic Engineering News 10:1, 54-55 (1990).
Polymerase Chain Reaction (PCR)
[0069] PCR is a nucleic acid amplification method described inter
alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR consists of
repeated cycles of DNA polymerase generated primer extension
reactions. The target DNA is heat denatured and two
oligonucleotides, which bracket the target sequence on opposite
strands of the DNA to be amplified, are hybridized. These
oligonucleotides become primers for use with DNA polymerase. The
primer may be the same chemically, or different from, the extended
sequence (for example, mammalian DNA polymerase is known to extend
a DNA sequence from an RNA primer). The DNA is copied by primer
extension to make a second copy of both strands. By repeating the
cycle of heat denaturation, primer hybridization and extension, the
target DNA can be amplified a million fold or more in about two to
four hours. PCR is a molecular biology tool, which must be used in
conjunction with a detection technique to determine the results of
amplification. An advantage of PCR is that it increases sensitivity
by amplifying the amount of target DNA by 1 million to 1 billion
fold in approximately 4 hours.
[0070] The polymerase chain reaction may be used in the selection
methods of our invention as follows. For example, PCR may be used
to select for variants of Taq polymerase having polymerase
activity. As described in further detail above, a library of
nucleic acids each encoding a DNA polymerase or a variant of the
DNA polymerase, for example, Taq polymerase, is generated and
subdivided into compartments. Each compartment comprises
substantially one member of the library together with the DNA
polymerase or variant encoded by that member.
[0071] The DNA polymerase or variant may be expressed in vivo
within a transformed bacterium or any other suitable expression
host, for example yeast or insect or mammalian cells, and the
expression host encapsulated within a compartment. Heat or other
suitable means is applied to disrupt the host and to release the
polymerase variant and its encoding nucleic acid within the
compartment. In the case of a bacterial host, timed expression of a
lytic protein, for example protein E from .PHI.X174, or use of an
inducible .lamda. lysogen, may be employed for disrupting the
bacterium.
[0072] It will be clear that the DNA polymerase need not be a
heterologous protein expressed in that host (e.g., a plasmid), but
may be expressed from a gene forming part of the host genome. Thus,
the polymerase may be for example an endogenous or native bacterial
polymerase. Thus, the methods of selection according to our
invention may be employed for the direct functional cloning of DNA
polymerases from diverse (and uncultured) microbial
populations.
[0073] Alternatively, the nucleic acid library may be
compartmentalised together with components of an in vitro
transcription/translation system (as described in further detail in
this document), and the polymerase variant expressed in vitro
within the compartment.
[0074] Each compartment also comprises humic acid. It is desirable
that humic acid is added at a concentration sufficient to provide a
selection pressure, so that humic acid resistant DNA polymerases
may be selected for. Importantly, the concentration of humic acid
should not be so great that total inhibition of the polymerase
activity occurs and accordingly no humic acid resistant polymerases
can be selected.
[0075] Each compartment also comprises components for a PCR
reaction, for example, nucleotide triphosphates (dNTPs), buffer,
magnesium, and oligonucleotide primers. The oligonucleotide primers
may have sequences corresponding to sequences flanking the
polymerase gene (i.e., within the genomic or vector DNA) or to
sequences within the polymerase gene. PCR thermal cycling is then
initiated to allow any polymerase variant having polymerase
activity to amplify the nucleic acid sequence.
[0076] Active polymerases will amplify their corresponding nucleic
acid sequences, while nucleic acid sequences encoding weakly active
or inactive polymerases will be weakly replicated or not be
replicated at all. In general, the final copy number of each member
of the nucleic acid library will be expected to be proportional to
the level of activity of the polymerase variant encoded by it.
Nucleic acids encoding active polymerases will be over-represented,
and nucleic acids encoding inactive or weakly active polymerases
will be under-represented. The resulting amplified sequences may
then be cloned and sequenced, etc, and replication ability of each
member assayed.
Reverse Transcriptase-PCR
[0077] RT-PCR is used to amplify RNA targets. In this process, the
reverse transcriptase enzyme is used to convert RNA to
complementary DNA (cDNA), which can then be amplified using PCR.
This method has proven useful for the detection of RNA viruses.
[0078] The methods of our invention may employ RT-PCR and the
engineered DNA polymerases of the present invention may be used in
RT-PCR. The pool of nucleic acids encoding the DNA polymerase or
its variants may be provided in the form of an RNA library. This
library could be generated in vivo in bacteria, mammalian cells,
yeast etc, which are compartmentalised, or by in-vitro
transcription of compartmentalised DNA. The RNA could encode a
co-compartmentalised DNA polymerase that has been expressed in vivo
(and released in emulsion along with the RNA by means disclosed
below) or in vitro. Other components necessary for amplification
(polymerase and/or reverse transcriptase, dNTPs, primers) are also
compartmentalised. Under the humic acid selection pressure, the
cDNA product of the reverse transcription reaction serves as a
template for PCR amplification.
Other Amplification Techniques
[0079] Alternative amplification technology may be exploited in the
present invention. For example, rolling circle amplification
(Lizardi et al., (1998) Nat Genet 19:225) is an amplification
technology available commercially (RCAT.TM.) which is driven by DNA
polymerase and can replicate circular oligonucleotide probes with
either linear or geometric kinetics under isothermal
conditions.
[0080] In the presence of two suitably designed primers, a
geometric amplification occurs via DNA strand displacement and
hyperbranching to generate 10.sup.12 or more copies of each circle
in 1 hour.
[0081] If a single primer is used, RCAT generates in a few minutes
a linear chain of thousands of tandemly linked DNA copies of a
target covalently linked to that target.
[0082] A further technique, strand displacement amplification (SDA;
Walker et al., (1992) PNAS (USA) 80:392) begins with a specifically
defined sequence unique to a specific target. But unlike other
techniques which rely on thermal cycling, SDA is an isothermal
process that utilizes a series of primers, DNA polymerase and a
restriction enzyme to exponentially amplify the unique nucleic acid
sequence.
[0083] SDA comprises both a target generation phase and an
exponential amplification phase.
[0084] In target generation, double-stranded DNA is heat denatured
creating two single-stranded copies. A series of specially
manufactured primers combine with DNA polymerase (amplification
primers for copying the base sequence and bumper primers for
displacing the newly created strands) to form altered targets
capable of exponential amplification.
[0085] The exponential amplification process begins with altered
targets (single-stranded partial DNA strands with restricted enzyme
recognition sites) from the target generation phase.
[0086] An amplification primer is bound to each strand at its
complimentary DNA sequence. DNA polymerase then uses the primer to
identify a location to extend the primer from its 3' end, using the
altered target as a template for adding individual nucleotides. The
extended primer thus forms a double-stranded DNA segment containing
a complete restriction enzyme recognition site at each end.
[0087] A restriction enzyme is then bound to the double stranded
DNA segment at its recognition site. The restriction enzyme
dissociates from the recognition site after having cleaved only one
strand of the double-sided segment, forming a nick. DNA polymerase
recognizes the nick and extends the strand from the site,
displacing the previously created strand. The recognition site is
thus repeatedly nicked and restored by the restriction enzyme and
DNA polymerase with continuous displacement of DNA strands
containing the target segment.
[0088] Each displaced strand is then available to anneal with
amplification primers as above. The process continues with repeated
nicking, extension and displacement of new DNA strands, resulting
in exponential amplification of the original DNA target.
Directed Evolution
[0089] In a preferred embodiment the present invention provides a
method for the generation of an engineered DNA polymerase which
comprises the steps of: [0090] (a) providing a pool of nucleic
acids-comprising members each encoding a DNA polymerase or a
variant of the DNA polymerase; [0091] (b) providing humic acid;
[0092] (c) subdividing the pool of nucleic acids into compartments,
such that each compartment comprises substantially a nucleic acid
member of the pool together with the DNA polymerase or variant
encoded by the nucleic acid member, and humic acid; [0093] (d)
allowing processing of the nucleic acid member to occur; and [0094]
(e) detecting processing of the nucleic acid member by the DNA
polymerase; and optionally repeating the series of steps (a) to (f)
one or more times.
[0095] The techniques of directed evolution and compartmentalised
self replication are detailed in GB 97143002 and GB 98063936 and GB
01275643, in the name of the present inventors. These documents are
herein incorporated by reference.
[0096] In its simplest form CSR involves the segregation of genes
coding for and directing the production of DNA polymerases within
discrete, spatially separated, aqueous compartments of a novel
heat-stable water-in-oil emulsion. Provided with nucleotide
triphosphates and appropriate flanking primers, polymerases
replicate only their own genes. Consequently, only genes encoding
active polymerases are replicated, while inactive variants that
cannot copy their genes disappear from the gene pool. By analogy to
biological systems, among differentially adapted variants, the most
active (the fittest) produce the most "offspring", hence directly
correlating post-selection copy number with enzymatic
turn-over.
[0097] Thus, by exposing repertoires of DNA polymerase genes
(diversified through targeted or random mutation) to
self-amplification and by altering the conditions under which
self-amplification can occur, the system can be used for the
isolation and engineering of polymerases with enhanced resistance
to humic acid.
[0098] Encapsulation of PCRs has been described previously for
lipid vesicles (Oberholzer, T., Albrizio, M. & Luisi, P. L.
(1995) Chem. Biol. 2, 677-82 and fixed cells and tissues (Haase, A.
T., Retzel, E. F. & Staskus, K. A. (1990) Proc. Natl. Acad.
Sci. USA 87, 4971-5; Embleton, M. J., Gorochov, G., Jones, P. T.
& Winter, G. (1992) Nucleic Acids) but with low
efficiencies.
Principles Underlying CST Technology
Microcapsules
[0099] The compartments or "microcapsules" used according to the
method of the invention require appropriate physical properties to
allow the working of the invention.
[0100] First, to ensure that the nucleic acids and gene products
may not diffuse between microcapsules, the contents of each
microcapsule must be isolated from the contents of the surrounding
microcapsules, so that there is no or little exchange of the
nucleic acids and gene products between the microcapsules over the
timescale of the experiment.
[0101] Second, the method of the present invention requires that
there are only a limited number of nucleic acids per microcapsule.
This ensures that the gene product of an individual nucleic acid
will be isolated from other nucleic acids. Thus, coupling between
nucleic acid and gene product will be highly specific. The
enrichment factor is greatest with on average one or fewer nucleic
acids per microcapsule, the linkage between nucleic acid and the
activity of the encoded gene product being as tight as is possible,
since the gene product of an individual nucleic acid will be
isolated from the products of all other nucleic acids. However,
even if the theoretically optimal situation of, on average, a
single nucleic acid or less per microcapsule is not used, a ratio
of 5, 10, 50, 100 or 1000 or more nucleic acids per microcapsule
may prove beneficial in sorting a large library. Subsequent rounds
of sorting, including renewed encapsulation with differing nucleic
acid distribution, will permit more stringent sorting of the
nucleic acids. Preferably, there is a single nucleic acid, or
fewer, per microcapsule.
[0102] Third, the formation and the composition of the
microcapsules must not abolish the function of the machinery the
expression of the nucleic acids and the activity of the gene
products.
[0103] Consequently, any microencapsulation system used must fulfil
these three requirements. The appropriate system(s) may vary
depending on the precise nature of the requirements in each
application of the invention, as will be apparent to the skilled
person.
[0104] A wide variety of microencapsulation procedures are
available (see Benita, 1996) and may be used to create the
microcapsules used in accordance with the present invention.
Indeed, more than 200 microencapsulation methods have been
identified in the literature (Finch, 1993).
[0105] These include membrane enveloped aqueous vesicles such as
lipid vesicles (liposomes) (New, 1990) and non-ionic surfactant
vesicles (van Hal et al., 1996). These are closed-membranous
capsules of single or multiple bilayers of non-covalently assembled
molecules, with each bilayer separated from its neighbour by an
aqueous compartment. In the case of liposomes the membrane is
composed of lipid molecules; these are usually phospholipids but
sterols such as cholesterol may also be incorporated into the
membranes (New, 1990). A variety of enzyme-catalysed biochemical
reactions, including RNA and DNA polymerisation, can be performed
within liposomes (Chakrabarti et al., 1994; Oberholzer et al.,
1995a; Oberholzer et al., 1995b; Walde et al., 1994; Wick &
Luisi, 1996).
[0106] With a membrane-enveloped vesicle system much of the aqueous
phase is outside the vesicles and is therefore
non-compartmentalised. This continuous, aqueous phase should be
removed or the biological systems in it inhibited or destroyed (for
example, by digestion of nucleic acids with DNase or RNase) in
order that the reactions are limited to the microcapsules (Luisi et
al., 1987).
[0107] Enzyme-catalysed biochemical reactions have also been
demonstrated in microcapsules generated by a variety of other
methods. Many enzymes are active in reverse-micellar solutions (Bru
& Walde, 1991; Bru & Walde, 1993; Creagh et al., 1993;
Haber et al., 1993; Kumar et al., 1989; Luisi & B., 1987; Mao
& Walde, 1991; Mao et al., 1992; Perez et al., 1992; Walde et
al., 1994; Walde et al., 1993; Walde et al., 1988) such as the
AOT-isooctane-water system (Menger & Yamada, 1979).
[0108] Microcapsules can also be generated by interfacial
polymerisation and interfacial complexation (Whateley, 1996).
Microcapsules of this sort can have rigid, nonpermeable membranes,
or semipermeable membranes. Semipermeable microcapsules bordered by
cellulose nitrate membranes, polyamide membranes and
lipid-polyamide membranes can all support biochemical reactions,
including multienzyme systems (Chang, 1987; Chang, 1992; Lim,
1984). Alginate/polylysine microcapsules (Lim & Sun, 1980),
which can be formed under very mild conditions, have also proven to
be very biocompatible, providing, for example, an effective method
of encapsulating living cells and tissues (Chang, 1992; Sun et al.,
1992).
[0109] Non-membranous microencapsulation systems based on phase
partitioning of an aqueous environment in a colloidal system, such
as an emulsion, may also be used.
[0110] Preferably, the microcapsules of the present invention are
formed from emulsions; heterogeneous systems of two immiscible
liquid phases with one of the phases dispersed in the other as
droplets of microscopic or colloidal size (Becher, 1957; Sherman,
1968; Lissant, 1974; Lissant, 1984).
Emulsions
[0111] Emulsions may be produced from any suitable combination of
immiscible liquids. Preferably the emulsion of the present
invention has water (containing the biochemical components) as the
phase present in the form of finely divided droplets (the disperse,
internal or discontinuous phase) and a hydrophobic, immiscible
liquid (an `oil`) as the matrix in which these droplets are
suspended (the nondisperse, continuous or external phase). Such
emulsions are termed `water-in-oil` (W/O). This has the advantage
that the entire aqueous phase containing the biochemical components
is compartmentalised in discreet droplets (the internal phase). The
external phase, being a hydrophobic oil, generally contains none of
the biochemical components and hence is inert.
[0112] The emulsion may be stabilised by addition of one or more
surface-active agents (surfactants). These surfactants are termed
emulsifying agents and act at the water/oil interface to prevent
(or at least delay) separation of the phases. Many oils and many
emulsifiers can be used for the generation of water-in-oil
emulsions; a recent compilation listed over 16,000 surfactants,
many of which are used as emulsifying agents (Ash and Ash, 1993).
Suitable oils include light white mineral oil and non-ionic
surfactants (Schick, 1966) such as sorbitan monooleate (Span.TM.80;
ICI) and polyoxyethylenesorbitan monooleate (Tween.TM. 80; ICI) and
Triton-X-100.
[0113] The use of anionic surfactants may also be beneficial.
Suitable surfactants include sodium cholate and sodium
taurocholate. Particularly preferred is sodium deoxycholate,
preferably at a concentration of 0.5% w/v, or below. Inclusion of
such surfactants can in some cases increase the expression of the
nucleic acids and/or the activity of the gene products. Addition of
some anionic surfactants to a non-emulsified reaction mixture
completely abolishes translation. During emulsification, however,
the surfactant is transferred from the aqueous phase into the
interface and activity is restored. Addition of an anionic
surfactant to the mixtures to be emulsified ensures that reactions
proceed only after compartmentalisation.
[0114] Creation of an emulsion generally requires the application
of mechanical energy to force the phases together. There are a
variety of ways of doing this which utilise a variety of mechanical
devices, including stirrers (such as magnetic stir-bars, propeller
and turbine stirrers, paddle devices and whisks), homogenisers
(including rotor-stator homogenisers, high-pressure valve
homogenisers and jet homogenisers), colloid mills, ultrasound and
`membrane emulsification` devices (Becher, 1957; Dickinson,
1994).
[0115] Aqueous microcapsules formed in water-in-oil emulsions are
generally stable with little if any exchange of nucleic acids or
gene products between microcapsules. Additionally, we have
demonstrated that several biochemical reactions proceed in emulsion
microcapsules. Moreover, complicated biochemical processes, notably
gene transcription and translation are also active in emulsion
microcapsules. The technology exists to create emulsions with
volumes all the way up to industrial scales of thousands of litres
(Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant, 1984).
[0116] The preferred microcapsule size will vary depending upon the
precise requirements of any individual selection process that is to
be performed according to the present invention. In all cases,
there will be an optimal balance between gene library size, the
required enrichment and the required concentration of components in
the individual microcapsules to achieve efficient expression and
reactivity of the gene products.
Expression within Microcapsules
[0117] The processes of expression must occur within each
individual microcapsule provided by the present invention. Both in
vitro transcription and coupled transcription-translation become
less efficient at sub-nanomolar DNA concentrations. Because of the
requirement for only a limited number of DNA molecules to be
present in each microcapsule, this therefore sets a practical upper
limit on the possible microcapsule size. Preferably, the mean
volume of the microcapsules is less that 5.2.times.10.sup.-16
m.sup.3, (corresponding to a spherical microcapsule of diameter
less than 10 .mu.m, more preferably less than 6.5.times.10.sup.-17
m.sup.3 (5 .mu.m), more preferably about 4.2.times.10.sup.-18
m.sup.3 (2 .mu.m) and ideally about 9.times.10.sup.-18 m.sup.3 (2.6
.mu.m).
[0118] The effective DNA or RNA concentration in the microcapsules
may be artificially increased by various methods that will be
well-known to those versed in the art. These include, for example,
the addition of volume excluding chemicals such as polyethylene
glycols (PEG) and a variety of gene amplification techniques,
including transcription using RNA polymerases including those from
bacteria such as E. coli (Roberts, 1969; Blattner and Dahlberg,
1972; Roberts et al., 1975; Rosenberg et al., 1975), eukaryotes
e.g. (Weil et al., 1979; Manley et al., 1983) and bacteriophage
such as T7, T3 and SP6 (Melton et al., 1984); the polymerase chain
reaction (PCR) (Saiki et al., 1988); Qb replicase amplification
(Miele et al., 1983; Cahill et al., 1991; Chetverin and Spirin,
1995; Katanaev et al., 1995); the ligase chain reaction (LCR)
(Landegren et al., 1988; Barany, 1991); and self-sustained sequence
replication system (Fahy et al., 1991) and strand displacement
amplification (Walker et al., 1992). Even gene amplification
techniques requiring thermal cycling such as PCR and LCR could be
used if the emulsions and the in vitro transcription or coupled
transcription-translation systems are thermostable (for example,
the coupled transcription-translation systems could be made from a
thermostable organism such as Thermus aquaticus).
[0119] Increasing the effective local nucleic acid concentration
enables larger microcapsules to be used effectively. This allows a
preferred practical upper limit to the microcapsule volume of about
5.2.times.10.sup.-16 m.sup.3 (corresponding to a sphere of diameter
10 um).
[0120] The microcapsule size must be sufficiently large to
accommodate all of the required components of the biochemical
reactions that are needed to occur within the microcapsule. For
example, in vitro, both transcription reactions and coupled
transcription-translation reactions require a total nucleoside
triphosphate concentration of about 2 mM.
[0121] For example, in order to transcribe a gene to a single short
RNA molecule of 500 bases in length, this would require a minimum
of 500 molecules of nucleoside triphosphate per microcapsule
(8.33.times.10.sup.-22 moles). In order to constitute a 2 mM
solution, this number of molecules must be contained within a
microcapsule of volume 4.17.times.10.sup.-19 litres
(4.17.times.10.sup.-22 m.sup.3 which if spherical would have a
diameter of 93 nm.
[0122] Furthermore, particularly in the case of reactions involving
translation, it is to be noted that the ribosomes necessary for the
translation to occur are themselves approximately 20 nm in
diameter. Hence, the preferred lower limit for microcapsules is a
diameter of approximately 100 nm.
[0123] Therefore, the microcapsule volume is preferably of the
order of between 5.2.times.10.sup.-22 m.sup.3 and
5.2.times.10.sup.-16 m.sup.3 corresponding to a sphere of diameter
between 0.1 um and 10 um, more preferably of between about
5.2.times.10.sup.-19 m.sup.3 and 6.5.times.10.sup.-17 m.sup.3 (1 um
and 5 um). Sphere diameters of about 2.6 um are most
advantageous.
[0124] It is no coincidence that the preferred dimensions of the
compartments (droplets of 2.6 um mean diameter) closely resemble
those of bacteria, for example, Escherichia are
1.1-1.5.times.2.0-6.0 um rods and Azotobacter are 1.5-2.0 um
diameter ovoid cells. In its simplest form, Darwinian evolution is
based on a `one genotype one phenotype` mechanism. The
concentration of a single compartmentalised gene, or genome, drops
from 0.4 nM in a compartment of 2 um diameter, to 25 pM in a
compartment of 5 um diameter. The prokaryotic
transcription/translation machinery has evolved to operate in
compartments of .about.1-2 um diameter, where single genes are at
approximately nanomolar concentrations. A single gene, in a
compartment of 2.6 um diameter is at a concentration of 0.2 nM.
This gene concentration is high enough for efficient translation.
Compartmentalisation in such a volume also ensures that even if
only a single molecule of the gene product is formed it is present
at about 0.2 nM, which is important if the gene product is to have
a modifying activity of the nucleic acid itself. The volume of the
microcapsule should thus be selected bearing in mind not only the
requirements for transcription and translation of the nucleic
acid/nucleic acid, but also the modifying activity required of the
gene product in the method of the invention.
[0125] The size of emulsion microcapsules may be varied simply by
tailoring the emulsion conditions used to form the emulsion
according to requirements of the selection system. The larger the
microcapsule size, the larger is the volume that will be required
to encapsulate a given nucleic acid/nucleic acid library, since the
ultimately limiting factor will be the size of the microcapsule and
thus the number of microcapsules possible per unit volume.
[0126] The size of the microcapsules is selected not only having
regard to the requirements of the transcription/translation system,
but also those of the selection system employed for the nucleic
acid/nucleic acid construct. Thus, the components of the selection
system, such as a chemical modification system, may require
reaction volumes and/or reagent concentrations which are not
optimal for transcription/translation. As set forth herein, such
requirements may be accommodated by a secondary re-encapsulation
step; moreover, they may be accommodated by selecting the
microcapsule size in order to maximise transcription/translation
and selection as a whole. Empirical determination of optimal
microcapsule volume and reagent concentration, for example as set
forth herein, is preferred.
[0127] A "nucleic acid" in accordance with the present invention is
as described above. Preferably, a nucleic acid is a molecule or
construct selected from the group consisting of a DNA molecule, an
RNA molecule, a partially or wholly artificial nucleic acid
molecule consisting of exclusively synthetic or a mixture of
naturally-occurring and synthetic bases, any one of the foregoing
linked to a polypeptide, and any one of the foregoing linked to any
other molecular group or construct. Advantageously, the other
molecular group or construct may be selected from the group
consisting of nucleic acids, polymeric substances, particularly
beads, for example polystyrene beads, magnetic substances such as
magnetic beads, labels, such as fluorophores or isotopic labels,
chemical reagents, binding agents such as macrocycles and the
like.
[0128] The nucleic acid portion of the nucleic acid may comprise
suitable regulatory sequences, such as those required for efficient
expression of the gene product, for example promoters, enhancers,
translational initiation sequences, polyadenylation sequences,
splice sites and the like.
Product Selection
[0129] A ligand or substrate can be connected to the nucleic acid
by a variety of means that will be apparent to those skilled in the
art (see, for example, Hermanson, 1996). Any tag will suffice that
allows for the subsequent selection of the nucleic acid. Sorting
can be by any method which allows the preferential separation,
amplification or survival of the tagged nucleic acid. Examples
include selection by binding (including techniques based on
magnetic separation, for example using Dynabeads.TM.), and by
resistance to degradation (for example by nucleases, including
restriction endonucleases).
[0130] One way in which the nucleic acid molecule may be linked to
a ligand or substrate is through biotinylation. This can be done by
PCR amplification with a 5'-biotinylation primer such that the
biotin and nucleic acid are covalently linked.
[0131] The ligand or substrate to be selected can be attached to
the modified nucleic acid by a variety of means that will be
apparent to those of skill in the art. A biotinylated nucleic acid
may be coupled to a polystyrene microbead (0.035 to 0.2 um in
diameter) that is coated with avidin or streptavidin, that will
therefore bind the nucleic acid with very high affinity. This bead
can be derivatised with substrate or ligand by any suitable method
such as by adding biotinylated substrate or by covalent
coupling.
[0132] Alternatively, a biotinylated nucleic acid may be coupled to
avidin or streptavidin complexed to a large protein molecule such
as thyroglobulin (669 Kd) or ferritin (440 Kd). This complex can be
derivatised with substrate or ligand, for example by covalent
coupling to the alpha-amino group of lysines or through a
non-covalent interaction such as biotin-avidin. The substrate may
be present in a form unlinked to the nucleic acid but containing an
inactive "tag" that requires a further step to activate it such as
photoactivation (e.g. of a "caged" biotin analogue, (Sundberg et
al., 1995; Pirrung and Huang, 1996)). The catalyst to be selected
then converts the substrate to product. The "tag" could then be
activated and the "tagged" substrate and/or product bound by a
tag-binding molecule (e.g. avidin or streptavidin) complexed with
the nucleic acid. The ratio of substrate to product attached to the
nucleic acid via the "tag" will therefore reflect the ratio of the
substrate and product in solution.
[0133] When all reactions are stopped and the microcapsules are
combined, the nucleic acids encoding active enzymes can be enriched
using an antibody or other molecule which binds, or reacts
specifically with the "tag". Although both substrates and product
have the molecular tag, only the nucleic acids encoding active gene
product will co-purify.
[0134] The terms "isolating", "sorting" and "selecting", as well as
variations thereof, are used herein. Isolation, according to the
present invention, refers to the process of separating an entity
from a heterogeneous population, for example a mixture, such that
it is free of at least one substance with which it was associated
before the isolation process. In a preferred embodiment, isolation
refers to purification of an entity essentially to homogeneity.
Sorting of an entity refers to the process of preferentially
isolating desired entities over undesired entities. In as far as
this relates to isolation of the desired entities, the terms
"isolating" and "sorting" are equivalent. The method of the present
invention permits the sorting of desired nucleic acids from pools
(libraries or repertoires) of nucleic acids which contain the
desired nucleic acid. Selecting is used to refer to the process
(including the sorting process) of isolating an entity according to
a particular property thereof.
Microcapsules/Sorting
[0135] In addition to the nucleic acids described above, the
microcapsules according to the invention will comprise further
components required for the sorting process to take place. Other
components of the system will for example comprise those necessary
for transcription and/or translation of the nucleic acid. These are
selected for the requirements of a specific system from the
following; a suitable buffer, an in vitro transcription/replication
system and/or an in vitro translation system containing all the
necessary ingredients, enzymes and cofactors, RNA polymerase,
nucleotides, nucleic acids (natural or synthetic), transfer RNAs,
ribosomes and amino acids, and the substrates of the reaction of
interest in order to allow selection of the modified gene
product.
[0136] A suitable buffer will be one in which all of the desired
components of the biological system are active and will therefore
depend upon the requirements of each specific reaction system.
Buffers suitable for biological and/or chemical reactions are known
in the art and recipes provided in various laboratory texts, such
as Sambrook et al., 1989.
[0137] The in vitro translation system will usually comprise a cell
extract, typically from bacteria (Zubay, 1973; Zubay, 1980; Lesley
et al., 1991; Lesley, 1995), rabbit reticulocytes (Pelham and
Jackson, 1976), or wheat germ (Anderson et al., 1983). Many
suitable systems are commercially available (for example from
Promega) including some which will allow coupled
transcription/translation (all the bacterial systems and the
reticulocyte and wheat germ TNT.TM. extract systems from Promega).
The mixture of amino acids used may include synthetic amino acids
if desired, to increase the possible number or variety of proteins
produced in the library. This can be accomplished by charging tRNAs
with artificial amino acids and using these tRNAs for the in vitro
translation of the proteins to be selected (Ellman et al., 1991;
Benner, 1994; Mendel et al., 1995).
[0138] After each round of selection the enrichment of the pool of
nucleic acids for those encoding the molecules of interest can be
assayed by non-compartmentalised in vitro transcription/replication
or coupled transcription-translation reactions. The selected pool
is cloned into a suitable plasmid vector and RNA or recombinant
protein is produced from the individual clones for further
purification and assay.
Microcapsule Identification
[0139] Microcapsules may be identified by virtue of a change
induced by the desired gene product which either occurs or
manifests itself at the surface of the microcapsule or is
detectable from the outside as described in the section
"Microcapsule Sorting". This change, when identified, is used to
trigger the modification of the gene within the compartment. In a
preferred aspect of the invention, microcapsule identification
relies on a change in the optical properties of the microcapsule
resulting from a reaction leading to luminescence, phosphorescence
or fluorescence within the microcapsule. Modification of the gene
within the microcapsules would be triggered by identification of
luminescence, phosphorescence or fluorescence. For example,
identification of luminescence, phosphorescence or fluorescence can
trigger bombardment of the compartment with photons (or other
particles or waves) which leads to modification of the nucleic
acid. A similar procedure has been described previously for the
rapid sorting of cells (Keij et al., 1994). Modification of the
nucleic acid may result, for example, from coupling a molecular
"tag", caged by a photolabile protecting group to the nucleic
acids: bombardment with photons of an appropriate wavelength leads
to the removal of the cage. Afterwards, all microcapsules are
combined and the nucleic acids pooled together in one environment.
Nucleic acids encoding gene products exhibiting the desired
activity can be selected by affinity purification using a molecule
that specifically binds to, or reacts specifically with, the
"tag".
Multi Step Procedure
[0140] It will be also be appreciated that according to the present
invention, it is not necessary for all the processes of
transcription/replication and/or translation, and selection to
proceed in one single step, with all reactions taking place in one
microcapsule. The selection procedure may comprise two or more
steps. First, transcription/replication and/or translation of each
nucleic acid of a nucleic acid library may take place in a first
microcapsule. Each gene product is then linked to the nucleic acid
which encoded it (which resides in the same microcapsule). The
microcapsules are then broken, and the nucleic acids attached to
their respective gene products optionally purified. Alternatively,
nucleic acids can be attached to their respective gene products
using methods which do not rely on encapsulation. For example phage
display (Smith, G. P., 1985), polysome display (Mattheakkis et al.,
1994), RNA-peptide fusion (Roberts and Szostak, 1997) or lac
repressor peptide fusion (Cull, et al., 1992).
[0141] In the second step of the procedure, each purified nucleic
acid attached to its gene product is put into a second microcapsule
containing components of the reaction to be selected. This reaction
is then initiated. After completion of the reactions, the
microcapsules are again broken and the modified nucleic acids are
selected. In the case of complicated multistep reactions in which
many individual components and reaction steps are involved, one or
more intervening steps may be performed between the initial step of
creation and linking of gene product to nucleic acid, and the final
step of generating the selectable change in the nucleic acid.
Libraries of Nucleic Acid Sequences
[0142] Herein, the terms "library", "repertoire" and "pool" are
used according to their ordinary signification in the art, such
that a library of nucleic acids encodes a repertoire of gene
products. Initial selection of a nucleic acid/nucleic acid from a
nucleic acid library (for example a mutant taq library) according
to the present invention will in most cases require the screening
of a large number of variant nucleic acids. Libraries of nucleic
acids can be created in a variety of different ways, including the
following.
[0143] Pools of naturally occurring nucleic acids can be cloned
from genomic DNA or cDNA (Sambrook et al., 1989); for example,
mutant Taq libraries or other DNA polymerase libraries, made by PCR
amplification repertoires of taq or other DNA polymerase genes have
proved very effective sources of DNA polymerase fragments.
[0144] Libraries of genes can also be made by encoding all (see for
example Smith, 1985; Parmley and Smith, 1988) or part of genes (see
for example Lowman et al., 1991) or pools of genes (see for example
Nissim et al., 1994) by a randomised or doped synthetic
oligonucleotide. Libraries can also be made by introducing
mutations into a nucleic acid or pool of nucleic acids `randomly`
by a variety of techniques in vivo, including; using `mutator
strains`, of bacteria such as E. coli mutD5 (Liao et al., 1986;
Yamagishi et al., 1990; Low et al., 1996). Random mutations can
also be introduced both in vivo and in vitro by chemical mutagens,
and ionising or UV irradiation (see Friedberg et al., 1995), or
incorporation of mutagenic base analogues (Freese, 1959; Zaccolo et
al., 1996). `Random` mutations can also be introduced into genes in
vitro during polymerisation for example by using error-prone
polymerases. Error-prone PCR introduces random copying errors by
imposing imperfect, and thus mutagenic, or "sloppy" reaction
conditions (for example by adding Mn.sup.2+ or Mg.sup.2+ to the
reaction mixture (Cadwell and Joyce, 1991, PCR Meth. Appl. 2:28-33;
Leung et al., 1989, Technique 1:11-13). This method has proven
useful for generation of randomised libraries of nucleotide
sequences. According to the method of the invention, the term
`random` may be in terms of random positions with random repertoire
of amino acids at those positions or it may be selected
(predetermined) positions with random repertoire of amino acids at
those selected positions.
[0145] Further diversification can be introduced by using
homologous recombination either in vivo (see Kowalczykowski et al.,
1994 or in vitro (Stemmer, 1994a; Stemmer, 1994b)). An example of
an in vitro homologous recombination technique to generate gene
diversity is gene shuffling.
[0146] Gene shuffling involves random fragmentation of several
mutant DNAs followed by their reassembly by PCR into full length
molecules (Smith, Nature, 370: 324 [1994]). Examples of various
gene shuffling procedures include, but are not limited to, assembly
following DNase treatment, the staggered extension process (STEP),
and random priming in vitro recombination. In the DNase mediated
method, DNA segments isolated from a pool of positive mutants are
cleaved into random fragments with DNaseI and subjected to multiple
rounds of PCR with no added primer. The lengths of random fragments
approach that of the uncleaved segment as the PCR cycles proceed,
resulting in mutations present in different clones becoming mixed
and accumulating in some of the resulting sequences. Multiple
cycles of selection and shuffling have led to the functional
enhancement of several enzymes (Stemmer, Nature, 370: 398 [1994];
Stemmer, Proc. Natl. Acad. Sci. USA, 91: 10747 [1994]; Crameri et
al., Nat. Biotech., 14: 315 [1996]; Zhang et al., Proc. Natl. Acad.
Sci. USA, 94: 4504 [1997]; and Crameri et al., Nat. Biotech., 15:
436 [1997]).
[0147] A modification of gene shuffling, the Staggered Extension
Protocol (StEP) has been described (WO 98/42832; Shao et al., 1998;
Zhao et al., 1997; Zhao et al., 1998). StEP involves priming
template polynucleotides with random or flanking primers. Extended
primers are reassembled in extremely fast cycles of PCR, generating
successively longer and longer extension products. In each cycle
the primers/extension products can anneal to different templates
based on sequence complementarity. The template switching between
different sequences creates "recombination cassettes". The process
is continued until full-length genes are created.
[0148] A modification of the StEP technology has also been
described (U.S. Pat. No. 5,965,408). Like StEP, random primers are
annealed to a target(s) to be shuffled. The random primers are
extended until stopped by "roadblocks" such as purine dimers. The
premature termination is facilitated by blocking the polymerase
with adducts associated with the template. Fragments are isolated
and used in a separate PCR reaction to create longer overlapping
fragments.
[0149] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations, and for screening cDNA libraries for gene products
having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by
the combinatorial mutagenesis or recombination of DNA polymerase
homologs or variants. The most widely used techniques for screening
large gene libraries typically comprises cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates relatively easy isolation of the
vector encoding the gene whose product was detected. Directed
evolution techniques for detection and selection of desired DNA
polymerase activity have already been described.
Vectors and Host Cells
[0150] Suitable vectors and host cells may be used to host nucleic
acid encoding candidate DNA polymerases, or libraries thereof, or
engineered DNA polymerases of the present invention. Host cells and
vectors may also be used to express and isolate candidate DNA
polymerase polypeptides or engineered DNA polymerases of the
present invention. Suitable host cells may also be used to isolate
wild type DNA polymerase genes and alternatively or additionally,
to express wild type DNA polymerase polypeptides for use in the
methods of the present invention.
Vectors
[0151] Expression vectors may be constructed from a starting vector
such as a commercially available vector. Preferred vectors are
those which are compatible with bacterial, insect, and mammalian
host cells. Such vectors include, inter alia, pCRII pCR3, and
pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSII (Stratagene, La
Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia
Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.),
pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO 90/14363)
and pFastBacDual (Gibco-BRL, Grand Island, N.Y.).
[0152] Additional suitable vectors include, but are not limited to,
cosmids, plasmids, or modified viruses, but it will be appreciated
that the vector system must be compatible with the selected host
cell. Such vectors include, but are not limited to plasmids such as
Bluescript.RTM. plasmid derivatives (a high copy number ColE1-based
phagemid, Stratagene Cloning Systems, La Jolla Calif.), PCR cloning
plasmids designed for cloning Taq-amplified PCR products (e.g.,
TOPO.TM. TA Cloning.RTM. Kit, PCR2.1.RTM. plasmid derivatives,
Invitrogen, Carlsbad, Calif.), pASK75, and mammalian, yeast or
virus vectors such as a baculovirus expression system (pBacPAK
plasmid derivatives, Clontech, Palo Alto, Calif.).
[0153] Vectors may also include a transcription regulatory element
(a promoter) operably linked to the DNA polymerase sequence. The
promoter may optionally contain operator portions and/or ribosome
binding sites. Non-limiting examples of bacterial promoters
compatible with E. coli include: trc promoter, alpha-lactamase
(penicillinase) promoter; lactose promoter; tryptophan (trp)
promoter; arabinose BAD operon promoter; lambda-derived PI promoter
and N gene ribosome binding site; and the hybrid tac promoter
derived from sequences of the trp and lac UV5 promoters.
[0154] After the vector has been constructed and a nucleic acid
molecule encoding a DNA polymerase polypeptide has been inserted
into the proper site of the vector, the completed vector may be
inserted into a suitable host cell for amplification and/or
polypeptide expression. The transformation of an expression vector
for a DNA polymerase polypeptide into a selected host cell may be
accomplished by well known methods including methods such as
transfection, infection, calcium chloride, electroporation,
microinjection, lipofection, DEAE-dextran method, or other known
techniques. The method selected will in part be a function of the
type of host cell to be used. These methods and other suitable
methods are well known to the skilled artisan, and are set forth,
for example, in Sambrook et al., supra.
Host Cells
[0155] By "host cell" or "recombinantly engineered cell" is meant a
cell, which contains a vector and supports the replication and/or
expression of the expression vector.
[0156] Host cells may be prokaryotic host cells (such as E. coli)
or eukaryotic host cells (such as a yeast, insect, or vertebrate
cell). The host cell, when cultured under appropriate conditions,
synthesizes a DNA polymerase polypeptide which can subsequently be
collected from the culture medium (if the host cell secretes it
into the medium) or directly from the host cell producing it (if it
is not secreted). The selection of an appropriate host cell will
depend upon various factors, such as desired expression levels,
polypeptide modifications that are desirable or necessary for
activity (such as glycosylation or phosphorylation) and ease of
folding into a biologically active molecule.
[0157] Of particular interest as host cells are bacterial cells.
For example, the various strains of E. coli (e.g., HB101,
DH5.alpha., DH10, and MC1061) are well known as host cells in the
field of biotechnology. Various strains of B. subtilis, Pseudomonas
spp., other Bacillus spp., Streptomyces spp., and the like may also
be employed in methods used in the present invention.
[0158] Host cells may be used to express heterologous candidate or
engineered DNA polymerases of the present invention. As used
herein, "heterologous" in reference to a nucleic acid is a nucleic
acid that originates from a foreign species, or, if from the same
species, is substantially modified from its native form in
composition and/or genomic locus by deliberate human intervention.
For example, a promoter operably linked to a heterologous
structural gene is from a species different from that from which
the structural gene was derived, or, if from the same species, one
or both are substantially modified from their original form. A
heterologous protein may originate from a foreign species or, if
from the same species, is substantially modified from its original
form by deliberate human intervention.
[0159] Preferred replication systems include M13, ColE1, SV40,
baculovirus, lambda, adenovirus, and the like. A large number of
transcription initiation and termination regulatory regions have
been isolated and shown to be effective in the transcription and
translation of heterologous proteins in the various hosts. Examples
of these regions, methods of isolation, manner of manipulation,
etc. are known in the art. Under appropriate expression conditions,
host cells can be used as a source of recombinantly produced DNA
polymerases or derived peptides and polypeptides.
EXAMPLES
Example 1
Libraries of Polymerase Chimeras
[0160] Libraries of chimeric polymerase gene variants were
constructed using the Step shuffling PCR technique (Zhao et al.,
(1998) Nature Biotechnol. 16, 258-261).
[0161] For a first library 3T: Thermus aquaticus (Taq) wild type
and T8 (a previously selected 11 fold more thermostable Taq variant
(Ghadessy et al. Proc Natl Acad Sci USA. 2001 Apr. 10;
98(8):4552-7), Thermus thermophilus (Tth) and Thermus flavus (Tfl)
polymerase genes were amplified from genomic DNA and cloned into
pASK75 (Skerra 1994) and tested for activity. These genes were
shuffled using Step, then recloned into pASK75 and transformed into
E. coli TG1 giving library 3T.
[0162] For a second more diverse library 8T, we amplified the Pol I
genes from the genomic DNA of Thermus brockianus, Thermus
filiformis, Thermus scotoductus and Thermus oshimai by PCR and
cloned them into the pAsk75 vector.
[0163] T8 was then generated by Step as above including the Pol I
genes of Thermus thermophilus, Thermus aquaticus, Thermus flavus,
Thermus brockianus, Thermus filiformis, Thermus scotoductus and
Thermus oshimai as well as Deinococcus radiodurans (a radiation
resistant bacterium) which had previously been cloned into pAsk75
in our laboratory.
[0164] The library size was scored by dilution assays and
determining the ratio of clones containing insert using PCR
screening and was approximately 1.times.10.sup.8 in both cases. A
diagnostic restriction digest of 20 clones produced 20 unique
restriction patterns, indicating that the library was diverse.
[0165] Subsequent sequencing of selected chimeras showed an average
of 4 to 6 crossovers per gene.
Example 2
Production of Humic Acid
[0166] A sample of peat soil was broken into small pieces and water
was added. The sample was then heated to 50.degree. C. for 1 hour
to aid solubilisation.
[0167] The resulting samples were spun down at 13000 rpm for 30
minutes and the water phase was recovered. The volume was then
reduced 10 fold by using a concentrator.
[0168] The inhibitory activity of the resulting humic acid was
tested by doing a 30 cycle PCR (94.degree. C. 10 min), then 30
cycles of 94.degree. C. 30 s, 50.degree. C. 30 s, 72.degree. C. 1
min then 65.degree. C. 10 min in the presence of a two fold
dilution series of humic acid from 60% humic acid to 0.03% (12
points). The PCR (1.times. SuperTaq buffer, 0.2 mM dNTP, 1 .mu.M
primers (AAA AAT CTA GAT AAC GAG GGC AA and ACC ACC GAA CTG CGG GTG
ACG CCA AGC G), 1 .mu.l SuperTaq, 2.5 .mu.l of an overnight growth
of E. coli cells and 0.01 .mu.l of pAsk75 as template (100 .mu.m
stock), water and humic acid as required) was performed in the
presence of E. coli cell debris as it is known that DNA and protein
counteract to an extent the inhibitor effect that humic acid has on
polymerases.
[0169] The humic acid solution was found to totally inhibit the PCR
a concentration of 5% and above.
Example 3
Selection of Humic Acid Resistant Clones
[0170] CSR emulsification and selection was performed on the StEP
Taq, Tth and Tfl library essentially as described (Ghadessy et al.
2001), but with the addition of humic acid to the water phase of
the emulsion as the source of selective pressure. The highest
amount of humic acid which produced a positive selection was
20%.
[0171] The primers used were (5'-GTA AAA CGA CGG CCA GTA CCA CCG
AAC TGC GGG TGA CGC CAA GCG-3', and 5'-CAG GAA ACA GCT ATG ACA AAA
ATC TAG ATA ACG AGG GCA A-3').
[0172] The aqueous phase was ether extracted, PCR-purified (Qiagen,
Chatsworth, Calif.) with an additional 35% GnHCl, digested with
DpnI to remove methylated plasmid DNA, treated with ExoSAP (USB) to
remove residual primers, reamplified with outnested primers (CAG
GAA ACA GCT ATG AC and GTA AAA CGA CGG CCA GT), recloned and
transformed into E. coli as above.
[0173] The resultant clones were screened and ranked in order using
a PCR assay. Briefly, 2.5 .mu.l of induced cells were added to 20
.mu.l of PCR mix ((1.times. SuperTaq buffer, 0.2 mM dNTP, 1 .mu.M,
0.01 .mu.l of pAsk75 (100 .mu.M stock), water and humic acid as
required) with the relevant primers (AAA AAT CTA GAT AAC GAG GGC AA
and ACC ACC GAA CTG CGG GTG ACG CCA AGC G). 6 Plates were screened
at varying concentrations of humic acid (10%, 5%) and a total of 14
polymerases were isolated that worked in PCR under conditions were
the WT did not (i.e. 5 or 10% humic acid): P1H2, P2E2, P3D5, P4D10,
P4F12, P5E1, P5H2, P6A9, P6A10, P6C10, P6D1, P6F3, P6F4.
Example 4
Ranking of Selected Clones
[0174] Polymerase clones: P1H2, P2E2, P3D5, P4D10, P4F8, P4F12,
P5E1, P5H2, P6A9, P6A10, P6C10, P6D1, P6F3 were streaked on
selective agar plates and grown overnight at 37.degree. C., diluted
1/100 into 2.times.TY/Amp incubated at 37.degree. C. until
O.D.sub.595=0.5 (ca. 2 hours). Anhydrotetracycline was added to a
final concentration of 0.04 .mu.g/ml and cultures were induced for
4 hours at 37.degree. C., shaking. Cells were spun down,
supernatant was discarded and cell pellet resuspended in 1/4 volume
of 1.times. Taq buffer followed by incubation at 85.degree. C. for
10 min. Lysate was cleared by centrifugation. Polymerases were
normalized and ranked for activity in PCR essentially as in
Ghadessy et al 2001 using PCR program ((94.degree. C. 1 min,
30.times.(94.degree. C. 30 sec, 50.degree. C. 30 sec, 72.degree. C.
1 min), 65.degree. C. for 2 min) using primers 1: 5'-ACC ACC GAA
CTG CGG GTG ACG CCA AG-3' and 2: 5'-GGG TAC GTG GAG ACC CTC TTC GGC
C-3' and 10 ng of pASK-Taq vector as template. Resistance to humic
acid inhibition was determined using serial dilution of peat
extract humic acid (HuAc P) (see above) and commercially available
humic acid (Fluka, product code: 53680; Lot: 1102067 34505220)
(HuAc F (dissolved in 1.times. Taq buffer to saturation (i.e. limit
of solubility)) (Table 1).
TABLE-US-00001 TABLE 1 Activity in humic acid from two different
sources Activity Activity in in Poly- HuAc P HuAc F f.sub.HuAcP/
merase activity f.sup.a 1/10 f.sub.HuAcP.sup.b 1/50
f.sub.HuAcF.sup.c f.sub.HuAcF.sup.d P2E2 1/128 4 1/16 0.25 1/64
0.03125 8 P1H2 1/64 2 1/8 1 1/32 0.25 4 P6A10 1/64 2 1/16 0.5 1/32
0.25 2 P3D5 1/8 0.25 1/32 2 1/128 0.5 4 P4D10 1/128 4 1/2 2 1/16
0.25 8 P4F8 1/32 1 1/32 0.5 1/64 0.25 2 P5H2 1/16 0.5 1/16 2 1/64
0.5 4 P6A9 1/16 0.5 1/16 2 1/64 0.5 4 P4F12 1/32 1 1/4 4 1/8 2 2
(Hu1) P5E1 1/64 2 1/8 1 1/16 0.5 2 P6C10 1/32 1 1/8 2 1/32 0.5 4
P6D1 1/32 1 1/16 1 1/64 0.25 4 P6F3 1/8 0.25 1/8 8 1/32 2 4 Taqwt
1/32 1 1/16 1 1/16 1 1 .sup.af: rel. activity vs Taqwt
.sup.bf.sub.HuAcP: rel. activity vs Taqwt in HuAc extracted from
peat (HuAc P) .sup.cf.sub.HuAcF: rel. activity vs Taqwt in HuAc
from Fluka (HuAc F) .sup.df.sub.HuAcP/f.sub.HuAcF: rel. activity in
HuAc P vs HuAc F
[0175] Clones show universally higher resistance to the inhibitory
effects of HuAc P for which they were selected. P4F12 (Hu1) and
P6F3 display the highest level of resistance (f.sub.HuAcP)
retaining activity at 4-resp. 8-fold the concentration of HuAc P at
which Taqwt is completely inhibited in PCR. Resistance to HuAc F is
low or absent. Only P4F12 (Hu1) and P6F3 display an increased
resistance (2.times.) compared to wtTaq to commercially available
humic acid (HuAc F).
[0176] This reflects the selection of polymerases for resistance to
HuAc P and not HuAcF. The relative activity in HuAc P vs HuAc F
(f.sub.HuAcP/f.sub.HuAcF) for Taqwt is 1, while most of the
selected clones display a f.sub.HuAcP/f.sub.HuAcF 2-8. HuAc P and
HuAC F are clearly distinct and reflect the heterogenous nature of
humic substances. Future selections may alternate between different
humic acid preparations to ensure a general resistance to humic
acids, although P4F12 (Hu1) and P6F3 already display a low level of
general resistance.
Example 5
Selection of a Polymerase Resistant to Inhibition by Soil
[0177] Using standard CSR selection as described (Ghadessy et al.,
2001), three polymerases were selected, which show an increased
resistance towards soil inhibition compared to the wildtype Thermus
aquaticus polymerase. The clones were selected after two rounds of
CSR.
[0178] The soil sample used in the experiments was collected in
Cambridge and showed a slightly alkaline pH.
[0179] For the first round of CSR an aliquot of the soil sample was
used to set up a soil slurry in 1.times. Supertaq buffer, which was
then added to the reaction as inhibitory reagent. In a control
reaction with Supertaq polymerase, the first product was observable
at a concentration of 0.3% of the soil slurry. The first round of
CSR was then carried out in the presence of 2.5% soil slurry.
[0180] For the second round an aliquot of the soil sample was
transferred into 1.times. Supertaq buffer and this soil slurry was
then incubated for 2 hours at 50.degree. C., followed by 20 minutes
at 90.degree. C. The extract was then centrifuged at 8.000 rpm for
10 minutes and the supernatant was kept as inhibitory solution
(-20.degree. C.).
[0181] The inhibitory concentration was then determined using
Supertaq polymerase. The polymerase starts to get inhibited at
around 3%, with an almost complete inhibition at 6% concentration.
For the second round of CSR the inhibitory concentration was set to
5%.
[0182] In the presence of the inhibitor the resulting clones soil3,
soil4 and soil5 appear to be twice (soil3, soil4) respectively
three times (soil5) more active than the wildtype Thermus aquaticus
polymerase.
[0183] All polymerases were stored in glycerol in liquid nitrogen.
Sequence CWU 1
1
1812502DNAArtificialSynthetic Polymerase 1atggcgatgc ttcccctctt
tgagcccaaa ggccgggtcc tcctggtgga cggccaccac 60ctggcctacc gcaccttctt
cgccctgaag ggcctcacca cgagccgggg cgaaccggtg 120caggcggtct
acggtttcgc caagagcctc ctcaaggccc tgaaggagga cgggtacaag
180gccgtcttcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc
ctacgaggcc 240tacaaggcgg ggagggcccc gacccccgag gacctccccc
ggcagctcgc cctcatcaag 300gagctggtgg acctcctggg gtttacccgc
ctcgaggtcc aaggctacga ggcggacgac 360gtcctcgcca ccctggccaa
gaaggcggaa aaagaagggt acgaggtgcg catcctcacc 420gccgaccggg
acctctacca gctcgtctcc gaccgcgtcg ccgtcctcca ccccgagggc
480cacctcatca ccccggagtg gctttgggag aagtacggcc tcaggccgga
gcagtgggtg 540gacttccgcg ccctcgtggg ggacccctcc gacaacctcc
ccggggtcaa gggcatcggg 600gagaagaccg ccctcaagct cctcaaggag
tggggaagcc tggaaaatct cctcaagaac 660ctggatcggg taaagccgga
aaacgtccgg gagaagatca aggcccacct ggaagacctc 720aggctctcct
tggagctctc ccgggtgcgt accgacctcc ccctggaggt ggacctcgcc
780caggggcggg agcccgaccg ggaagggctt agggccttcc tggagaggct
ggagttcggc 840agcctcctcc atgagttcgg ccttctggaa agccccaagg
ccctggagga ggccccctgg 900cccccaccgg aaggggcctt cgtgggcttt
gtgctttccc gcaaggagcc catgtgggcc 960gatcttctgg ccctggccgc
cgccaggggt ggtcgggtcc accgggcccc cgagccttat 1020aaagccctca
gggacttgaa ggaggcgcgg gggcttctcg ccaaagacct gagcgttctg
1080gccctaaggg aaggccttgg cctcccgccc ggcgacgacc ccatgctcct
cgcctacctc 1140ctggaccctt ccaacaccac ccccgagggg gtggcccggc
gctacggcgg ggagtggacg 1200gaggaggcgg gggagcgggc cgccctttcc
gagaggctct tcgccaacct gtgggggagg 1260cttgaggggg aggagaggct
cctttggctt taccgggagg tggataggcc cctttccgct 1320gtcctggccc
acatggaggc cacaggggtg cgcctggacg tggcctatct cagggccttg
1380tccctggagg tggccgagga gatcgcccgc ctcgaggccg aggtcttccg
cctggccggc 1440caccccttca acctcaactc ccgggaccag ctggaaaggg
tcctctttga cgagctaggg 1500cttcccgcca tcggcaagac ggagaggacc
ggcaagcgct ccaccagcgc cgccgtcctg 1560gaggccctcc gcgaggccca
ccccatcgtg gagaagatcc tgcagtaccg ggagctcacc 1620aagctgaaga
gcacctacat tgaccccttg ccggacctca tccaccccag gacgggccgc
1680ctccacaccc gcttcaacca gacggccacg gccacgggca ggctaagtag
ctccgatccc 1740aacctccaga acatccccgt ccgcaccccg cttgggcaga
ggatccgccg ggccttcatc 1800gccgaggagg ggtggctatt ggtggccctg
gactatagcc agatagagct cagggtgctg 1860gcccacctct ccggcgacga
gaacctgatc cgggtcttcc aggaggggcg ggacatccac 1920acggagaccg
ccagctggat gttcggtgtc cccccggagg ccgtggaccc cctgatgcgc
1980cgggcggcca agacggtgaa cttcggcgtc ctctacggca tgtccgccca
taggctctcc 2040caggagcttt ccatccccta cgaggaggcg gtggccttta
tagagcgcta cttccaaagc 2100ttccccaagg tgcgggcctg gatagaaaag
accctggagg aggggaggaa gcggggctac 2160gtggaaaccc tcttcggaag
aaggcgctac gtgcccgacc tcaacgcccg ggtgaagagc 2220gtcagggagg
ccgcggagcg catggccttc aacatgcccg tccagggcac cgccgccgac
2280ctcatgaagc tcgccatggt gaagctcttc ccccgcctcc gggagatggg
ggcccgcatg 2340ctcctccagg tccacgacga gctcctcctg gaggcccccc
aagcgcgggc cgaggaggtg 2400gcggctttgg ccaaggaggc catggagaag
gcctatcccc tcgccgtacc cctggaggtg 2460gaggtgggga tcggggagga
ctggctttcc gccaagggtt ag 25022833PRTArtificialSynthetic Polymerase
2Met Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val1 5
10 15Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly
Leu 20 25 30Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala Lys 35 40 45Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala
Val Phe Val 50 55 60Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu
Ala Tyr Glu Ala65 70 75 80Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu
Asp Leu Pro Arg Gln Leu 85 90 95Ala Leu Ile Lys Glu Leu Val Asp Leu
Leu Gly Phe Thr Arg Leu Glu 100 105 110Val Gln Gly Tyr Glu Ala Asp
Asp Val Leu Ala Thr Leu Ala Lys Lys 115 120 125Ala Glu Lys Glu Gly
Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg Asp 130 135 140Leu Tyr Gln
Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu Gly145 150 155
160His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser
Asp Asn 180 185 190Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala
Leu Lys Leu Leu 195 200 205Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu
Lys Asn Leu Asp Arg Val 210 215 220Lys Pro Glu Asn Val Arg Glu Lys
Ile Lys Ala His Leu Glu Asp Leu225 230 235 240Arg Leu Ser Leu Glu
Leu Ser Arg Val Arg Thr Asp Leu Pro Leu Glu 245 250 255Val Asp Leu
Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala 260 265 270Phe
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 275 280
285Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu
290 295 300Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met
Trp Ala305 310 315 320Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly
Arg Val His Arg Ala 325 330 335Pro Glu Pro Tyr Lys Ala Leu Arg Asp
Leu Lys Glu Ala Arg Gly Leu 340 345 350Leu Ala Lys Asp Leu Ser Val
Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365Pro Pro Gly Asp Asp
Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375 380Asn Thr Thr
Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr385 390 395
400Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn
405 410 415Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu
Tyr Arg 420 425 430Glu Val Asp Arg Pro Leu Ser Ala Val Leu Ala His
Met Glu Ala Thr 435 440 445Gly Val Arg Leu Asp Val Ala Tyr Leu Arg
Ala Leu Ser Leu Glu Val 450 455 460Ala Glu Glu Ile Ala Arg Leu Glu
Ala Glu Val Phe Arg Leu Ala Gly465 470 475 480His Pro Phe Asn Leu
Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe 485 490 495Asp Glu Leu
Gly Leu Pro Ala Ile Gly Lys Thr Glu Arg Thr Gly Lys 500 505 510Arg
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro 515 520
525Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser
530 535 540Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr
Gly Arg545 550 555 560Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala
Thr Gly Arg Leu Ser 565 570 575Ser Ser Asp Pro Asn Leu Gln Asn Ile
Pro Val Arg Thr Pro Leu Gly 580 585 590Gln Arg Ile Arg Arg Ala Phe
Ile Ala Glu Glu Gly Trp Leu Leu Val 595 600 605Ala Leu Asp Tyr Ser
Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615 620Gly Asp Glu
Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His625 630 635
640Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp
645 650 655Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val
Leu Tyr 660 665 670Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ser
Ile Pro Tyr Glu 675 680 685Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe
Gln Ser Phe Pro Lys Val 690 695 700Arg Ala Trp Ile Glu Lys Thr Leu
Glu Glu Gly Arg Lys Arg Gly Tyr705 710 715 720Val Glu Thr Leu Phe
Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn Ala 725 730 735Arg Val Lys
Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750Pro
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys 755 760
765Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln Val
770 775 780His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu
Glu Val785 790 795 800Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala
Tyr Pro Leu Ala Val 805 810 815Pro Leu Glu Val Glu Val Gly Ile Gly
Glu Asp Trp Leu Ser Ala Lys 820 825 830Gly
32502DNAArtificialSynthetic Polymerase 3atggcgatgc ttcccctctt
tgagcccaaa ggccgggtcc tcctggtgga cggccaccac 60ctggcctacc gcaccttctt
cgccctgaag ggcctcacca cgagccgggg cgaaccggtg 120caggcggtct
acggcttcgc caagagcctc ctcaaggccc tgaaggagga cgggtacaag
180gccgtcttcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc
ctacgaggcc 240tacaaggcgg ggagggcccc gacccccgag gacttccccc
ggcagctcgc cctcatcaag 300gagctggtgg acctcctggg gtttacccgt
ctcgaggtcc ccggctacga ggcggacgac 360gttctcgcca ccctggccaa
gaaggcggaa aaggaggggt acgaggtgcg catcctcacc 420gccgaccgcg
acctctacca actcgtctcc gaccgcgtcg ccgtcctcca ccccgagggc
480cacctcatca ccccggagtg gctttgggag aagtacggcc tcaggccgga
gcagtgggtg 540gacttccgcg ccctcgtggg ggacccctcc gacaacctcc
ccggggtcaa gggcatcggg 600gagaagaccg ccctcaagct cctcaaggag
tggggaagcc tggaaaacct cctcaagaac 660ctggaccggg taaagccaga
aaacgtccgg gagaagatca aggcccacct ggaagacctc 720aggctctcct
tggagctctc ccgggtgcgc accgacctcc ccctggaggt ggacctcgcc
780caggggcggg agcccgaccg ggaaaggctt agggcctttc tggagaggct
tgagtttggc 840agcctcctcc acgagttcgg ccttctggaa agccccaagg
ccctggagga ggccccctgg 900cccccgccgg aaggggcctt cgtgggcttt
gtgctttccc gcaaggcgcc catgtgggcc 960gatcttctgg ccctggccgc
cgccaggggt ggtcgggtct accgggcccc cgagccttat 1020aaagccctca
gggacttgaa ggaggcgcgg gggcttctcg ccaaagacct gagcgttctg
1080gccctaaggg aaggccttgg cctcccgccc ggcgacgacc ccatgctcct
cgcctacctc 1140ctggaccctt ccaacaccac ccccgagggg gtggcccggc
gctacggcgg ggagtggacg 1200gaggaggcgg gggagcgggc cgccctttcc
gagaggctct tcgccaacct gtgggggagg 1260cttgaggggg aggagaggct
cctttggctt taccgggagg tggataggcc cctttccgct 1320gtcctggccc
acatggaggc cacaggggtg cgcctggacg tggcctatct cagggccttg
1380tccctggagg tggccgagga gatcgcccgc ctcgaggccg aggtcttccg
cctggccggc 1440caccccttca acctcaactc ccgggaccag ctggaaaggg
tcctctttga cgagctaggg 1500cttcccgcca tcggcaagac ggagaagacc
ggcaagcgct ccaccagcgc cgccgtcctg 1560gaggccctcc gcgaggccca
ccccatcgtg gagaagatcc tgcagtaccg ggagctcacc 1620aagctgaaga
gcacctacat tgaccccttg ccggacctca tccaccccag gacgggccgc
1680ctccacaccc gcttcaacca gacggccacg gccacgggca ggctaagtag
ctccgatccc 1740aacctccaga acatccccgt ccgcaccccg ctcgggcaga
ggatccgccg ggccttcatc 1800gctgaggagg ggtggctatt ggtggtcctg
gactatagcc agatagagct cagggtgctg 1860gcccacctct ccggcgacga
gaacctgatc cgggtcttcc aggaggggcg ggacatccac 1920acggaaaccg
ccagctggat gttcggcgtc ccccgggagg ccgtggaccc cctgatgcgc
1980cgggcggcca agaccatcaa cttcggggtt ctctacggca tgtcggccca
ccgcctctcc 2040caggagctag ccatccctta cgaggaggcc cgggccttca
ttgagcgcta ctttcagagc 2100ttccccaagg tgcgggcctg gattgagaag
accctggagg agggcaggag gcgggggtac 2160gtggagaccc tcttcggccg
ccgccgctac gtgccagacc tagaggcccg ggtgaagagc 2220gtgcgggagg
cggccgagcg catggccttc aacatgcctg tccagggcac cgccgccgac
2280ctcatgaagc tggctatggt gaagctcttc cccaggctgg aggaaacggg
ggccaggatg 2340ctccttcagg tccacgacga gctggtcctc gagaccccaa
aagagagggc ggaggccgtg 2400gcccggctgg ccaaggaggt catggagggg
gtgtatcccc tggccgtgcc cctggaggtg 2460gaggtgggga taggggagga
ctggctctcc gccaaggagt ga 25024833PRTArtificialSynthetic Polymerase
4Met Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val1 5
10 15Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly
Leu 20 25 30Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala Lys 35 40 45Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala
Val Phe Val 50 55 60Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu
Ala Tyr Glu Ala65 70 75 80Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu
Asp Phe Pro Arg Gln Leu 85 90 95Ala Leu Ile Lys Glu Leu Val Asp Leu
Leu Gly Phe Thr Arg Leu Glu 100 105 110Val Pro Gly Tyr Glu Ala Asp
Asp Val Leu Ala Thr Leu Ala Lys Lys 115 120 125Ala Glu Lys Glu Gly
Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg Asp 130 135 140Leu Tyr Gln
Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu Gly145 150 155
160His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser
Asp Asn 180 185 190Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala
Leu Lys Leu Leu 195 200 205Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu
Lys Asn Leu Asp Arg Val 210 215 220Lys Pro Glu Asn Val Arg Glu Lys
Ile Lys Ala His Leu Glu Asp Leu225 230 235 240Arg Leu Ser Leu Glu
Leu Ser Arg Val Arg Thr Asp Leu Pro Leu Glu 245 250 255Val Asp Leu
Ala Gln Gly Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala 260 265 270Phe
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 275 280
285Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu
290 295 300Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys Ala Pro Met
Trp Ala305 310 315 320Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly
Arg Val Tyr Arg Ala 325 330 335Pro Glu Pro Tyr Lys Ala Leu Arg Asp
Leu Lys Glu Ala Arg Gly Leu 340 345 350Leu Ala Lys Asp Leu Ser Val
Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365Pro Pro Gly Asp Asp
Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375 380Asn Thr Thr
Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr385 390 395
400Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn
405 410 415Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu
Tyr Arg 420 425 430Glu Val Asp Arg Pro Leu Ser Ala Val Leu Ala His
Met Glu Ala Thr 435 440 445Gly Val Arg Leu Asp Val Ala Tyr Leu Arg
Ala Leu Ser Leu Glu Val 450 455 460Ala Glu Glu Ile Ala Arg Leu Glu
Ala Glu Val Phe Arg Leu Ala Gly465 470 475 480His Pro Phe Asn Leu
Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe 485 490 495Asp Glu Leu
Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys 500 505 510Arg
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro 515 520
525Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser
530 535 540Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr
Gly Arg545 550 555 560Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala
Thr Gly Arg Leu Ser 565 570 575Ser Ser Asp Pro Asn Leu Gln Asn Ile
Pro Val Arg Thr Pro Leu Gly 580 585 590Gln Arg Ile Arg Arg Ala Phe
Ile Ala Glu Glu Gly Trp Leu Leu Val 595 600 605Val Leu Asp Tyr Ser
Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615 620Gly Asp Glu
Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His625 630 635
640Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp
645 650 655Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val
Leu Tyr 660 665 670Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala
Ile Pro Tyr Glu 675 680 685Glu Ala Arg Ala Phe Ile Glu Arg Tyr Phe
Gln Ser Phe Pro Lys Val 690 695 700Arg Ala Trp Ile Glu Lys Thr Leu
Glu Glu Gly Arg Arg Arg Gly Tyr705 710 715 720Val Glu Thr Leu Phe
Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala 725 730 735Arg Val Lys
Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750Pro
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys 755 760
765Leu Phe Pro Arg Leu Glu Glu Thr Gly Ala Arg Met Leu Leu Gln Val
770 775 780His Asp Glu Leu Val Leu Glu Thr
Pro Lys Glu Arg Ala Glu Ala Val785 790 795 800Ala Arg Leu Ala Lys
Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val 805 810 815Pro Leu Glu
Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830Glu
52499DNAArtificialSynthetic Polymerase 5atgcgtggta tgcttcctct
ttttgagccc aagggccgcg tcctcctggt ggacggccac 60cacctggcct accgcacctt
ccacgccctg aagggcctca ccaccagccg gggggagccg 120gtgcaggcgg
tctacggctt cgccaagagc ctcctcaagg tcctcaagga ggacggggac
180gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc
ctacgggggg 240tacaaggcgg gccgggcccc cacgccggag gactttcccc
ggcaactcgc cctcatcaag 300gagctggtgg acctcctggg gctggcgcgc
ctcgaggtcc cgggctacga ggcggacgac 360gtcctggcca gcctggccaa
gaaggcggaa aaggagggct acgaggtccg catcctcacc 420gccgacaaag
acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg
480tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga
ccagtgggcc 540gactaccggg ccctgaccgg ggacgagtcc gacaaccttc
ccggggtcaa gggcatcggg 600gagaagacgg cgaggaagct tctggaggag
tgggggagcc tggaagccct cctcaagaac 660ctggaccggc tgaagcccgc
catccgggag aagatcctgg cccacatgga cgatctgaag 720ctctcctggg
acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa
780aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga
gtttggcagc 840ctcctccacg agttcggcct tctggaaagc cccaaggccc
tggaggaggc cccctggccc 900ccgccggaag gggccttcgt gggctttgtg
ctttcccgca aggagcccat gtgggccgat 960cttctggctc tggccgccgc
cagggggggc cgggtccacc gggcccccga gccttataaa 1020gccctcaggg
acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc
1080ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc
ctacctcctg 1140gacccttcca acaccacccc cgagggggtg gcccggcgct
acggcgggga gtggacggag 1200gaggcggggg agcgggccgc cctttccgag
aggctcttcg ccaacctgtg ggggaggctt 1260gagggggagg agaggctcct
ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320ctggcccaca
tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc
1380ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct
ggccggccac 1440cccttcaacc tcaactcccg ggaccagctg gaaagggtcc
tctttgacga gctagggctt 1500cccgccatcg gcaagacgga gaagaccggc
aagcgctcca ccagcgccgc cgtcctggag 1560gccctccgcg aggcccaccc
catcgtggag aagatcctgc agtaccggga gctcaccaag 1620ctgaagagca
cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc
1680cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc
cgatcccaac 1740ctccagaaca tccccgtccg caccccgctt gggcagagga
tccgccgggc cttcatcgcc 1800gaggaggggt ggctattggt ggccctggac
tatagccaga tagagctcag ggtgctggcc 1860cacctctccg gcgacgagaa
cctgatccgg gtcttccagg aggggcggga catccacacg 1920gagaccgcca
gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg
1980gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg
cctctcccag 2040gagctagcca tcccttacga ggaggcccag gccttcattg
agcgctactt tcagagcttc 2100cccaaggtgc gggcctggat tgagaagacc
ctggaggagg gcaggaggcg ggggtacgtg 2160gagaccctct tcggccgccg
ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220cgggaggcgg
ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc
2280atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc
caggatgctc 2340cttcaggtcc acgacgagct ggtcctcgag gccccaaaag
agagggcgga ggccgtggcc 2400cggctggcca aggaggtcat ggagggggtg
tatcccctgg ccgtgcccct ggaggtggag 2460gtggggatag gggaggactg
gctttccgcc aagggttag 24996832PRTArtificialSynthetic Polymerase 6Met
Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu1 5 10
15Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala 35 40 45Lys Ser Leu Leu Lys Val Leu Lys Glu Asp Gly Asp Ala Val
Ile Val 50 55 60Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala
Tyr Gly Gly65 70 75 80Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp
Phe Pro Arg Gln Leu 85 90 95Ala Leu Ile Lys Glu Leu Val Asp Leu Leu
Gly Leu Ala Arg Leu Glu 100 105 110Val Pro Gly Tyr Glu Ala Asp Asp
Val Leu Ala Ser Leu Ala Lys Lys 115 120 125Ala Glu Lys Glu Gly Tyr
Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140Leu Tyr Gln Leu
Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly145 150 155 160Tyr
Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170
175Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn
180 185 190Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys
Leu Leu 195 200 205Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn
Leu Asp Arg Leu 210 215 220Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala
His Met Asp Asp Leu Lys225 230 235 240Leu Ser Trp Asp Leu Ala Lys
Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255Asp Phe Ala Lys Arg
Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270Leu Glu Arg
Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285Glu
Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295
300Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala
Asp305 310 315 320Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val
His Arg Ala Pro 325 330 335Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys
Glu Ala Arg Gly Leu Leu 340 345 350Ala Lys Asp Leu Ser Val Leu Ala
Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365Pro Gly Asp Asp Pro Met
Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380Thr Thr Pro Glu
Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu385 390 395 400Glu
Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410
415Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala
Thr Gly 435 440 445Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser
Leu Glu Val Ala 450 455 460Glu Glu Ile Ala Arg Leu Glu Ala Glu Val
Phe Arg Leu Ala Gly His465 470 475 480Pro Phe Asn Leu Asn Ser Arg
Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495Glu Leu Gly Leu Pro
Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510Ser Thr Ser
Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525Val
Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535
540Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg
Leu545 550 555 560His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly
Arg Leu Ser Ser 565 570 575Ser Asp Pro Asn Leu Gln Asn Ile Pro Val
Arg Thr Pro Leu Gly Gln 580 585 590Arg Ile Arg Arg Ala Phe Ile Ala
Glu Glu Gly Trp Leu Leu Val Ala 595 600 605Leu Asp Tyr Ser Gln Ile
Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620Asp Glu Asn Leu
Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr625 630 635 640Glu
Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650
655Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr
Glu Glu 675 680 685Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe
Pro Lys Val Arg 690 695 700Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly
Arg Arg Arg Gly Tyr Val705 710 715 720Glu Thr Leu Phe Gly Arg Arg
Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735Val Lys Ser Val Arg
Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750Val Gln Gly
Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765Phe
Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775
780Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val
Ala785 790 795 800Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro
Leu Ala Val Pro 805 810 815Leu Glu Val Glu Val Gly Ile Gly Glu Asp
Trp Leu Ser Ala Lys Gly 820 825 83072499DNAArtificialSynthetic
Polymerase 7atggcgatgc ttcccctctt tgagcccaaa ggccgggtcc tcctggtgga
cggccactac 60ctggcctacc gcaccttctt cgccctgaag ggcctcacca cgagccgggg
cgaaccggtg 120caggcggtct acggcttcgc caagagcctc ctcaaggccc
tgaaggagga cgggtacaag 180gccgtcttcg tggtctttga cgccaaggcc
ccctccttcc gccacgaggc ctacgaggcc 240tacaaggcgg ggagggcccc
gacccccgag gacttccccc ggcagctcgc cctcatcaag 300gagctggtgg
acctcctggg gtttacccgc ctcgaggtcc ccggctacga ggcggacgac
360gttctcgcca ccctggccaa gaaggcggaa aaggaggggt acgaggtgcg
catcctcacc 420gccgaccgtg acctctacca actcgtctcc gaccgcgtcg
ccgtcctcca ccccgagggc 480cacctcatca ccccggagtg gctttgggag
aagtacggcc tcaggccgga gcagtgggtg 540gacttccgcg ccctcgtggg
ggacccctcc gacaacctcc ccggggtcaa gggcatcggg 600gagaagaccg
ccctcaagct cctcaaggag tggggaagcc tggaaaacct cctcaagaac
660ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga
cgatctgaag 720ctctcctggg acctggccaa ggtgcgcacc gacctgcccc
tggaggtgga cttcgccaaa 780aggcgggagc ccgaccggga gaggcttagg
gcctttctgg agaggcttga gtttggcagc 840ctcctccacg agttcggcct
tctggaaagc cccaaggccc tggaggaggc cccctggccc 900ccgccggaag
gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat
960cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga
gccttacaaa 1020gccctcaggg acctgaagga ggcgcggggg cttctcgcca
aagacctgag cgttctggcc 1080ctgagggaag gccttggcct cccgcccggc
gacgacccca tgctcctcgc ctacctcctg 1140gacccttcca acaccacccc
cgagggggtg gcccggcgct acggcgggga gtggacggag 1200gaggcggggg
agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt
1260gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct
ttccgctgtc 1320ctggcccaca tggaggccac gggggtgcgc ctggacgtgg
cctatctcag ggccttgtcc 1380ctggaggtgg ccgaggagat cgcccgcctc
gaggccgagg tcttccgcct ggccggccac 1440cccttcaacc tcaactcccg
ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500cccgccatcg
gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag
1560gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga
gctcaccaag 1620ctgaagagca cctacattga ccccttgccg gacctcatcc
accccaggac gggccgcctc 1680cacacccgct tcaaccagac ggccacggcc
acgggcaggc taagtagctc cgatcccaac 1740ctccagaaca tccccgtccg
caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800gaggaggggt
ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc
1860cacctctctg gcgacgagaa cctgatccgg gtcttccagg aggggcggga
catccacacg 1920gagaccgcca gctgggtgtt cggcgtcccc cgggaggccg
tggaccccct gatgcgccgg 1980gcggccaaga ccatcaactt cggggtcctc
tacggcatgt cggcccaccg cctctcccag 2040gagctagcca tcccttacga
ggaggcccag gccttcattg agcgctactt ccagagcttc 2100cccaaggtgc
gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg
2160gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt
gaagagcgtg 2220cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc
agggcaccgc cgccgacctc 2280atgaagctgg ctatggtgaa gctcttcccc
aggctggagg aaatgggggc caggatgctc 2340cttcaggtcc acgacgagct
ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400cggctggcca
aggaggtcct ggagggggtg tatcccctgg ccgtgcccct ggaggtggag
2460gtggggatag gggaggactg gctctccgcc aaggagtga
24998832PRTArtificialSynthetic Polymerase 8Met Ala Met Leu Pro Leu
Phe Glu Pro Lys Gly Arg Val Leu Leu Val1 5 10 15Asp Gly His Tyr Leu
Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly Leu 20 25 30Thr Thr Ser Arg
Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys 35 40 45Ser Leu Leu
Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe Val 50 55 60Val Phe
Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu Ala65 70 75
80Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu
Glu 100 105 110Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu
Ala Lys Lys 115 120 125Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu
Thr Ala Asp Arg Asp 130 135 140Leu Tyr Gln Leu Val Ser Asp Arg Val
Ala Val Leu His Pro Glu Gly145 150 155 160His Leu Ile Thr Pro Glu
Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175Glu Gln Trp Val
Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp Asn 180 185 190Leu Pro
Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu Leu 195 200
205Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp
Leu Lys225 230 235 240Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp
Leu Pro Leu Glu Val 245 250 255Asp Phe Ala Lys Arg Arg Glu Pro Asp
Arg Glu Arg Leu Arg Ala Phe 260 265 270Leu Glu Arg Leu Glu Phe Gly
Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285Glu Ser Pro Lys Ala
Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300Ala Phe Val
Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp305 310 315
320Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly
Leu Leu 340 345 350Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly
Leu Gly Leu Pro 355 360 365Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr
Leu Leu Asp Pro Ser Asn 370 375 380Thr Thr Pro Glu Gly Val Ala Arg
Arg Tyr Gly Gly Glu Trp Thr Glu385 390 395 400Glu Ala Gly Glu Arg
Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415Trp Gly Arg
Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430Val
Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440
445Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala
Gly His465 470 475 480Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu
Arg Val Leu Phe Asp 485 490 495Glu Leu Gly Leu Pro Ala Ile Gly Lys
Thr Glu Lys Thr Gly Lys Arg 500 505 510Ser Thr Ser Ala Ala Val Leu
Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525Val Glu Lys Ile Leu
Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540Tyr Ile Asp
Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu545 550 555
560His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu
Gly Gln 580 585 590Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp
Leu Leu Val Ala 595 600 605Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val
Leu Ala His Leu Ser Gly 610 615 620Asp Glu Asn Leu Ile Arg Val Phe
Gln Glu Gly Arg Asp Ile His Thr625 630 635 640Glu Thr Ala Ser Trp
Val Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655Leu Met Arg
Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670Met
Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680
685Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly
Tyr Val705 710 715 720Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro
Asp Leu Glu Ala Arg 725
730 735Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met
Pro 740 745 750Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met
Val Lys Leu 755 760 765Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met
Leu Leu Gln Val His 770 775 780Asp Glu Leu Val Leu Glu Ala Pro Lys
Glu Arg Ala Glu Ala Val Ala785 790 795 800Arg Leu Ala Lys Glu Val
Leu Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815Leu Glu Val Glu
Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825
83092502DNAArtificialSynthetic Polymerase 9atggcgatgc ttcccctctt
tgagcccaaa ggccgggtcc tcctggtgga cggccaccac 60ctggcctacc gcaccttctt
cgccctgaag ggcctcacca cgagccgggg cgaaccggtg 120caggcggtct
acggcttcgc caagagcctc ctcaaggccc tgaaggagga cgggtacaag
180gccgtcttcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc
ctacgaggcc 240tacaaggcgg ggagggcccc gacccccgag gacttccccc
ggcagctcgc cctcatcaag 300gagctggtgg acctcctggg gtttacccgc
ctcgaggtcc aaggctacga ggcggacgac 360gtcctcgcca ccctggccaa
gaaggcggaa aaagaagggt acgaggtgcg cgtcctcacc 420gccgaccggg
acctctacca gctcgtctcc gaccgcgtcg ccgtcctcca ccccgagggc
480cacctcatca ccccggagtg gctttgggag aagtacggcc tcaggccgga
gcagtgggtg 540gacttccgcg ccctcgtggg ggacccctcc gacaacctcc
ccggggtcaa gggcatcggg 600gagaagaccg ccctcaagct cctcaaggag
tggggaagcc tggaaaatct cctcaagaac 660ctggatcggg taaagccgga
aaacgtccgg gagaagatca aggcccacct ggaagacctc 720aggctctcct
tggagctctc ccgggtgcgc accgacctcc ccctggaggt ggacctcgcc
780caggggcggg agcccgaccg ggaagggctt agggccttcc tggagaggct
ggagttcggc 840agcctcctcc atgagttcgg ccttctggaa agccccaagg
ccctggagga ggccccctgg 900cccccgccgg aaggggcctt cgtgggcttt
gtgctttccc gcaaggagcc catgtgggcc 960gatcttctgg ccctggccgc
cgccaggggg ggccgggtcc accgggcccc cgagccttat 1020aaagccctca
gggacctgaa ggaggcgcgg gggcttctcg ccaaagacct gagcgttctg
1080gccctgaggg aaggccttgg cctcccgccc gccgacgacc ccatgctcct
cgcctacctc 1140ctggaccctt ccaacaccac ccccgagggg gtggcccggc
gctacggcgg ggagtggacg 1200gaggaggcgg gggagcgggc cgccctttcc
gagaggctct tcgccaacct gtgggggagg 1260cttgaggggg aggagaggct
cctttggctt taccgggagg tggagaggcc cctttccgct 1320gccctggccc
acatggaggc cacgggggtg cgcctggacg tggcctatct cagggccttg
1380tccctggagg tggccgagga gatcgcccgc ctcgaggccg aggtcttccg
cctggccggc 1440caccccttca acctcaactc ccgggaccag ctggaaaggg
tcctctttga cgagctaggg 1500cttcccgcca tcggcaagac ggagaagacc
ggcaagcgct ccaccagcgc cgccgtcctg 1560gaggccctcc gcgaggccca
ccccatcgtg gagaagatcc tgcagtaccg ggagctcacc 1620aagctgaaga
gcacctacat tgaccccttg ccggacctca tccaccccag gacgggccgc
1680ctccacaccc gcttcaacca gacggccacg gccacgggca ggctaagtag
ctccgatccc 1740aacctccaga acatccccgt ccgcaccccg cttgggcaga
ggatccgccg ggccttcatc 1800gccgaggagg ggtggctatt ggtggccctg
gactatagcc agatagagct cagggtgctg 1860gcccacctct ccggcgacga
gaacctgatc cgggtcttcc aggaggggcg ggacatccac 1920acggagaccg
ccagctggat gttcggcgtc ccccgggagg ccgtggaccc cctgatgcgc
1980cgggcggcca agaccatcaa cttcggggtc ctctacggca tgtcggccca
ccgcctctcc 2040caggagctag ccatccctta cgaggaggcc caggccctca
ttgagcgcta cttccagagc 2100ttccccaagg tgcgggcctg gattgagaag
accctggagg agggcaggag gcgggggtac 2160gtggagaccc tcctcggccg
ccgccgctac gtgccagacc tagaggcccg ggtgaagagc 2220gtgcgggagg
cggccgagcg catggccttc aacatgcccg tccagggcac cgccgccgac
2280ctcatgaagc tggctatggt gaagctcttc cccaggctgg aggaaatggg
ggccaggatg 2340ctccttcagg tccacgacga gctggtcctc gaggccccaa
aagagagggc ggaggccgtg 2400gcccggctgg ccaaggaggt catggagggg
gtgtatcccc tggccgtgcc cctggaggtg 2460gaggtgggga taggggagga
ctggctctcc gccaaggagt ga 250210833PRTArtificialSynthetic Polymerase
10Met Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val1
5 10 15Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly
Leu 20 25 30Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe
Ala Lys 35 40 45Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala
Val Phe Val 50 55 60Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu
Ala Tyr Glu Ala65 70 75 80Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu
Asp Phe Pro Arg Gln Leu 85 90 95Ala Leu Ile Lys Glu Leu Val Asp Leu
Leu Gly Phe Thr Arg Leu Glu 100 105 110Val Gln Gly Tyr Glu Ala Asp
Asp Val Leu Ala Thr Leu Ala Lys Lys 115 120 125Ala Glu Lys Glu Gly
Tyr Glu Val Arg Val Leu Thr Ala Asp Arg Asp 130 135 140Leu Tyr Gln
Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu Gly145 150 155
160His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser
Asp Asn 180 185 190Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala
Leu Lys Leu Leu 195 200 205Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu
Lys Asn Leu Asp Arg Val 210 215 220Lys Pro Glu Asn Val Arg Glu Lys
Ile Lys Ala His Leu Glu Asp Leu225 230 235 240Arg Leu Ser Leu Glu
Leu Ser Arg Val Arg Thr Asp Leu Pro Leu Glu 245 250 255Val Asp Leu
Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala 260 265 270Phe
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 275 280
285Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu
290 295 300Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met
Trp Ala305 310 315 320Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly
Arg Val His Arg Ala 325 330 335Pro Glu Pro Tyr Lys Ala Leu Arg Asp
Leu Lys Glu Ala Arg Gly Leu 340 345 350Leu Ala Lys Asp Leu Ser Val
Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365Pro Pro Ala Asp Asp
Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375 380Asn Thr Thr
Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr385 390 395
400Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn
405 410 415Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu
Tyr Arg 420 425 430Glu Val Glu Arg Pro Leu Ser Ala Ala Leu Ala His
Met Glu Ala Thr 435 440 445Gly Val Arg Leu Asp Val Ala Tyr Leu Arg
Ala Leu Ser Leu Glu Val 450 455 460Ala Glu Glu Ile Ala Arg Leu Glu
Ala Glu Val Phe Arg Leu Ala Gly465 470 475 480His Pro Phe Asn Leu
Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe 485 490 495Asp Glu Leu
Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys 500 505 510Arg
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro 515 520
525Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser
530 535 540Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr
Gly Arg545 550 555 560Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala
Thr Gly Arg Leu Ser 565 570 575Ser Ser Asp Pro Asn Leu Gln Asn Ile
Pro Val Arg Thr Pro Leu Gly 580 585 590Gln Arg Ile Arg Arg Ala Phe
Ile Ala Glu Glu Gly Trp Leu Leu Val 595 600 605Ala Leu Asp Tyr Ser
Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615 620Gly Asp Glu
Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His625 630 635
640Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp
645 650 655Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val
Leu Tyr 660 665 670Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala
Ile Pro Tyr Glu 675 680 685Glu Ala Gln Ala Leu Ile Glu Arg Tyr Phe
Gln Ser Phe Pro Lys Val 690 695 700Arg Ala Trp Ile Glu Lys Thr Leu
Glu Glu Gly Arg Arg Arg Gly Tyr705 710 715 720Val Glu Thr Leu Leu
Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala 725 730 735Arg Val Lys
Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750Pro
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys 755 760
765Leu Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val
770 775 780His Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu
Ala Val785 790 795 800Ala Arg Leu Ala Lys Glu Val Met Glu Gly Val
Tyr Pro Leu Ala Val 805 810 815Pro Leu Glu Val Glu Val Gly Ile Gly
Glu Asp Trp Leu Ser Ala Lys 820 825 830Glu
1123DNAArtificialSynthetic Oligonucleotide Primer 11aaaaatctag
ataacgaggg caa 231228DNAArtificialSynthetic Oligonucleotide Primer
12accaccgaac tgcgggtgac gccaagcg 281345DNAArtificialSynthetic
Oligonucleotide Primer 13gtaaaacgac ggccagtacc accgaactgc
gggtgacgcc aagcg 451440DNAArtificialSynthetic Oligonucleotide
14caggaaacag ctatgacaaa aatctagata acgagggcaa
401517DNAArtificialSynthetic Oligonucleotide Primer 15caggaaacag
ctatgac 171617DNAArtificialSynthetic Oligonucleotide Primer
16gtaaaacgac ggccagt 171726DNAArtificialSynthetic Oligonucleotide
Primer 17accaccgaac tgcgggtgac gccaag 261825DNAArtificialSynthetic
Oligonucleotide Primer 18gggtacgtgg agaccctctt cggcc 25
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