U.S. patent application number 10/770989 was filed with the patent office on 2005-01-27 for thermostable dna polymerase from anaerocellum thermophilum.
This patent application is currently assigned to Roche Diagnostics GMBH. Invention is credited to Angerer, Bernhard, Ankenbauer, Waltraud, Bonch-Osmolovskaya, Elizaveta, Markau, Ursula, Reiser, Astrid, Schmitz-Agheguian, Gudrun, Svetlichny, Vitaly.
Application Number | 20050019789 10/770989 |
Document ID | / |
Family ID | 8223264 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019789 |
Kind Code |
A1 |
Ankenbauer, Waltraud ; et
al. |
January 27, 2005 |
Thermostable DNA polymerase from anaerocellum thermophilum
Abstract
A thermostable enzyme is provided which is derived from the
microorganism Anaerocellum thermophilum. The enzyme has a molecular
weight of 96 to 100 kDa, shows DNA polymerase activity and reverse
transcriptase activity in the presence of magnesium ions. The
enzyme may be native or recombinant, and may be used with selected
primers and nucleoside triphosphates in a temperature cycling
polymerase chain reaction on DNA or RNA as template with or without
additional DNA polymerases as an enzyme mixture.
Inventors: |
Ankenbauer, Waltraud;
(Penzberg, DE) ; Schmitz-Agheguian, Gudrun;
(Bernried, DE) ; Bonch-Osmolovskaya, Elizaveta;
(Moscow, RU) ; Svetlichny, Vitaly; (Bayreuth,
DE) ; Markau, Ursula; (Polling, DE) ; Angerer,
Bernhard; (Rosenheim, DE) ; Reiser, Astrid;
(Antdorf, DE) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
2 EMBARCADERO CENTER, 8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
Roche Diagnostics GMBH
Mannheim
DE
|
Family ID: |
8223264 |
Appl. No.: |
10/770989 |
Filed: |
February 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10770989 |
Feb 2, 2004 |
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09269858 |
Jun 10, 1999 |
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6692932 |
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09269858 |
Jun 10, 1999 |
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PCT/EP97/05390 |
Oct 1, 1997 |
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Current U.S.
Class: |
435/134 ;
435/199; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1252
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/252.3; 435/320.1; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/22; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 1996 |
EP |
96 115 877.1 |
Claims
1-21. (Cancelled)
22. An isolated DNA encoding a purified thermostable DNA polymerase
obtainable from Anaerocellum thermophilum that catalyses the
template directed polymerisation of DNA, possesses 5'-3'polymerase
activity, lacks 3'-5'-exonuclease activity, has a
magnesium-dependent reverse transcriptase activity, and has a
manganese-dependent reverse transcriptase activity.
23. The isolated DNA sequence of claim 22 that has the nucleotide
sequence shown in SEQ ID NO. 7.
24. A vector containing the isolated DNA sequence of claim 23.
25. A microbial host transformed with the vector of claim 26.
Description
[0001] The present invention relates to a thermostable enzyme which
is a DNA polymerase obtainable from Anaerocellum thermophilum.
[0002] Heat stable DNA polymerases (EC 2.7.7.7. DNA
nucleotidyltransferase, DNA-directed) have been isolated from
numerous thermophilic organisms (for example: Kaledin et al., 1980,
Bio-kimiya Vol. 45, p. 644-651; Kaledin et al., 1981, Biokimiya
Vol. 46, p. 1247-1254; Kaledin et al., 1982, Biokimiya Vol. 47, p.
1515-1521; Ruttimann, et al., 1985, Eur. J. Biochem. Vol. 149, p.
41-46; Neuner et al., 1990, Arch. Microbiol. Vol. 153, p.
205-207.)
[0003] For some organisms, the polymerase gene has been cloned and
expressed (Lawyer et al., 1989, J. Biol. Chem. Vol. 264, p.
6427-6437; Engelke et al., 1990, Anal. Biochem. Vol. 191, p.
396-400; Lundberg et al., 1991, Gene, Vol. 108, p. 1-6; Kaledin et
al., 1980 Biokimiya Vol. 44, p. 644-651; Kaledin et al., 1981,
Biokimiya Vol. 46, p. 1247-1254; Kaledin et al., 1982, Biokimiya
Vol. 47, p. 1515-1521; Ruttimann, et al., 1985, Eur. J. Biochem.
Vol. 149, p. 41-46; Neuner et al., 1990, Arch. Microbiol. Vol. 153,
p. 205-207; Perler et al., 1992, Proc. Natl. Acad. Sci. USA Vol.
89, p. 5577).
[0004] Thermophilic DNA polymerases are increasingly becoming
important tools for use in molecular biology and there is growing
interest in finding new polymerases which have more suitable
properties and activities for use in diagnostic detection of RNA
and DNA, gene cloning and DNA sequencing. At present, the
thermophilic DNA polymerases mostly used for these purposes are
from Thermus species like Taq polymerase from T. aquaticus (Brock
et al 1969, J. Bacteriol. Vol. 98, p. 289-297).
[0005] Reverse transcription is commonly performed with viral
reverse transcriptases like the enzymes isolated from Avian
myeloblastosis virus or Moloney murine leukemia virus, which are
active in the presence of Magnesium ions but have the disadvantages
to possess RNase H-activity, which destroys the template RNA during
the reverse transcription reaction and have a temperature optimum
at 42.degree. C. or 37.degree. C., respectively.
[0006] Alternative methods are described using the reverse
transcriptase activity of DNA polymerases of thermophilic organisms
which are active at higher temperatures. Reverse transcription at
higher temperatures is of advantage to overcome secondary
structures of the RNA template which could result in premature
termination of products. Thermostable DNA polymerases with reverse
transcriptase activities are commonly isolated from Thermus
species. These DNA polymerases however, show reverse transcriptase
activity only in the presence of Manganese ions. These reaction
conditions are suboptimal, because the presence of Manganese ions
lowers the fidelity of the DNA polymerase transcribing the template
RNA.
[0007] Therefore, it is desirable to develop a reverse
transcriptase which acts at higher temperatures to overcome
secondary structures of the template and is active in the presence
of Magnesium ions in order to prepare cDNA from RNA templates with
higher fidelity.
[0008] The present invention addresses these needs and provides a
purified DNA polymerase enzyme (EC 2.7.7.7.) active at higher
temperatures which has reverse transcriptase activity in the
presence of magnesium ions. The invention comprises a DNA
polymerase isolated from Anaerocellum thermophilum DSM 8995,
deposited on the Deutsche Sammlung von Mikro-organismen und
Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. In a
further aspect the invention comprises a DNA polymerase that
catalyses the template directed polymerisation of DNA and posess
5'-3'-polymerase activity, 5'-3'-exonuclease activity and no
substantial 3'-5'-exonuclease activity.
[0009] The polymerase according to the present invention retains at
least 90% of its activity after incubation for 30 Minutes at
80.degree. C. in absence of stabilizing detergents.
[0010] In a further aspect the invention comprises a DNA polymerase
having a molecular mass of about 96 to 100 kDa as determined by in
situ activity PAGE analysis.
[0011] In a further aspect the invention comprises a DNA polymerase
having reverse transcriptase activity in the presence of magnesiums
ions and in the substantial absence of manganese ions. The
polymerase according to the present invention exhibits a Mg.sup.2+
dependent reverse transcriptase activity of more than 30% relative
to the DNA polymerase activity which is set to 100%. In a further
aspect the present invention comprises a thermostable DNA
polymerase wherein said polymerase exhibits a reverse transcriptase
activity which is Mn.sup.2+ dependent. The Mn.sup.2+ dependent
reverse transcriptase activity is more than 60% relative to the DNA
polymerase activity.
[0012] In a further aspect the invention comprises a thermostable
reverse transcriptase. The thermostable reverse transcriptase
retains more than 80% after incubation for 60 minutes at 80.degree.
C.
[0013] Moreover, DNA encoding the 96.000-100.000 daltons
thermostable DNA polymerase obtainable from Anearocellum
thermophilum has been isolated and which allows to obtain the
thermostable enzyme of the present invention by expression in
E.coli. The entire Anearocellum thermophilum DNA polymerase coding
sequence is depicted below as SEQ ID NO. 7. The recombinant
Anearocellum thermophilum DNA polymerase also possesses
5'-3'polymerase activity, no substantial 3'-5'-exonuclease
activity, 5'-3'-exonuclease activity and a reverse transcriptase
activity which is a Mg.sup.2+ dependent.
[0014] Anaerocellum thermophilum was isolated from a hot spring in
the Valley of Geysers in Kamchatka (V. Svetlichny et al.
Mikrobilogiya, Vol. 59, No. 5 p. 871-879, 1990). Anaerocellum
thermophilum was deposited with the Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124
Braunschweig under the terms of the Budapest Treaty and received
DSM Accession Number 8995. The thermostable polymerase isolated
from Anaerocellum thermophilum has a molecular weight of 96 to 100
kDa and retains more than 90% of activity after heating to
80.degree. C. for 30 minutes in absence of stabilizing detergents.
The thermostable enzyme possesses a 5'-3' polymerase activity and a
reverse transcriptase activity which is Mn.sup.++ as well as
Mg.sup.++-dependent. The thermostable enzyme may be native or
recombinant and may be used for first and second strand cDNA
synthesis, in cDNA cloning, DNA sequencing, DNA labeling and DNA
amplification.
[0015] The present invention provides improved methods for the
replication and amplification of deoxyribonucleic (DNA) and
ribonucleic acid (RNA) sequences. These improvements are achieved
by the discovery and application of previously unknown properties
of thermoactive DNA polymerases. In a preferred embodiment, the
invention provides a method for synthesizing a complementary DNA
copy from an RNA template with a thermoreactive DNA polymerase. In
another aspect, the invention provides methods for amplifying a DNA
segment from an RNA or DNA template using a thermostable DNA
polymerase (RT-PCR or PCR).
[0016] The term "reverse transcriptase" describes a class of
polymerases characterized as RNA-dependent DNA polymerases. All
known reverse transcriptases require a primer to synthesize a DNA
transcript from an RNA template. Historically, reverse
transcriptase has been used primarily to transcribe MRNA into cDNA
which can then be cloned into a vector for further
manipulation.
[0017] For recovering the native protein Anaerocellum thermophilum
may be grown using any suitable technique, such as the technique
described by Svetlichny et al., 1991, System. Appl. Microbiol. Vol.
14, p. 205-208. After cell growth one preferred method for
isolation and purification of the enzyme is accomplished using the
multi-step process as follows:
[0018] The cells are thawed, suspended in buffer A (40 mM Tris-HCl,
pH 7.5, 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 0.4 M NaCl, 10 mM
Pefabloc.TM. SC (4-(2-Aminoethyl)-benzolsulfonyl-fluorid,
Hydrochlorid) and lysed by twofold passage through a Gaulin
homogenizer. The raw extract is cleared by centrifugation, the
supernatant dialyzed against buffer B (40 mM Tris-HCl, pH 7.5, 0.1
mM EDTA, 7 mM 2-mercaptoethanol, 10% Glycerol) and applied onto a
column filled with Heparin-Sepharose (Pharmacia). In each case the
columns are equilibrated with the starting solvent and after
application of the sample the columns are washed with the threefold
of their volume with this solvent. Eluation of the first column is
performed with a linear gradient of 0 to 0.5 M NaCl in Buffer B.
The fractions showing polymerase activity are pooled and ammonium
sulfate is added to a final concentration of 20%. This solution is
applied to a hydrophobic column containing Butyl-TSK-Toyopearl
(TosoHaas). This column is eluted with a falling gradient of 20 to
0% ammonium sulfate. The pool containing the activity is dialyzed
and again transferred to a column of DEAE-Sepharose (Pharmacia) and
eluted with a linear gradient of 0-0.5 M NaCl in buffer B. The
fourth column contains Tris-Acryl-Blue (Biosepra) and is eluted as
in the preceding case. Finally the active fractions are dialyzed
against buffer C (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7.0 mM
2-mercaptoethanol, 100 mM NaCl, 50% Glycerol).
[0019] DNA polymerase activity was either measured by incorporation
of .sup.32P-dCTP or by incorporation of digoxigenin labeled dUTP
into the synthesized DNA. Detection and quantification of the
incorporated digoxigenin was performed essentially as described in
Holtke, H.-J.; Sagner, G. Kessler, C. and Schmitz, G., 1992,
Biotechniques Vol. 12, p. 104-113.
[0020] Reverse transcriptase activity was measured using oligo dT
primed poly A template by incorporation of either .sup.32P-dTTP or
digoxigenin-labeled dUTP into the complementary strand. Detection
of the incorporated digoxigenin was perfomed in analogy to the
procedure used for detection of DNA polymerase activity.
[0021] In situ PAGE analysis of polymerase activity and reverse
transcriptase activity was performed essentially according to the
method described by Spanos A. and Hubscher U., 1983, Methods in
Enzymology Vol. 91 p. 263-277. Some minor, but essential
modifications to the original method are, that the renaturation of
the SDS-denatured polypeptides is performed in the presence of
magnesium ions (3 mM) and dATP (0.5-1 .mu.M) to assist
refolding.
[0022] The thermostable enzyme of this invention may also be
produced by recombinant DNA techniques, as the gene encoding this
enzyme has been cloned from Anaerocellum thermophilum genomic DNA.
In a further aspect the invention includes a recombinant plasmid
comprising the vector pASK75 carrying the Anaerocellum thermophilum
DNA polymerase gene and designated pAR10.
[0023] The isolation of the recombinant clone expressing DNA
polymerase from Anaerocellum thermophilum includes the following
steps: chromosomal DNA from Anaerocellum thermophilum is isolated
by treating the cells with detergent e.g. SDS and a proteinase e.g.
Proteinase K. The solution is extracted with phenol and chloroform
and the DNA purified by precipitation with ethanol. The DNA is
dissolved in Tris/EDTA buffer and the gene encoding the DNA
polymerase is specifically amplified by the PCR technique using two
mixed oligonucleotides (primer 1 and 2). These oligonucleotides,
described in SEQ ID NO.: 1 and SEQ ID NO.: 2, were designed on the
basis of conserved regions of family A DNA polymerases as published
by Braithwaite D. K. and Ito J., 1993, Nucl. Acids Res. Vol. 21, p.
787-802. The specifically amplified fragment is ligated into an
vector, preferably the pCR.TM.II vector (Invitrogen) and the
sequence is determined by cycle-sequencing. Complete isolation of
the coding region and the flanking sequences of the DNA polymerase
gene can be performed by restriction fragmentation of the
Anaerocellum thermophilum DNA with another restriction enzyme as in
the first round of screening and by inverse PCR (Innis et al.,
(1990) PCR Protocols; Academic Press, Inc., p. 219-227). This can
be accomplished with synthesized oligonucleotide primers binding at
the outer DNA sequences of the gene part but in opposite
orientation. These oligonucleotides, described by SEQ ID Nos. 3 and
4, were designed on the basis of the sequences which were
determined by the first above described PCR. As template
Anaerocellum thermophilum DNA is used which is cleaved by
restriction digestion and circularized by contacting with T4 DNA
ligase. To isolate the coding region of the whole polymerase gene,
another PCR is performed using primers as shown in SEQ ID Nos. 5
and 6 to amplify the complete DNA polymerase gene directly from
genomic DNA and introducing ends compatible with the linearized
expression vector.
1 Primer 1: 5'-WSN GAY AAY ATH CCN GGN GT-3': SEQ ID NO. 1 Primer
2: 5'-NCC NAC YTC NAC YTC NAR NGG-3': SEQ ID NO. 2 Primer 3: 5'-CAA
TTC AGG GCA GTG CTG CTG ATA SEQ ID NO. 3 TC-3': Primer 4: 5'-GAG
CTT CTG GGC ACT CTT TTC GCC- SEQ ID NO. 4 3': Primer 5: 5'-CGA ATT
CGG CCG TCA TGA AAC TGG SEQ ID NO. 5 TTA TAT TCG ATG GAA ACA G-3':
Primer 6: 5'-CGA ATT GGA TCC GTT TTG TCT CAT SEQ ID NO. 6 ACC AGT
TCA GTC CTT C-3':
[0024] The gene is operably linked to appropriate control sequences
for expression in either prokaryotic or eucaryotic host/vector
systems. The vector preferably encodes all functions required for
transformation and maintenance in a suitable host, and may encode
selectable markers and/or control sequences for polymerase
expression. Active recombinant thermostable polymerase can be
produced by transformed host cultures either continuously or after
induction of expression. Active thermostable polymerase can be
recovered either from host cells or from the culture media if the
protein is secreted through the cell membrane.
[0025] It is also preferable that Anaerocellum thermophilum
thermostable polymerase expression is tightly controlled in E. coli
during cloning and expression. Vectors useful in practicing the
present invention should provide varying degrees of controlled
expression of Anaerocellum thermophilum polymerase by providing
some or all of the following control features: (1) promoters or
sites of initiation of transcription, either directly adjacent to
the start of the polymerase gene or as fusion proteins, (2)
operators which could be used to turn gene expression on or off,
(3) ribosome binding sites for improved translation, and (4)
transcription or translation termination sites for improved
stability. Appropriate vectors used in cloning and expression of
Anaerocellum thermophilum polymerase include, for example, phage
and plasmids. Example of phage include lambda gt11 (Promega),
lambda Dash (Stratagene) lambda ZapII (Stratagene). Examples of
plasmids include pBR322, pBTac2 (Boehringer Mannheim), pBluescript
(Stratagene), pET3A (Rosenberg, A. H. et al., (1987) Gene
56:125-135), pASK75 (Biometra) and pET11C (Studier, F. W. et al.
(1990) Methods in Enzymology, 185:60-89). According to the present
invention the use of a plasmid has shown to be advantageously,
particularly pASK75 (Biometra). The Plasmid pASK75 carrying the
Anaerocellum thermophilum DNA polymerase gene is then designated
pAR10.
[0026] Standard protocols exist for transformation, phage infection
and cell culture (Maniatis, et al. (1982) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory Press). Of the
numerous E. coli strains which can be used for plasmid
transformation, the preferred strains include JM110 (ATCC 47013),
LE392 pUBS 520 (Maniatis et al. supra; Brinkmann et al., (1989)
Gene 85:109-114;), JM101 (ATCC No. 33876), XL1 (Stratagene), and
RR1 (ATCC no. 31343), and BL21 (DE3) plysS (Studier, F. W. et al.,
(1990) Methods in Enzymology, supra). According to the present
invention the use of the E. coli strain LE392 pUBS 520 has shown to
be advantageously. The E. coli strain LE392 pUBS 520 transformed
with the plasmid pASK75 carrying the Anaerocellum thermophilum DNA
polymerase gene (designated pAR10) is then designated E. coli AR220
(DSM No. 11177). E. coli strain XL1. Blue (Stratagene) is among the
strains that can be used for lambda phage, and Y1089 can be used
for lambda gt11 lysogeny. The transformed cells are preferably
grown at 37.degree. C. and expression of the cloned gene is induced
with anhydrotetracycline.
[0027] Isolation of the recombinant DNA polymerase can be performed
by standard techniques. Separation and purification of the DNA
polymerase from the E. coli extract can be performed by standard
methods. These methods include, for example, methods utilizing
solubility such as salt precipitation and solvent precipitation,
methods utilizing the difference in molecular weight such as
dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide
gel electrophoresis, methods utilizing a difference in electric
charge such as ion-exchange column chromatography, methods
utilizing specific interaction such as affinity chromatography,
methods utilizing a difference in hydrophobicity such as
reversed-phase high performance liquid chromatography and methods
utilizing a difference in isoelectric point such as isoelectric
focussing electrophoresis.
[0028] The thermostable enzyme of this invention may be used for
any purpose in which such enzyme activity is necessary or desired.
In a particularly preferred embodiment, the enzyme catalyzes the
nucleic acid amplification reaction known as PCR. This process for
amplifying nucleic acid sequences is disclosed and claimed in EP 0
201 189. The PCR nucleic acid amplification method involves
amplifying at least one specific nucleic acid sequence contained in
a nucleic acid or a mixture of nucleic acids and produces double
stranded DNA. Any nucleic acid sequence, in purified or nonpurified
form, can be utilized as the starting nucleic acid(s), provided it
contains or is suspected to contain the specific nucleic acid
sequence desired. The nucleic acid to be amplified can be obtained
from any source, for example, from plasmids such as pBR322, from
cloned DNA or RNA, from natural DNA or RNA from any source,
including bacteria, yeast, viruses, organelles, and higher
organisms such as plants and animals, or from preparations of
nucleic acids made in vitro. DNA or RNA may be extracted from
blood, tissue material such as chorionic villi, or amniotic cells
by a variety of techniques. See, e.g., Maniatis T. et al., 1982,
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.) pp. 280-281. Thus the process
may employ, for example, DNA or RNA, including messenger RNA, which
DNA or RNA may be single-stranded or double-stranded. In addition,
a DNA-RNA hybrid which contains one strand of each may be
utilized.
[0029] The amplification of target sequences in DNA or from RNA may
be performed to proof the presence of a particular sequence in the
sample of nucleic acid to be analyzed or to clone a specific gene.
DNA polymerase from Anaerocellum thermophilum is very useful for
these processes. Due to the fact that the DNA polymerase from
Anaerocellum thermophilum requires Mg.sup.++ ions as a cofactor
instead of Mn.sup.++ like the other DNA polymerases from
thermophilic organisms with reverse transcriptase activity of the
state of the art the RNA templates can be copied with higher
fidelity. These properties make DNA polymerase from Anaerocellum
thermophilum a very useful tool for the molecular biologist.
[0030] DNA polymerase from Anaerocellum thermophilum may also be
used to simplify and improve methods for detection of RNA target
molecules in a sample. In these methods DNA polymerase from
Anaerocellum thermophilum catalyzes: (a) reverse transcription, (b)
second strand cDNA synthesis, and, if desired, (c) amplification by
PCR. The use of DNA polymerase from Anaerocellum thermophilum in
the described methods eliminates the previous requirement of two
sets of incubation conditions which were necessary due to the use
of different enzymes for each step. The use of DNA polymerase from
Anaerocellum thermophilum provides RNA reverse transcription and
amplification of the resulting complementary DNA with enhanced
specificity and with fewer steps than previous RNA cloning and
diagnostic methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows a photograph of a DNA polymerase assay
performed in situ. The DNA polymerase activity of DNA polymerase
from Anaerocellum thermophilum is analysed in comparison with DNA
polymerase I and Klenow fragment of E. coli and DNA polymerase from
Thermus thermophilus. A fraction of DNA polymerase from
Anaerocellum thermophilum was submitted to electrophoresis on a
SDS-polyacrylamide gel containing activated (DNAseI treated) DNA.
After electrophoresis the SDS was removed, the proteins were
renatured over night and incubated at 72.degree. C. in the presence
of magnesium salt, dNTPs and digoxigenin labeled dUTPs to allow
synthesis of the complementary strand. The nucleic acid was blotted
to a nylon membrane and the newly synthesized DNA detected by a
chemiluminescence reaction.
[0032] As control proteins DNA polymerase I and Klenow fragment of
E. coli and DNA polymerase from Thermus thermophilus were analyzed
on the same gel. Using these proteins as standards the apparent
molecular weight of DNA polymerase from Anaerocellum thermophilum
of 96.000 to 100.000 Daltons can be deduced.
[0033] FIG. 2 shows results obtained from assays determining the
relative activity of the reverse transcriptase in dependence of
varying concentrations of magnesium and manganese ions.
[0034] FIG. 3 shows the thermostability of DNA polymerase from
Anaerocellum thermophilum. Aliquots of the DNA polymerase were
incubated at 80.degree. C. and the activity measured at the times
indicated in the figure.
[0035] FIG. 4 shows the DNA sequence (SEQ ID No. 7) of the
polymerase gene of Anaerocellum thermophilum and the derived
peptide sequence (SEQ ID No. 8) for Anaerocellum thermophilum
polymerase.
[0036] FIG. 5 shows the comparison ot the reverse transcriptase
activity of Anaerocellum thermophilum polymerase with Thermus
filiformis and Thermus thermophilus.
EXAMPLE 1
[0037] Isolation of DNA Polymerase
[0038] For recovering the native protein Anaerocellum thermophilum
may be grown using any suitable technique, such as the technique
described by Svetlichny et al., 1991, System. Appl. Microbiol. Vol.
14, p. 205-208. After cell growth one preferred method for
isolation and purification of the enzyme is accomplished using the
multi-step process as follows:
[0039] The cells are thawed, suspended in buffer A (40 mM Tris-HCl,
pH 7.5, 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 0.4 M NaCl, 10 mM
Pefabloc.TM. SC (4-(2-Aminoethyl)-benzolsulfonyl-fluorid,
Hydrochlorid) and lysed by twofold passage through a Gaulin
homogenizer. The raw extract is cleared by centrifugation, the
supernatant dialyzed against buffer B (40 mM Tris-HCl, pH 7.5, 0.1
mM EDTA, 7 mM 2-mercaptoethanol, 10% Glycerol) and applied onto a
column filled with Heparin-Sepharose (Pharmacia). In each case the
columns are equilibrated with the starting solvent and after
application of the sample the columns are washed with the threefold
of their volume with this solvent. Eluation of the first column is
performed with a linear gradient of 0 to 0.5 M NaCl in Buffer B.
The fractions showing polymerase activity are pooled and ammonium
sulfate is added to a final concentration of 20%. This solution is
applied to a hydrophobic column containing Butyl-TSK-Toyopearl
(TosoHaas). This column is eluted with a falling gradient of 20 to
0% ammonium sulfate. The pool containing the activity is dialyzed
and again transferred to a column of DEAE-Sepharose (Pharmacia) and
eluted with a linear gradient of 0-0.5 M NaCl in buffer B. The
fourth column contains Tris-Acryl-Blue (Biosepra) and is eluted as
in the preceding case. Finally the active fractions are dialyzed
against buffer C (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7.0 mM
2-mercaptoethanol, 100 mM NaCl, 50% Glycerol).
EXAMPLE 2
[0040] Detection of Endonuclease, Exonuclease and Ribonuclease
Activities:
[0041] Absence of endonuclease activity: 1 .mu.g of plasmid DNA is
incubated for 4 hours with an excess of purified DNA polymerase in
50 .mu.l of test buffer with a paraffin oil overlay at 72.degree.
C.
[0042] Absence of nonspecific exonuclease activity: 1 .mu.g of
EcoRI/HindIII-fragments of lambda DNA are incubated in 50 .mu.l of
test buffer in the absence and presence of dNTPs (1 mM final
concentration each) with an excess of purified DNA polymerase for 4
hours at 72.degree. C. with a paraffin overlay.
[0043] Absence of ribonuclease activity: 3 .mu.g of MS2 RNA are
incubated with an excess of DNA polymerase in 20 .mu.l of test
buffer for 4 hours at 72.degree. C. The RNA is subsequently
analyzed by electrophoresis in a MOPS gel (Maniatis et al., 1982,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y.).
EXAMPLE 3
[0044] Determination of DNA Polymerase Activity
[0045] DNA polymerase activity was either measured by incorporation
of .sup.32P-dCTP or by incorporation of digoxigenin labeled dUTP
into the synthesized DNA.
[0046] Detection and quantification of .sup.32P-dCTP incorporation
was measured as follows: The reaction mixture contained 50 mM
Tris-HCl, pH 8.5; 12.5 mM (NH.sub.4).sub.2SO.sub.4; 10 mM KCl; 5 mM
MgCl.sub.2; 10 mM 2-mercaptoethanol, 200 .mu.g/ml BSA, 200 .mu.M of
dATP, dGTP and dTTP, 100 .mu.M dCTP, 12 .mu.g of DNAse activated
DNA from calf thymus and 0.1 .mu.l of .sup.32P-dCTP (10 mCi/ml,
3000 Ci/mmol). After incubation for 30 min. at 70.degree. C. the
samples were placed on ice, 250 .mu.l of 10% trichloroacetic acid
were added, the samples mixed and incubated for 10 more min. on
ice. 150 .mu.l of the samples were filtrated through nylon
membranes, the filters washed four times with 5% trichloroacetic
acid. The filters were dried for 30 minutes at 80.degree. C. and
the radioactivity bound to the filters determined in a Packard
Matrix 96 Direct Beta Counter.
[0047] Detection and quantification of the incorporated digoxigenin
was performed essentially as described in Holtke, H.-J.; Sagner, G;
Kessler, C. and Schmitz, G., 1992, Biotechniques Vol. 12, p.
104-113. Typically, this assay is performed in a total volume of 50
.mu.l of a reaction mixture composed of 1 or 2 .mu.l of diluted
(0.05 U-0.01 U) DNA polymerase and 50 mM Tris-HCl, pH 8.5; 12.5 mM
(NH.sub.4).sub.2SO.sub.4; 10 mM KCl; 5 mM MgCl.sub.2; 10 mM
2-mercaptoethanol; 33 .mu.M dNTPs; 200 .mu.g/ml BSA; 12 .mu.g of
DNAse activated DNA from calf thymus and 0.036 .mu.M
digoxigenin-dUTP.
[0048] The samples are incubated for 30 min. at 72.degree. C., the
reaction is stopped by addition of 2 .mu.l 0.5 M EDTA and the tubes
placed on ice. After addition of 8 .mu.l 5 M NaCl and 150 .mu.l of
Ethanol (precooled to -20.degree. C.) the DNA is precipitated by
incubation for 15 min. on ice and pelleted by centrifugation for 10
min. at 13000.times.rpm and 4.degree. C. The pellet is washed with
100 .mu.l of 70% Ethanol (precooled to -20.degree. C.) and 0.2 M
NaCl, centrifuged again and dried under vacuum. The pellets are
dissolved in 50 .mu.l Tris-EDTA (10 mM/0.1 mM; pH 7.5). 5 .mu.l of
the sample are spotted into a well of a nylon membrane bottomed
white microwell plate (Pall Filtrationstechnik GmbH, Dreieich, FRG,
product no: SM045BWP). The DNA is fixed to the membrane by baking
for 10 min. at 70.degree. C. The DNA loaded wells are filled with
100 .mu.l of 0.45 .mu.m-filtrated 1% blocking solution (100 mM
maleic acid, 150 mM NaCl, 1% (w/v) casein, pH 7.5). All following
incubation steps are done at room temperature. After incubation for
2 min. the solution is sucked through the membrane with a suitable
vacuum manifold at -0.4 bar. After repeating the washing step, the
wells are filled with 100 .mu.l of a 1:10000-dilution
anti-digoxigenin-AP, Fab fragments (Boehringer Mannheim, FRG, no:
1093274) diluted in the blocking solution described above. After
incubation for 2 min. and sucking this step is repeated once. The
wells are washed twice under vacuum with 200 .mu.l of washing
buffer 1 (100 mM maleic acid, 150 mM NaCl, 0.3% (v/v) Tween.TM. 20,
pH7.5). After washing another two times under vacuum with 200 .mu.l
washing buffer 2 (10 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl.sub.2, pH
9.5), 50 .mu.l of CSPD.TM. (Boehringer Mannheim, no: 1655884)
diluted 1:100 in washing buffer 2, which serves as a
chemiluminescent substrate for the alkaline phosphatase, are added
to the wells and the microwell plate is incubated for 5 min. at
room temperature. The solution is then sucked through the membrane
and after 10 min. further incubation at room temperature the RLU/s
(Relative Light Unit per second) are detected in a Luminometer e.g.
MicroLumat LB 96 P (EG&G Berthold, Wildbad, FRG).
[0049] With a serial dilution of Taq DNA polymerase a standard
curve is prepared from which the linear range serves as a standard
for the activity determination of the DNA polymerase to be
analyzed.
EXAMPLE 4
[0050] Determination of Reverse Transcriptase Activity
[0051] Reverse transcriptase activity was measured using oligo dT
primed poly A template by incorporation of either .sup.32P-dTTP or
digoxigenin-labeled dUTP into the complementary strand.
Incorporation of 32P-dTTP was measured in a mixture containing 1
.mu.g of poly A.multidot.(dT).sub.15, 500 .mu.M of dTTP, 100 mg/ml
BSA, 10 mM Tris-HCl, pH 8.5, 20 mM KCl, 0.5-10 mM MgCl.sub.2 or
0.1-5 mM MnCl.sub.2, 10 mM DTE, 0.5 .mu.g, of .sup.32P-dTTP (10 mM
Ci/ml, 3000 Ci/mmol) and various amounts of DNA polymerase. The
incubation temperature used was 50.degree. C. The incorporated
radioactivity was determined as described in the assay for
determination of DNA polymerase activity.
[0052] Incorporation of digoxigenin-dUTP was measured in a mixture
containing 1 .mu.g of poly A.multidot.(dT).sub.15, 330 .mu.M of
dTTP, 0.36 .mu.M of digoxigenin-dUTP, 200 mg/ml BSA, 10 mM
Tris-HCl, pH 8.5, 20 mM KCl, 0.5-10 mM MgCl.sub.2 or 0.1-5 mM
MnCl.sub.2, 10 mM DTE and various amounts of DNA polymerase. The
incubation temperature used was 50.degree. C. Detection of the
radioactivity incorporated was performed in analogy to the
procedure used for detection of DNA polymerase activity.
EXAMPLE 5
[0053] Detection of DNA Polymerase and Reverse Transcriptase
Activity in situ
[0054] In situ PAGE analysis of polymerase activity and reverse
transcriptase activity was performed essentially according to the
method described by Spanos A. and Hubscher U., 1983, Methods in
Enzymology Vol. 91 p. 263-277. Some minor, but essential
modifications to the original method are, that the renaturation of
the SDS-denatured polypeptides is performed in the presence of
magnesium ions (3 mM) and dATP (0.5-1 .mu.M) to assist
refolding.
[0055] In brief the method is as follows:
[0056] After separation of polypeptides from either crude cell
extracts or purified samples on a denaturing 8% polyacrylamide gel
(stacking gel 5% acrylamide) which contains 150 .mu.g activated
calf thymus DNA per ml gel volume, the gel is washed four times
(15-30 min. each at room temperature with moderate shaking) in
excess renaturation buffer (Tris-HCl, 50 mM, pH 8.4; EDTA, 1 mM;
2-mercaptoethanol, 3 mM; KCl, 50 mM; Glycerol, 5-10%) to remove
SDS. Then the gel is incubated overnight in the same buffer,
including 3 mM MgCl.sub.2 and 0.5-1 .mu.M dATP at 4.degree. C.
without agitation. The first four washes are repeated the next day
with renaturation buffer. After the removal of SDS and renaturation
of the proteins the gel is transferred into the reaction mixture
consisting of Tris-HCl, 50 mM, pH 8.4; KCl, 50 mM; DTT, 3 mM;
MgCl.sub.2, 7 mM; 12 .mu.M of dATP, dCTP, dGTP (each), 8 .mu.M dTTP
and 4 .mu.M Dig-dUTP; 10% (v/v) glycerol. The gel is first
incubated under shaking at room temperature (30 min.) and then
slowly warmed up to 72.degree. C. by temperature increments of
5.degree. C. At each temperature interval DNA synthesis is allowed
to proceed for 30 min., in order to detect also polymerase activity
of mesophile control polymerases. After DNA synthesis, the DNA is
transferred either electrophoretically (0.25.times.TBE) or by
capillary blotting (15.times.SSC) to nylon membranes (Boehringer
Mannheim) and UV crosslinked. Newly synthesized Dig-labeled DNA is
detected according to the procedure described for analysis of DNA
polymerase activity.
EXAMPLE 6
[0057] Cloning of the Anaerocellum Thermophilum DNA Polymerase
Gene.
[0058] Preparation of chromosomal DNA from Anaerocellum
thermophilum. 0.8 g biomass of Anaerocellum thermophilum was
suspended in 20 ml 1M KCl and centrifuged. Then the pellet was
resuspended in 4.8 ml SET-buffer (150 mM NaCl, 15 mM EDTA, pH 8.0,
60 mM Tris-HCl, pH 8.0, 50 .mu.g/.mu.l RNaseA), after which 1 ml
20% SDS and 50 .mu.l of proteinase K (10 mg/ml) were added. The
mixture was kept at 37.degree. C. for 45 min. After extraction with
phenol and chloroform the DNA was precipitated with ethanol and
dissolved in H.sub.2O. Thus about 3.8 mg of DNA were obtained.
[0059] Amplification of Specific DNA by PCR.
[0060] For amplification of the gene encoding the DNA polymerase of
Anaerocellum thermophilum by the PCR technique two mixed
oligonucleotides (primer 1 and 2) were designed on the basis of
conserved regions of family A DNA polymerases as published by
Braithwaite D. K. and Ito J., 1993, Nucl. Acids Res. Vol.21, p.
787-802.
2 Primer 1: 5'-WSN GAY AAY ATH CCN GGN GT-3': SEQ ID NO. 1 Primer
2: 5'-NCC NAC YTC NAC YTC NAR NGG-3': SEQ ID NO. 2
[0061] The PCR amplification was performed in 100 .mu.l buffer
containing 750 ng of genomic DNA from Anaerocellum thermophilum, 10
mM Tris-HCl, pH 8.8, 2.5 mM MgCl.sub.2, 50 mM KCl, 200 .mu.M dNTPs,
100 pmoles of each primer and 2.5 units of Taq polymerase
(Boehringer Mannheim GmbH). The target sequence was amplified by
first denaturing at 95.degree. C. for 2 min. followed by 30 cycles
of 95.degree. C. for 0.5 min, 50.degree. C. for 1 min. and
72.degree. C. for 2 min. Thermal cycling was performed in a Perkin
Elmer GenAmp 9600 thermal cycler. Agarose gel electrophoresis
showed, that a fragment of approximately 1,900 base pairs was
amplified specifically. This fragment was ligated into the
pCR.TM.II vector (Invitrogen) and the sequence determined by
cycle-sequencing. The amino acid sequence deduced from this
nucleotide sequence was very similar to that of other known DNA
polymerases, so that primer 3 and 4 could be designed for inverse
PCR.
3 Primer 3: 5'-CAA TTC AGG GCA GTG CTG CTG ATA SEQ ID NO. 3 TC-3':
Primer 4: 5'-GAG CTT CTG GGC ACT CTT TTC GCC- SEQ ID NO. 4 3':
[0062] Inverse PCR was performed essentially as described in
Triglia T. et al., 1988, Nucleic Acids Research Vol. 16, p. 8186. 5
.mu.g genomic DNA from Anaerocellum thermophilum were cleaved by
EcoRI according to supplier's specifications (Boehringer Mannheim
GmbH) and treated with an equal volume of phenol/chloroform
mixture. The aqueous phase was removed, the DNA precipitated with
ethanol and collected by centrifugation.
[0063] For circularization the digested DNA was diluted to a
concentration of 50 ng/.mu.l in ligation buffer (Boehringer
Mannheim GmbH). The ligation reaction was initiated by the addition
of T4 DNA Ligase (Boehringer Mannheim GmbH) to a concentration of
0.2 units/.mu.l and the reaction was allowed to proceed for 15 hrs
at 15.degree. C. The ligated DNA was then precipitated with ethanol
and collected by centrifugation.
[0064] The PCR was performed in 50 .mu.l buffer containing 50 mM
Tris-Cl, pH 9.2, 16 mM (NH.sub.4).sub.2SO.sub.4, 2.25 mM
MgCl.sub.2, 2% (v/v) DMSO, 0.1% (v/v) Tween.TM. 20
(Poly(oxyethylen).sub.n-sorbitan-mono-laura- t), 700 ng of
circularized DNA obtained as described above, 50 pmoles of each
primer, 500 .mu.M dNTP and 0.75 .mu.l enzyme mix (Expand Long
Template PCR System, Boehringer Mannheim GmbH).
[0065] The cycle conditions were as follows: 1 1 .times.
denaturation of template for 2 min . at 92 .degree. C . 10 .times.
[ denaturation at 92 .degree. C . for 10 sec . annealing at 64
.degree. C . for 30 sec . elongation at 68 .degree. C . for 2 min .
20 .times. [ denaturation at 92 .degree. C . for 10 sec . annealing
at 64 .degree. C . for 30 sec . elongation at 68 .degree. C . for 2
min . + cycle elongation of 20 sec . for each cycle
[0066] Agarose gel electrophoresis revealed a specifically
amplified DNA fragment 6,500 base pairs long. The DNA fragment was
ligated into the pCR.TM.II vector (Invitrogen) and sequenced.
Deduced from this sequence primer 5 and 6 coding for the 5'- and
3'-ends, respectively, of the polymerase region could be designed.
Primer 5 contained a EclX1 site and primer 6 contained a BamHI
site.
[0067] The PCR was performed under the same conditions as described
above (inverse PCR) using 750 ng genomic DNA from Anaerocellum
thermophilum as template.
4 Primer 5: 5'-CGA ATT CGG CCG TCA TGA AAC TGG SEQ ID NO. 5 TTA TAT
TCG ATG GAA ACA G-3': Primer 6: 5'-CGA ATT GGA TCC GTT TTG TCT CAT
SEQ ID NO. 6 ACC AGT TCA GTC CTT C-3':
[0068] Cloning and Expression.
[0069] The PCR product was purified by electrophoresis of 20 .mu.l
of the PCR mixture on a 0.8% agarose gel. The 2.552 kb band of the
polymerase coding region was purified from the agarose by phenol
extraction. The DNA was then treated with chloroform and
precipitated with ethanol. The pellet was resuspended and digested
with EclXI and BamHI according to supplier's specifications
(Boehringer Mannheim GmbH) to give cohesive ends for directional
cloning. The DNA was ligated into the expression vector pASK75
(Biometra) that had also been digested with EclXI and BamHI. The
ligated products were introduced into E. coli strain LE392 pUBS520
(Brinkmann U., et al., 1989, Gene Vol. 85, p. 109-114) by
transformation. Transformants were grown on L-agar containing 100
.mu.g/ml ampicillin and 50 .mu.g/ml kanamycin to allow selection of
recombinants. Colonies were picked and grown in L-broth containing
100 .mu.g/ml ampicillin and 50 .mu.g/ml kanamycin, and plasmid DNA
was prepared by alkaline lysis. The plasmids were screened for
insertions by digestion with BamHI. Those recombinants containing
inserts were grown in L-broth containing ampicillin and kanamycin
and tested for the expression of thermophilic DNA polymerase by
induction of exponentially growing culture with 0.2 .mu.g/ml
anhydrotetracycline and assaying the heat-treated extracts for DNA
polymerase activity as described above (determination of DNA
polymerase activity). A recombinant expressing the DNA polymerase
from Anaerocellum thermophilum was obtained. The strain was
designated E. coli AR220 (DSM No. 11177) and the plasmid pAR10.
EXAMPLE 7
[0070] DNA polymerase from Anaerocellum thermophilum was compared
with DNA polymerases from Thermus thermophilus and Thermus
filiformis.
[0071] Similar amounts (units) of the DNA polymerases were
analyzed. Each enzyme was tested for DNA polymerase activity, for
reverse transcriptase activity in the presence of Mg++ (5 mM) and
reverse transcriptase activity in the presence of Mn++ (1 mM) under
the reaction conditions optimal for the individual enzymes. In
order to compare the ratio of DNA polymerase to reverse
transcriptase activity, the relative light units (RLU) measured in
the DNA polymerase assay were set to 100. The RLUs measured in the
reverse transcriptase activity tests are expressed as percent of
the polymerase activity. Results are shown in FIG. 5.
Sequence CWU 1
1
9 1 20 DNA Artificial Sequence Description of Artificial
SequencePCR amplification oligonucleotide Primer 1 based on
conserved regions of family A DNA polymerases 1 wsngayaaya
thccnggngt 20 2 21 DNA Artificial Sequence Description of
Artificial SequencePCR amplification oligonucleotide Primer 2 based
on conserved regions of family A DNA polymerases 2 nccnacytcn
acytcnarng g 21 3 26 DNA Artificial Sequence Description of
Artificial Sequencesynthesized inverse PCR amplification
oligonucleotide Primer 3 binding at outer DNA sequences of the gene
3 caattcaggg cagtgctgct gatatc 26 4 24 DNA Artificial Sequence
Description of Artificial Sequencesynthesized inverse PCR
amplification oligonucleotide Primer 4 binding at outer DNA
sequences of the gene 4 gagcttctgg gcactctttt cgcc 24 5 43 DNA
Artificial Sequence Description of Artificial SequencePCR
amplification oligonucleotide Primer 5 coding for 5' end of
polymerase region containing EclX1 site 5 cgaattcggc cgtcatgaaa
ctggttatat tcgatggaaa cag 43 6 40 DNA Artificial Sequence
Description of Artificial SequencePCR amplification oligonucleotide
Primer 6 coding for 3' end of polymerase region containing BamHI
site 6 cgaattggat ccgttttgtc tcataccagt tcagtccttc 40 7 2553 DNA
Anaerocellum thermophilum entire DNA polymerase gene coding
sequence 7 atg aaa ctg gtt ata ttc gat gga aac agc att ttg tac aga
gcc ttt 48 Met Lys Leu Val Ile Phe Asp Gly Asn Ser Ile Leu Tyr Arg
Ala Phe 1 5 10 15 ttt gct ctt cct gaa ctg aca acc tca aat aat att
cca aca aac gct 96 Phe Ala Leu Pro Glu Leu Thr Thr Ser Asn Asn Ile
Pro Thr Asn Ala 20 25 30 ata tat gga ttt gta aat gtg ata ttg aaa
tat tta gaa caa gaa aaa 144 Ile Tyr Gly Phe Val Asn Val Ile Leu Lys
Tyr Leu Glu Gln Glu Lys 35 40 45 cct gat tat gtt gct gta gca ttt
gat aaa aga gga aga gag gca cga 192 Pro Asp Tyr Val Ala Val Ala Phe
Asp Lys Arg Gly Arg Glu Ala Arg 50 55 60 aaa agc gag tac gaa gaa
tat aaa gct aac aga aaa cct atg cca gat 240 Lys Ser Glu Tyr Glu Glu
Tyr Lys Ala Asn Arg Lys Pro Met Pro Asp 65 70 75 80 aac ctt caa gta
caa atc cct tat gtt cga gag att ctt tat gcc ttt 288 Asn Leu Gln Val
Gln Ile Pro Tyr Val Arg Glu Ile Leu Tyr Ala Phe 85 90 95 aac att
cca ata att gag ttt gaa gga tat gaa gca gat gat gta atc 336 Asn Ile
Pro Ile Ile Glu Phe Glu Gly Tyr Glu Ala Asp Asp Val Ile 100 105 110
ggt tca ctt gtt aac cag ttc aaa aat act ggt ttg gat att gtt att 384
Gly Ser Leu Val Asn Gln Phe Lys Asn Thr Gly Leu Asp Ile Val Ile 115
120 125 att acg ggt gac agg gat act ctt cag ttg ctc gac aaa aat gta
gtt 432 Ile Thr Gly Asp Arg Asp Thr Leu Gln Leu Leu Asp Lys Asn Val
Val 130 135 140 gtg aag att gtt tca aca aaa ttt gat aaa aca gta gaa
gat ttg tac 480 Val Lys Ile Val Ser Thr Lys Phe Asp Lys Thr Val Glu
Asp Leu Tyr 145 150 155 160 act gtg gaa aat gtt aaa gaa aaa tat ggg
gtt tgg gca aat caa gtg 528 Thr Val Glu Asn Val Lys Glu Lys Tyr Gly
Val Trp Ala Asn Gln Val 165 170 175 cct gat tac aaa gcg ctt gtt gga
gac caa tca gat aac att ccc ggg 576 Pro Asp Tyr Lys Ala Leu Val Gly
Asp Gln Ser Asp Asn Ile Pro Gly 180 185 190 gta aag gga att ggc gaa
aag agt gcc cag aag ctc ttg gaa gag tac 624 Val Lys Gly Ile Gly Glu
Lys Ser Ala Gln Lys Leu Leu Glu Glu Tyr 195 200 205 tca tcc tta gaa
gag ata tac caa aat tta gat aaa att aaa agt tcc 672 Ser Ser Leu Glu
Glu Ile Tyr Gln Asn Leu Asp Lys Ile Lys Ser Ser 210 215 220 att cgt
gaa aag tta gaa gca gga aaa gat atg gcg ttt tta tcc aag 720 Ile Arg
Glu Lys Leu Glu Ala Gly Lys Asp Met Ala Phe Leu Ser Lys 225 230 235
240 cgc tta gca aca att gta tgt gat tta cca cta aat gtt aaa ctt gaa
768 Arg Leu Ala Thr Ile Val Cys Asp Leu Pro Leu Asn Val Lys Leu Glu
245 250 255 gac cta aga aca aaa gag tgg aac aag gaa agg ctc tat gag
att ttg 816 Asp Leu Arg Thr Lys Glu Trp Asn Lys Glu Arg Leu Tyr Glu
Ile Leu 260 265 270 gtg cag tta gag ttc aaa agc ata ata aaa cgg tta
gga cta tca gaa 864 Val Gln Leu Glu Phe Lys Ser Ile Ile Lys Arg Leu
Gly Leu Ser Glu 275 280 285 gtt gtt caa ttt gaa ttt gtt cag cag cga
acc gat ata cct gac gtt 912 Val Val Gln Phe Glu Phe Val Gln Gln Arg
Thr Asp Ile Pro Asp Val 290 295 300 gaa caa aaa gag ctt gaa agt att
tca caa ata aga tca aaa gag att 960 Glu Gln Lys Glu Leu Glu Ser Ile
Ser Gln Ile Arg Ser Lys Glu Ile 305 310 315 320 cca tta atg ttt gta
cag ggc gaa aaa tgt ttt tat tta tat gat caa 1008 Pro Leu Met Phe
Val Gln Gly Glu Lys Cys Phe Tyr Leu Tyr Asp Gln 325 330 335 gaa agt
aat act gta ttt ata aca agt aat aaa ctt ttg ata gag gag 1056 Glu
Ser Asn Thr Val Phe Ile Thr Ser Asn Lys Leu Leu Ile Glu Glu 340 345
350 att tta aaa agt gat act gtg aaa att atg tat gat ttg aaa aat ata
1104 Ile Leu Lys Ser Asp Thr Val Lys Ile Met Tyr Asp Leu Lys Asn
Ile 355 360 365 ttt cat caa ctc aac ctg gaa gac act aat aat att aaa
aat tgc gaa 1152 Phe His Gln Leu Asn Leu Glu Asp Thr Asn Asn Ile
Lys Asn Cys Glu 370 375 380 gat gta atg att gct tcc tat gtt ctt gac
agc aca aga agt tca tat 1200 Asp Val Met Ile Ala Ser Tyr Val Leu
Asp Ser Thr Arg Ser Ser Tyr 385 390 395 400 gag tta gaa acg ttg ttt
gta tct tac ttg aac act gac ata gaa gct 1248 Glu Leu Glu Thr Leu
Phe Val Ser Tyr Leu Asn Thr Asp Ile Glu Ala 405 410 415 gta aaa aaa
gac aag aag ata gtc tct gtg gta ctt cta aaa cgg tta 1296 Val Lys
Lys Asp Lys Lys Ile Val Ser Val Val Leu Leu Lys Arg Leu 420 425 430
tgg gac gag ctt ttg aga tta ata gat tta aat tca tgc cag ttt tta
1344 Trp Asp Glu Leu Leu Arg Leu Ile Asp Leu Asn Ser Cys Gln Phe
Leu 435 440 445 tat gag aat ata gaa aga cct ctt atc cca gtt cta tat
gaa atg gaa 1392 Tyr Glu Asn Ile Glu Arg Pro Leu Ile Pro Val Leu
Tyr Glu Met Glu 450 455 460 aaa aca gga ttt aag gtg gat aga gat gcc
ctc atc caa tat acc aaa 1440 Lys Thr Gly Phe Lys Val Asp Arg Asp
Ala Leu Ile Gln Tyr Thr Lys 465 470 475 480 gag att gaa aac aaa ata
tta aaa ctt gaa acg cag ata tac cag att 1488 Glu Ile Glu Asn Lys
Ile Leu Lys Leu Glu Thr Gln Ile Tyr Gln Ile 485 490 495 gca ggt gag
tgg ttt aac ata aat tca ccg aaa cag ctt tct tac att 1536 Ala Gly
Glu Trp Phe Asn Ile Asn Ser Pro Lys Gln Leu Ser Tyr Ile 500 505 510
ttg ttt gaa aag cta aaa ctt cct gta ata aag aag aca aaa aca gga
1584 Leu Phe Glu Lys Leu Lys Leu Pro Val Ile Lys Lys Thr Lys Thr
Gly 515 520 525 tat tcc act gat gcc gag gtt tta gaa gag ctt ttt gac
aaa cat gaa 1632 Tyr Ser Thr Asp Ala Glu Val Leu Glu Glu Leu Phe
Asp Lys His Glu 530 535 540 ata gtt cct ctt att ttg gat tac agg atg
tat aca aag ata ctg aca 1680 Ile Val Pro Leu Ile Leu Asp Tyr Arg
Met Tyr Thr Lys Ile Leu Thr 545 550 555 560 act tac tgt cag gga tta
cta cag gca ata aat cct tct tcg ggt aga 1728 Thr Tyr Cys Gln Gly
Leu Leu Gln Ala Ile Asn Pro Ser Ser Gly Arg 565 570 575 gtt cat aca
acc ttt atc caa aca ggt aca gcc aca gga aga ctt gca 1776 Val His
Thr Thr Phe Ile Gln Thr Gly Thr Ala Thr Gly Arg Leu Ala 580 585 590
agc agc gat cct aat tta caa aat ata cct gta aaa tat gat gag ggg
1824 Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Lys Tyr Asp Glu
Gly 595 600 605 aaa ttg ata cga aag gtt ttt gta cct gag ggt gga cat
gta ctg att 1872 Lys Leu Ile Arg Lys Val Phe Val Pro Glu Gly Gly
His Val Leu Ile 610 615 620 gat gca gat tat tcc caa att gag ctg aga
ata ctt gcc cat att tct 1920 Asp Ala Asp Tyr Ser Gln Ile Glu Leu
Arg Ile Leu Ala His Ile Ser 625 630 635 640 gaa gat gaa aga ctt ata
agt gct ttc aaa aat aat gtt gac att cat 1968 Glu Asp Glu Arg Leu
Ile Ser Ala Phe Lys Asn Asn Val Asp Ile His 645 650 655 tcg cag aca
gca gct gag gtt ttt ggt gta gac ata gcc gat gtt act 2016 Ser Gln
Thr Ala Ala Glu Val Phe Gly Val Asp Ile Ala Asp Val Thr 660 665 670
cca gag atg aga agt caa gct aaa gca gta aat ttt ggt ata gtt tat
2064 Pro Glu Met Arg Ser Gln Ala Lys Ala Val Asn Phe Gly Ile Val
Tyr 675 680 685 ggg att tct gat tat ggt ctt gca agg gat att aaa att
tcc agg aaa 2112 Gly Ile Ser Asp Tyr Gly Leu Ala Arg Asp Ile Lys
Ile Ser Arg Lys 690 695 700 gaa gct gca gag ttt ata aat aag tat ttt
gag cgt tat ccc aaa gtt 2160 Glu Ala Ala Glu Phe Ile Asn Lys Tyr
Phe Glu Arg Tyr Pro Lys Val 705 710 715 720 aaa gag tat tta gat aat
act gtt aag ttt gct cgt gat aat gga ttt 2208 Lys Glu Tyr Leu Asp
Asn Thr Val Lys Phe Ala Arg Asp Asn Gly Phe 725 730 735 gtt ttg act
tta ttt aat aga aag aga tat ata aaa gac ata aaa tct 2256 Val Leu
Thr Leu Phe Asn Arg Lys Arg Tyr Ile Lys Asp Ile Lys Ser 740 745 750
aca aac aga aac tta agg ggt tat gca gaa agg att gca atg aat tcg
2304 Thr Asn Arg Asn Leu Arg Gly Tyr Ala Glu Arg Ile Ala Met Asn
Ser 755 760 765 cca att cag ggc agt gct gct gat atc atg aaa ttg gca
atg att aag 2352 Pro Ile Gln Gly Ser Ala Ala Asp Ile Met Lys Leu
Ala Met Ile Lys 770 775 780 gtt tat cag aaa ctt aaa gaa aac aat ctc
aaa tca aaa ata att ttg 2400 Val Tyr Gln Lys Leu Lys Glu Asn Asn
Leu Lys Ser Lys Ile Ile Leu 785 790 795 800 cag gta cac gat gag ctt
tta att gaa gcc cca tac gaa gaa aag gat 2448 Gln Val His Asp Glu
Leu Leu Ile Glu Ala Pro Tyr Glu Glu Lys Asp 805 810 815 ata gta aag
gaa ata gta aaa aga gaa atg gaa aat gcg gta gct tta 2496 Ile Val
Lys Glu Ile Val Lys Arg Glu Met Glu Asn Ala Val Ala Leu 820 825 830
aaa gta cct ttg gta gtt gaa gtg aaa gaa gga ctg aac tgg tat gag
2544 Lys Val Pro Leu Val Val Glu Val Lys Glu Gly Leu Asn Trp Tyr
Glu 835 840 845 aca aaa tag 2553 Thr Lys 850 8 850 PRT Anaerocellum
thermophilum DNA polymerase 8 Met Lys Leu Val Ile Phe Asp Gly Asn
Ser Ile Leu Tyr Arg Ala Phe 1 5 10 15 Phe Ala Leu Pro Glu Leu Thr
Thr Ser Asn Asn Ile Pro Thr Asn Ala 20 25 30 Ile Tyr Gly Phe Val
Asn Val Ile Leu Lys Tyr Leu Glu Gln Glu Lys 35 40 45 Pro Asp Tyr
Val Ala Val Ala Phe Asp Lys Arg Gly Arg Glu Ala Arg 50 55 60 Lys
Ser Glu Tyr Glu Glu Tyr Lys Ala Asn Arg Lys Pro Met Pro Asp 65 70
75 80 Asn Leu Gln Val Gln Ile Pro Tyr Val Arg Glu Ile Leu Tyr Ala
Phe 85 90 95 Asn Ile Pro Ile Ile Glu Phe Glu Gly Tyr Glu Ala Asp
Asp Val Ile 100 105 110 Gly Ser Leu Val Asn Gln Phe Lys Asn Thr Gly
Leu Asp Ile Val Ile 115 120 125 Ile Thr Gly Asp Arg Asp Thr Leu Gln
Leu Leu Asp Lys Asn Val Val 130 135 140 Val Lys Ile Val Ser Thr Lys
Phe Asp Lys Thr Val Glu Asp Leu Tyr 145 150 155 160 Thr Val Glu Asn
Val Lys Glu Lys Tyr Gly Val Trp Ala Asn Gln Val 165 170 175 Pro Asp
Tyr Lys Ala Leu Val Gly Asp Gln Ser Asp Asn Ile Pro Gly 180 185 190
Val Lys Gly Ile Gly Glu Lys Ser Ala Gln Lys Leu Leu Glu Glu Tyr 195
200 205 Ser Ser Leu Glu Glu Ile Tyr Gln Asn Leu Asp Lys Ile Lys Ser
Ser 210 215 220 Ile Arg Glu Lys Leu Glu Ala Gly Lys Asp Met Ala Phe
Leu Ser Lys 225 230 235 240 Arg Leu Ala Thr Ile Val Cys Asp Leu Pro
Leu Asn Val Lys Leu Glu 245 250 255 Asp Leu Arg Thr Lys Glu Trp Asn
Lys Glu Arg Leu Tyr Glu Ile Leu 260 265 270 Val Gln Leu Glu Phe Lys
Ser Ile Ile Lys Arg Leu Gly Leu Ser Glu 275 280 285 Val Val Gln Phe
Glu Phe Val Gln Gln Arg Thr Asp Ile Pro Asp Val 290 295 300 Glu Gln
Lys Glu Leu Glu Ser Ile Ser Gln Ile Arg Ser Lys Glu Ile 305 310 315
320 Pro Leu Met Phe Val Gln Gly Glu Lys Cys Phe Tyr Leu Tyr Asp Gln
325 330 335 Glu Ser Asn Thr Val Phe Ile Thr Ser Asn Lys Leu Leu Ile
Glu Glu 340 345 350 Ile Leu Lys Ser Asp Thr Val Lys Ile Met Tyr Asp
Leu Lys Asn Ile 355 360 365 Phe His Gln Leu Asn Leu Glu Asp Thr Asn
Asn Ile Lys Asn Cys Glu 370 375 380 Asp Val Met Ile Ala Ser Tyr Val
Leu Asp Ser Thr Arg Ser Ser Tyr 385 390 395 400 Glu Leu Glu Thr Leu
Phe Val Ser Tyr Leu Asn Thr Asp Ile Glu Ala 405 410 415 Val Lys Lys
Asp Lys Lys Ile Val Ser Val Val Leu Leu Lys Arg Leu 420 425 430 Trp
Asp Glu Leu Leu Arg Leu Ile Asp Leu Asn Ser Cys Gln Phe Leu 435 440
445 Tyr Glu Asn Ile Glu Arg Pro Leu Ile Pro Val Leu Tyr Glu Met Glu
450 455 460 Lys Thr Gly Phe Lys Val Asp Arg Asp Ala Leu Ile Gln Tyr
Thr Lys 465 470 475 480 Glu Ile Glu Asn Lys Ile Leu Lys Leu Glu Thr
Gln Ile Tyr Gln Ile 485 490 495 Ala Gly Glu Trp Phe Asn Ile Asn Ser
Pro Lys Gln Leu Ser Tyr Ile 500 505 510 Leu Phe Glu Lys Leu Lys Leu
Pro Val Ile Lys Lys Thr Lys Thr Gly 515 520 525 Tyr Ser Thr Asp Ala
Glu Val Leu Glu Glu Leu Phe Asp Lys His Glu 530 535 540 Ile Val Pro
Leu Ile Leu Asp Tyr Arg Met Tyr Thr Lys Ile Leu Thr 545 550 555 560
Thr Tyr Cys Gln Gly Leu Leu Gln Ala Ile Asn Pro Ser Ser Gly Arg 565
570 575 Val His Thr Thr Phe Ile Gln Thr Gly Thr Ala Thr Gly Arg Leu
Ala 580 585 590 Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Lys Tyr
Asp Glu Gly 595 600 605 Lys Leu Ile Arg Lys Val Phe Val Pro Glu Gly
Gly His Val Leu Ile 610 615 620 Asp Ala Asp Tyr Ser Gln Ile Glu Leu
Arg Ile Leu Ala His Ile Ser 625 630 635 640 Glu Asp Glu Arg Leu Ile
Ser Ala Phe Lys Asn Asn Val Asp Ile His 645 650 655 Ser Gln Thr Ala
Ala Glu Val Phe Gly Val Asp Ile Ala Asp Val Thr 660 665 670 Pro Glu
Met Arg Ser Gln Ala Lys Ala Val Asn Phe Gly Ile Val Tyr 675 680 685
Gly Ile Ser Asp Tyr Gly Leu Ala Arg Asp Ile Lys Ile Ser Arg Lys 690
695 700 Glu Ala Ala Glu Phe Ile Asn Lys Tyr Phe Glu Arg Tyr Pro Lys
Val 705 710 715 720 Lys Glu Tyr Leu Asp Asn Thr Val Lys Phe Ala Arg
Asp Asn Gly Phe 725 730 735 Val Leu Thr Leu Phe Asn Arg Lys Arg Tyr
Ile Lys Asp Ile Lys Ser 740 745 750 Thr Asn Arg Asn Leu Arg Gly Tyr
Ala Glu Arg Ile Ala Met Asn Ser 755 760 765 Pro Ile Gln Gly Ser Ala
Ala Asp Ile Met Lys Leu Ala Met Ile Lys 770 775 780 Val Tyr Gln Lys
Leu Lys Glu Asn Asn Leu Lys Ser Lys Ile Ile Leu 785 790 795 800 Gln
Val His Asp Glu Leu Leu Ile Glu Ala Pro Tyr Glu Glu Lys Asp 805 810
815 Ile Val Lys Glu Ile Val Lys Arg Glu Met Glu Asn Ala Val Ala Leu
820 825 830 Lys Val Pro Leu Val Val Glu Val Lys Glu Gly Leu Asn Trp
Tyr Glu 835
840 845 Thr Lys 850 9 15 DNA Artificial Sequence Description of
Artificial Sequence(dT)-15, oligo dT primer 9 tttttttttt ttttt
15
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