Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1999-06-10
2004-02-17
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000, C435S091100, C435S091200, C435S091500, C536S023200, C536S023100
Reexamination Certificate
active
06692932
ABSTRACT:
The present invention relates to a thermostable enzyme which is a DNA polymerase obtainable from
Anaerocellum thermophilum.
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
, Biokimiya
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.)
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).
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).
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° C. or 37° C., respectively.
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.
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.
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 Samnulung 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.
The polymerase according to the present invention retains at least 90% of its activity after incubation for 30 Minutes at 80° C. in absence of stablilizing detergents.
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.
In a futther aspect the invention comprises a DNA a polymerase having reverse transcriptase activity in the presence of magnesiums ions and in the substantial absence of maganese ions. The polymerase according to the present invention exhibits a Mg
2+
dependent reverse transcriptase activity of more than 30% relative to the DNA polymerase activity which is set to 100%. In further aspect the present invention comprises a thermostable DAN polymerase wherein said polymerase exhibits a reverse transcriptaqse activity which is Mn
2+
dependent. The Mn
2+
dependent reverse transcriptase activity is more than 60% relative to the DNA polymerase activity.
In further aspect the invention comprises a thermostable reverse transcriptase. The thermostable reverse transcriptase retains more than 80% after incubation for 60 minutes at 80° C.
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
2+
dependent.
Anaerocullum thermophilum was isolated from a hot spring in the Valley of Geyser in Kamchatka (V. svetlichny et al.
Mikrobilogiya,
Vol. 59, No. 5 p. 871-879, 1990).
Anaerocullum 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° 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
++
as well as Mg
++
-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.
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).
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.
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:
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™ SC (4-(2-Aminoethyl)-benzolsulfonyl-fluorid, Hydrochlorid) and lysed by twofold passage through a Gaulin homogenizer. The raw extract is c
Angerer Bernhard
Ankenbauer Waltraud
Bonch-Osmolovskaya Elizaveta
Markau Ursula
Reiser Astrid
Hutson Richard
Pennie & Edmonds LLP
Prouty Rebecca E.
LandOfFree
Thermostable DNA polymerase from anaerocellum thermophilum does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Thermostable DNA polymerase from anaerocellum thermophilum, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Thermostable DNA polymerase from anaerocellum thermophilum will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3328005