Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-11-22
2002-10-22
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S320100, C435S252100, C435S252300, C435S325000, C435S252330, C536S023100, C536S023200, C530S350000
Reexamination Certificate
active
06468775
ABSTRACT:
The present invention relates to a thermostable enzyme which is a DNA polymerase obtainable from
Carboxydothermus hydrogenoformans.
Furthermore, the present invention relates to the field of molecular biology and provides improved methods for the replication and amplification of deoxyribonucleic (DNA) and ribonucleic acid (RNA) sequences. 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).
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). Biokimiva 45, 644-651; Kaledin et al. (1981) Biokimiva46, 1247-1254; Kaledin et al. (1982) Biokimiya 47, 1515-1521; Ruttimann et al. (1985) Eur. J. Biochem. 149, 41-46; Neuner et al. (1990) Arch. Microbiol. 153, 205-207). For some organisms, the polymerase gene has been cloned and expressed (Lawyer et al. (1989) J. Biol. Chem. 264, 6427-6437; Engelke et al. (1990) Anal. Biochem. 191, 396-400; Lundberg et al. (1991) Gene 108, 1-6; Perler et al. (1992) Proc. Natl. Acad. Sci. USA 89, 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. 98. 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 at 42° C. or 37° C., respectively.
Alternative methodes 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 in the presence of Manganese ions the polymerase copies the template RNA with low fidelity.
Another feature of the commonly used reverse transcriptases is that they do not contain 3′-5′ exonuclease activity. Therefore, misincorporated nucleotides cannot be removed and thus the cDNA copies from the template RNA may contain a significant degree of mutations.
Therefore, it is desirable to develop a reverse transcriptase
which acts at higher temperatures to overcome secondary structures in the template to avoid premature termination of the reaction and to assure the production of cDNA without deletions
which is active in the presence of Magnesium ions in order to prepare cDNA from RNA templates with higher fidelity and
which has 3′-5′-exonuclease in order to remove misincorporated nucleotides before continuation of DNA synthesis and to produce a product with a low mutation frequency.
The present invention adresses these needs and provides a heat stable DNA polymerase active at higher temperatures which has reverse transcriptase activity in the presence of magnesium ions and and which has 3′-5′ exonuclease activity.
It is an object of this invention to provide a polymerase enzyme (EC 2.7.7.7.), characterised in that it has reverse transcriptase activity in the presence of magnesium ions as well as in the presence of manganese ions. In another aspect the invention comprises a DNA polymerase isolated from
Carboxydothermus hydrogenoformans
(Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, DSM No. 8979). 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. In a further aspect the invention comprises a DNA polymerase having a molecular mass of about 100 to 105 kDa as determined by in situ PAGE analysis. In a further aspect the invention comprises a reverse transcriptase which is thermostable. In a further aspect the invention comprises a DNA polymerase having 3′-5′-exonuclease activity. In a further aspect the invention comprises a recombinant DNA sequence that encodes DNA polymerase activity of the microorganism
Carboxidothermus hydrogenoformans.
In a related aspect the DNA sequence is depicted as SEQ ID No. 7. In a second related aspect the invention comprises a recombinant DNA sequence that encodes essentially amino acid residues 1 to 831. In a further aspect the invention comprises a recombinant DNA plasmid that comprises the DNA sequence of the invention inserted into plasmid vectors and which can be used to drive the expression of the thermostable DNA polymerase of
Carboxydothermus hydrogenoformans
in a host cell transformed with the plasmid. In a further aspect the invention includes a recombinant strain comprising the vector pDS56 carrying the
Carboxydothermus hydrogenoformans
DNA polymerase gene and designated pAR 4. The
E.coli
strain (BL21 DE3)pUBS520) carrying the plasmid pAR4 was deposited on the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DSM No. 11179) and is designated AR96.
In referring to a peptide chain as being comprised of a series of amino acids “substantially or effectively” in accordance with a list offering no alternatives within itself, we include within that reference any versions of the peptide chain bearing substitutions made to one or more amino acids in such a way that the overall structure and the overall function of the protein composed of that peptide chain is substantially the same as—or undetectably different to—that of the unsubstituted version. For example it is generally possible to exchange alanine and valine without greatly changing the properties of the protein, especially if the changed site or sites are at positions not critical to the morphology of the folded protein.
The DNA polymerase is “thermostable” meaning that it is stable to heat and preferentially active at higher temperatures, especially the high temperatures used for denaturation of DNA strands. More particularly, the thermostable DNA polymerases are not substantially inactivated at the high temperatures used in polymerase chain reactions.
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.
Other definitions are used in a manner consistent with the art.
Carboxydothermus hydrogenoformans
was isolated from a hot spring in Kamchatka by V. Svetlichny. A sample of
C. hydrogenoformans
was deposited on the Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH (DSM) under the terms of the BudaPest Treaty and received Accession Number DSM 8979. The thermostable polymerase isolated from
Carboxydothermus hydrogenoformans
has a molecular weight of 100 to 105 KDa and retains more than 60% of its initial activity after heating to 95° C. for 30 minutes. The thermostable enzyme possesses a 5′-3&prim
Angerer Bernhard
Ankenbauer Waltraud
Bonch-Osmolovskaya Elizaveta
Ebenbichler Christine
Laue Frank
Hutson Richard G
Pennie & Edmonds LLP
Prouty Rebecca E.
Roche Molecular Systems Inc.
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