Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2002-02-05
2004-09-21
Hutson, Richard (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S194000, C435S183000, C435S320100, C536S023100, C536S023200, C536S023700, C536S023500, C530S350000
Reexamination Certificate
active
06794177
ABSTRACT:
This application claims priority to European patent application No. 97.121151.1, filed Dec. 2, 1997.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a modified DNA-polymerase having reverse transcriptase activity and reduced 5′-3′ exonuclease activity derived from a native polymerase which is obtainable from
Carboxydothermus hydrogenoformans
. Furthermore the invention relates to the field of molecular biology and provides methods for amplifying a DNA segment from an RNA template using an enzyme with reverse transcriptase activity (RT-PCR). In another aspect, the invention provides a kit for Coupled High Temperature Reverse Transcription and Polymerase Chain Reaction.
2. Description of Related Art
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),
Biokhimiya
45, 644-651; Kaledin et al. (1981)
Biokhimiya
46, 1576-1584; Kaledin et al. (1982)
Biokhimiya
47, 1785-1791; 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, 5577-5581).
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).
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.
Reverse transcription is commonly performed with viral reverse transcriptases like the enzymes isolated from Avian myeloblastosis virus or Moloney murine leukemia virus. Both enzymes mentioned 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. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA-polymerase (Verma (1977)
Biochem. Biophys. Acta
473, 1). The enzyme has 5′-3′ RNA-directed DNA polymerase activity, 5′-3′ DNA directed DNA polymerase activity, and RNaseH activity. RNaseH is a processive 5′-3′ ribonuclease specific for the RNA strand of RNA-DNA hybrids (Perbal (1984), A Practical Guide to Molecular Cloning, Wiley & Sons New York). Errors in transcription cannot be corrected because known viral reverse transcriptases lack the 3′-5′ exonuclease activity necessary for proofreading (Saunders and Saunders (1987) Microbial Genetics Applied to Biotechnology, Croom Helm, London). A detailed study of the activity of AMV reverse transcriptase and its associated RNaseH activity has been presented by Berger et al., (1983)
Biochemistry
22, 2365-2372.
DNA polymerases isolated from mesophilic microorganisms such as
E. coli
have been extensively characterized (see, for example, Bessmann et al. (1957)
J. Biol. Chem.
233, 171-177 and Buttin and Kornberg (1966)
J. Biol. Chem.
241, 5419-5427).
E. coli
DNA polymerase I (Pol I) is useful for a number of applications including: nick-translation reactions, DNA sequencing, in vitro mutagenesis, second strand cDNA synthesis, polymerase chain reactions (PCR), and blunt end formation for linker ligation (Maniatis et al., (1982) Molecular Cloning: A Laboratory Manual Cold Spring Harbor, N.Y.).
Several laboratories have shown that some polymerases are capable of in vitro reverse transcription of RNA (Karkas (1973)
Proc. Nat. Acad. Sci. USA
70, 3834-3838; Gulati et al. (1974)
Proc. Nat. Acad. Sci
USA 71, 1035-1039; and Wittig and Wittig, (1978)
Nuc. Acids Res.
5, 1165-1178). Gulati et al. found that
E. coli
Pol I could be used to transcribe Q&bgr; viral RNA using oligo(dT)
10
as a primer. Wittig and Wittig have shown that
E. coli
Pol I can be used to reverse transcribe tRNA that has been enzymatically elongated with oligo(dA). However, as Gulati et al. demonstrated, the amount of enzyme required and the small size of cDNA product suggest that the reverse transcriptase activity of
E. coli
Pol I has little practical value.
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 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.
One of the known DNA polymerases having high reverse transcriptase activity is obtainable from
Thermus thermophilus
(Tth polymerase) (WO 91/09944). Tth polymerase, as well as Taq polymerase, lacks 3′ to 5′ exonucleolytic proofreading activity. This 3′ to 5′ exonuclease activity is generally considered to be desirable because it allows removal of misincorporated or unmatched bases in the newly synthesized nucleic acid sequences. Another thermophilic pol I-type DNA polymerase isolated from
Thermotoga maritima
(Tma pol) has 3′ to 5′ exonuclease activity. U.S. Pat. No. 5,624,833 provides means for isolating and producing Tma polymerase. However, both DNA polymerases, Tth as well as Tma polymerase, show reverse transcriptase activity only in the presence of manganese ions.
The DNA polymerase of
Carboxydothermus hydrogenoformans
shows reverse transcription activity in the presence of magnesium ions and in the substantial absence of manganese ions and can be used to reverse transcribe RNA, to detect and amplify (in combination with a thermostable DNA polymerase like Taq) specific sequences of RNA. Using DNA polymerase of
Carboxydothermus hydrogenoformans
polymerase a high specificity of transcription is observed with short incubation times. A high specificity is observed using e.g. 5 min of incubation time and 33 units of DNA polymerase protein. With longer incubation times also with lower amounts of
Carboxydothermus hydrogenoformans
polymerase specific products can be obtained. However an unspecific smear of products is occurring. These unspecific products might be caused by the 5′-3′ exonuclease activity of the polymerase which enables the enzyme to cleave the template at secondary structures (“RNaseH”-activity) and to create additional primers which can be elongated by the DNA polymerase activity. The thermostable DNA polymerase from
Carboxydothermus hydrogenoformans
has been identified and cloned and is described in the copending European application with the Application No. 96115873.0, filed Oct. 3, 1996, and incorporated herein by reference.
In summary, rever
Achhammer Gunthar
Ankenbauer Waltraud
Ebenbichler Christine
Markau Ursula
Hutson Richard
Pennie & Edmonds
Roche Diagnostics GmbH
LandOfFree
Modified DNA-polymerase from carboxydothermus... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Modified DNA-polymerase from carboxydothermus..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Modified DNA-polymerase from carboxydothermus... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3228583