Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-08
2003-08-19
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100
Reexamination Certificate
active
06607883
ABSTRACT:
The invention concerns polymerase chimeras which are composed of amino acid fragments representing domains and which combine properties of naturally occurring polymerases that are advantageous with regard to a particular application. It has surprisingly turned out that the domains from the various enzymes are active in the chimeras and exhibit a cooperative behaviour. The present invention especially concerns those polymerase chimeras in which the domains having polymerase activity and domains having 3′-5′exonuclease activity are derived from different enzymes. Such chimeras can also have RT activity. In addition the present invention concerns a process for the production of the chimeras according to the invention and the use of these chimeras for the synthesis of nucleic acids e.g. during a polymerase chain reaction. Moreover the present invention concerns a kit which contains the polymerase chimeras according to the invention.
According to Braithwaite, D. K. and Ito, J. (1993) Nucl. Acids Res. 21, 787-802 DNA polymerases are divided according to the correspondence in their amino acid sequences into three main families with subclasses. Joyce, C. M. and Steitz, T. A. (1994) Annu. Rev. Biochem. 63, 777-822 give a summary of the motifs and conserved amino acids that were found. In prokaryotes the main distinction is made between three polymerases: polymerase I, II and III. These polymerases differ with regard to their function in the cell and with regard to their properties. DNA polymerase I is considered to be a repair enzyme and frequently has 5′-3′ as well as 3′-5′ exonuclease activity. Polymerase II appears to facilitate DNA synthesis which starts from a damaged template strand and thus preserves mutations. Polymerase III is the replication enzyme of the cell, it synthesizes nucleotides at a high rate (ca. 30,000 per minute) and is considered to be very processive. Polymerase III has no 5′-3′ exonuclease activity. Other properties of polymerases are due to their origin such as e.g. thermostability or processivity.
Particular properties of polymerases are desirable depending on the application. For example thermostable, high-fidelity (i.e. polymerases with proof-reading activity), processive and rapidly synthesizing polymerases are preferred for PCR. Enzymes are preferred for sequencing which do not discriminate much between dideoxy and deoxy nucleotides. In contrast the proof-reading activity of polymerases, i.e. 3′-5′ exonuclease activity, is not desirable for sequencing. For some applications e.g. PCR it is desirable that the polymerase has no or little 5′-3′ exonuclease activity (5′ nuclease activity).
Polymerases can also differ in their ability to accept RNA as a template i.e. with regard to their reverse transcriptase (RT) activity. The RT activity may be dependent on the presence of manganese or/and magnesium ions. It is often desirable that the RT activity of the polymerase is independent of manganese ions since the reading accuracy of polymerase is decreased in the presence of manganese ions. Polymerases additionally differ in their processivity which is also a desirable property for many applications.
There is therefore a need to optimize the properties of polymerases with regard to a particular application. In the past this was often achieved by introducing mutations or by deleting functions of the polymerases.
Thus for example the 5′-3′ exonuclease activity was abolished by introducing mutations (Merkens, L. S. (1995)
Biochem. Biophys. Acta
1264, 243-248) as well as by truncation (Jacobsen, H. (1974)
Eur. J. Biochem.
45, 623-627; Barnes, W. M. (1992)
Gene
112, 29-35). The ability of polymerases to discriminate between dideoxy and deoxynucleotides was reduced by introducing point mutations (Tabor S. and Richardson, C. C. (1995)
Proc. Natl. Acad. Sci.
92, 6339-6343). Tabor and Richardson describe the construction of active site hybrids.
The object to provide polymerases with optimized properties was achieved by the present invention for the first time by producing polymerase chimeras by exchanging domains that are structurally and functionally independent of one another. Domains in the sense of the present invention are understood as regions which contain all essential centres or all functionally important amino acids such that the domains essentially retain their function. It is therefore also possible to exchange only parts i.e. functioning fragments of domains. Thus these domains can be referred to as functional amino acid fragments in the sense of the present invention. Furthermore the chimeras can be additionally modified by mutations or truncations. If it appears to be advantageous it is also possible to introduce mutations into the chimeras which further optimize their properties with regard to the respective application. Thus for example mutations can be introduced which reduce the ability of the polymerases to discriminate between dideoxy and deoxy nucleotides. Alternatively desired properties such as processivity can be strengthened or introduced by introducing mutations or by truncation. The introduction of mutations or truncations can also abolish undesired properties e.g. the 5′ nuclease activity.
Thus polymerase chimeras are a subject matter of the present invention which combine advantageous properties of naturally occurring polymerases with regard to a particular application. The polymerase chimeras according to the invention are composed of functional amino acid fragments of different enzymes which preferably represent domains of different enzymes. The invention surprisingly showed that the domains from the different enzymes are active in the chimera and exhibit a cooperative behaviour between the domains. The present invention also concerns general processes for the production of polymerase chimeras with optimized properties. This process according to the invention thus enables a chimera to be designed from an arbitrary combination of enzymes by exchanging domains. It is additionally preferred that the interactions at the sites of contact between the domains are further harmonized by various methods. This can for example lead to an increase in the thermostability of the chimeras. A further subject matter of the invention is a kit for the synthesis of nucleic acids which contains a chimera according to the invention.
Thermostable DNA polymerases with proof-reading function are being increasingly used in practice for PCR. The use of mixtures of Taq polymerase and thermostable proof-reading DNA polymerase (such as Pfu, Pwo, Vent polymerase) has proven to be particularly successful for the amplification of long DNA molecules. Thus a further subject matter of the present invention was to combine the high processivity and thermostability of Taq polymerase with the 3′-5′ exonuclease activity of another DNA polymerase in one enzyme. Hence the present invention especially concerns thermostable polymerase chimeras which have a processivity which corresponds to at least that of Taq polymerase and have a low error rate when incorporating nucleotides into the polymer chain during amplification due to the presence of a 3′-5′ exonuclease activity (proof-reading activity). The combination of these two properties enables for example a chimera to be generated which is able to make long PCR products i.e. nucleic acid fragments which are larger than 2 kb. The chimera according to the invention is also suitable for amplifying shorter fragments.
The present invention therefore concerns in particular a polymerase chimera which is composed of functional amino acid fragments of two different polymerases wherein the first or the second polymerase has 3′-5′ exonuclease activity and the polymerase chimera has 5′-3′ polymerase activity as well as 3′-5′ exonuclease activity. The polymerases can be naturally occurring or recombinant polymerases. The polymerase chimera according to the invention can be composed of fu
Ankenbauer Waltraud
Frey Bruno
Schomburg Dietmar
Sobek Harald
Villbrandt Britta
Benzion Gary
Pennie & Edmonds LLP
Roche Diagnostics GmbH
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