Method for reversible modification of thermostable enzymes

Details Method for reversible modification of thermostable enzymes Method for reversible modification of thermostable enzymes

1998-10-31

2001-02-06

Brusca, John S. (Department: 1634)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C530S350000

Reexamination Certificate

active

06183998

ABSTRACT:

FEDERALLY SPONSORED RESEARCH
Research relating to the invention described below was supported under German BMBF Project Number 0311018.
FIELD OF THE INVENTION
The present invention provides a method for reversible inactivation of thermostable enzymes by chemical modification under aqueous conditions. This chemical modification of thermostable enzymes has surprising effects in applications in the field of molecular biology such as nucleic acid amplification.
BACKGROUND OF THE INVENTION
The most important nucleic acid amplification technology is the Polymerase Chain Reaction (PCR) which was first described by Saiki et al.,
Science,
230:1350-54 (1985) and is also disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,965,188. Commercial vendors, such as QIAGEN GmbH, Hilden, Germany, market PCR reagents and kits, and provide protocols for PCR.
The principle of PCR is basically described by the amplification of specific nucleic acid sequences of a nucleic acid target template, using at least one, two or several target-specific oligonucleotides (primers), a thermostable nucleic acid polymerase, deoxyribonucleoside triphosphates and a reaction buffer. DNA synthesis initiates at the accessible 3 ′—OH group of the target-specific oligonucleotides flanking the DNA sequence to be copied, thereby generating an identical copy of the target template nucleic acid sequence. The enzymatic reaction is repeated for a substantial number of thermal cycles consisting of the denaturation of the target nucleic acid, annealing of the primer oligonucleotides to complementary nucleic acid sequences and the subsequent extension of these primer-template complexes using a thermostable nucleic acid polymerase in a sequence-dependent manner. Hybridization of primers occurs usually at sufficiently high temperature to provide annealing conditions that ensure binding of the oligonucleotide primers mainly to its complementary target nucleic acid sequence. However, PCR reaction mixtures are often assembled at room temperature, thus providing much less stringent oligonucleotide hybridization conditions, at a temperature at which most thermostable nucleic acid polymerases possess DNA synthesis activity. Since non-specifically annealed and extended oligonucleotides lead to formation of non-specific amplification products, these mis-primed non-specific PCR products can compete during subsequent PCR cycles with the specific PCR product for primer molecules, polymerase and nucleotides, thereby severely interfering or even abrogating the amplification reaction of the specific amplification product (see, Chou et al.,
Nucleic Acids Research,
20(7): 1717-1723 (1992)).
To overcome difficulties related to non-specific amplification products caused by the extension of mis-primed oligonucleotides during the reaction set-up or the initial heating phase of PCR, an essential PCR component such as the oligonucleotide primers, nucleotide triphosphates, magnesium ions or thermostable nucleic acid polymerase could be added only at higher temperatures, thereby reducing the probability of having non-specific hybridization or extending mis-primed oligonucleotides. This technique is commonly known as “hot-start PCR”, or more specifically “manual hot-start PCR”.
Another method, described in U.S. Pat. No. 5,411,876, employs a solid wax-barrier between the template-primer mix and the remaining reaction mixture. This wax-barrier melts only at elevated temperature, so that all of the reaction components are mixed only at high temperature, preventing mis-priming and extension of mis-primed oligonucleotides. However, as in the case of the manual hot start PCR, the wax-mediated hot start procedure carries a higher risk of contamination and is less convenient, due to increased time necessary for sample processing and due to the solid wax-barrier that forms above the reaction mixture after finishing PCR.
Extension of mis-primed oligonucleotides can also be prevented by pre-incubating the primers with a compound that binds specifically to single-stranded DNA in a heat-reversible manner, such as a single-strand binding protein. Such a compound would prevent the oligonucleotide primer from hybridizing to any template sequence at ambient temperature. For instance, the use of Gene 32 protein, a single stranded DNA binding protein, was shown to improve the yield of PCR products in Schwarz et al.,
Nucleic Acid Research,
18(4): 10 (1990).
Another method of reducing formation of extension products from mis-primed oligonucleotides during the reaction set-up is a reversible non-covalent modification of the nucleic acid polymerase. U.S. Pat. No. 5,338,671 discloses the use of antibodies specific for the nucleic acid polymerase to inhibit the polymerase's activity. Pre-mixing of nucleic acid polymerase and polymerase-specific antibodies results in the formation of an antibody-polymerase complex. Under these conditions substantially no oligonucleotide extension activity can be detected. At elevated temperatures, the antibody dissociates from the complex, thus releasing the nucleic acid polymerase, which can then function in DNA synthesis during the Polymerase Chain Reaction. However, this method carries the risk of contamination due to an increased number of handling steps and the possible presence of residual nucleic acids derived from the antibody preparation. Another method to reduce non-specific amplification products involves the use of a chemically modified thermostable DNA polymerase that becomes active only after incubation of the DNA polymerase for a certain period of time at elevated temperature, thus preventing production of non-specific DNA synthesis products during reaction set-up and the initial heating phase of PCR. U.S. Pat. No. 5,677,152 and corresponding European patent publication EP 0 771 870 A1 describe a method for amplification of a target nucleic acid using a thermostable polymerase reversibly inactivated using a dicarboxylic acid anhydride.
Standard protocols of molecular biology applications, enzymology, protein and nucleic acid chemistry are well described in printed publications such as
Molecular Cloning
-A Laboratory Manual, Cold Spring Harbor, N.Y. (Sambrook et al. 1989); PCR Protocols-
A Guide to Methods and Applications,
Academic Press, N.Y. (Innis et al., eds, 1990), PCR Primer-
A Laboratory Manual,
CSHL Press (Dieffenbach and Dveksler, eds., 1995); and
Methods in Enzymology,
Academic Press, Inc. All of the patents, patent applications, and publications cited herein are incorporated by reference.
SUMMARY OF THE INVENTION
The present invention provides methods and reagents for reversible inactivation of thermostable enzymes using a chemical modification under essentially aqueous conditions. In particular, the thermostable enzymes of the present invention are reversibly modified in the presence of an aldehyde. The modified thermostable enzymes of the present invention do not show significant increase in enzyme activity at 37° C., even when incubated for periods of an hour or more. On the other hand, enzymatic activity of the present chemically modified enzymes is increased at least two-fold within thirty minutes when incubated at a more elevated temperature, i.e., above 50° C., preferably at a temperature of 75° C. to 100° C., and most preferably at 95° C. Such chemically modified enzymes may be employed in all applications involving manipulation of nucleic acids, such as amplification, ligation, exonucleolytic or endonucleolytic reactions, or nucleic acid topology changing enzymatic reactions, wherein the inactivated enzyme becomes reactivated by incubating the reaction mixture prior or as part of the intended enzymatic reaction at an elevated temperature.
One major aspect of the modification is crosslinking of enzyme molecules, thereby limiting enzyme structure flexibility and accessibility of functional core region(s) of the enzyme. The great advantage of this method is its broad applicability, since the inactivation and reactivation of the thermostable enzyme is essentially independent of pH, which in g

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