Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-13
2003-08-26
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S174000, C435S177000, C435S320100, C435S440000, C435S810000
Reexamination Certificate
active
06610479
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the activation of enzymes by their release from an immobilizing moiety, and in particular heat-mediated release.
BACKGROUND OF THE INVENTION
The increasing availability of enzymes from various organisms with specific defined activities has led to the use of these catalysts as reagents in many in vitro and in vivo systems. Notably, methods of detection and analysis in the area of molecular biology require the use of a least one of the enzymes involved in DNA/RNA replication, transcription and/or translation. Precise control of the activity of these enzymes is generally achieved through precise knowledge of their pH, temperature, ionic strength and cofactor requirements and the consideration of other criteria essential for their working. Not only is the ability to control activation of these enzymes important, but also the ability to inactivate the enzymes, generally reversibly. The capacity to turn the activity of an enzyme on and off is often crucial to the correct functioning of a particular analytical or diagnostic assay.
For many years a limitation of molecular biological methods was the difficulty in obtaining sufficient amounts of homogeneous DNA for further analysis. This problem was largely overcome by the development of the Polymerase chain reaction (PCR) method which has, since its inception in the late 1980s, been responsible for many of the advancements in the genetic engineering field. A number of related amplification techniques employing the same principle as PCR have evolved from the basic concept of PCR, namely cycles of replication, denaturation and reannealing with suitable primers.
In order to obtain an amplified DNA product which is homogeneous, strict regulation of the cycles is required, in terms of time and temperature, the activity of the enzymes employed, for example the Taq polymerase, the choice of primers and the conditions of hybridization and denaturation. Thus, ideally, primers which anneal to complementary strands of target double stranded DNA are added to the DNA which has been denatured. The primers are then annealed under suitable conditions of stringency to prevent binding to non-complementary sequences. DNA extension along the length of the DNA template 3' of the annealed primer is then performed using a suitable DNA polymerase. After a desired extended product is achieved, denaturation conditions are effected to allow separation of the parent and daughter strands which may then reenter the cycle.
In the laboratory situation, temporal control of each consecutive event is not routinely performed and a reaction mix is employed in which the change of temperature during the course of the cycle is the initiator of the consecutive steps. However, reliance on such temperature control can lead to problems resulting in a heterogeneous product. For example, in experimental procedures in which a reaction mix is heated to a temperature of around 90° C. to effect denaturation, if all the components necessary for the polymerase reaction are present during the heating step, spurious annealing of the primers to non-complementary strands and subsequent extension may occur, resulting in amplification of non-target DNA. Although efficiencies of thermostable DNA polymerases are greatly reduced at ambient temperature relative to their peak efficiencies at higher temperatures, sufficient activity may be present at ambient temperatures to cause PCR side-products. Commonly, dimerized primer-amplified fragments (“primer dimer”) as well as larger non-specific side-reaction products (mis-primed products) are obtained. The non-specific fragments can vary in size and yield, are primer sequence dependent and are most likely to arise from reactions using complex (e.g. genomic) DNA. Such non-specific fragments have been observed to reduce the yield of desired specific fragments through competition with the specific target in the reaction. Furthermore, non-specific products that are approximately the same size as the specific product can cause confusion when interpreting results. PCR amplifications particularly prone to generation of a variety of side reaction products include those involving one or more of the following: complex genomic DNA or cDNA templates; degenerate primers; very low-copy-number targets; large numbers of thermal cycles (i.e. >35); more than one target sequence in the same tube (i.e. multiplex PCR).
This has led to the introduction of a number of techniques for initiating the cycle at the temperature of denaturation, the so-called “hot start” method (Chou et al. (1992) Nucl. Acids Res., 20, 1717-1723; D'Aquila et al. (1991) Nucl. Acids Res., 19, 3749; Faloona et al. (1990) 6th International conference on AIDS,. San Francisco, Calif., USA, Abstract No. 1019). “Hot start” methods have found particular utility for long range PCR. For many years PCR was restricted to amplification of a few thousand bases. However, successful amplification of up to 40 kbp has been achieved using a mixture of different thermostable enzymes employing the “hot start” procedure. If the “hot start” procedure is not used in this case, comparatively short non-specific products are preferentially amplified. The “hot start” procedure is also beneficial when low-copy-number targets are to be amplified or for in situ PCR.
The original approach to achieve “hot start” of the polymerase reaction was to withhold an essential reagent of the reaction (for example the DNA polymerase, MgCl
2
, primers, deoxyribonucleoside triphosphates and/or DNA sample) until the reaction mixture was heated to a high temperature (e.g. >55° C.), followed by the addition of the missing component. Another approach is the use of a heat-labile wax or jelly barrier that melts and permits mixing of aqueous components at an elevated temperature. However, both these “hot start” methods suffer from the drawback that they have increased probability of crossover contamination on reopening the reaction tubes and that they are cumbersome and time-consuming when working with multiple samples.
An alternative to “hot start” approaches which prevents PCR product carryover to subsequent cycles and allows the addition of all components of the PCR reaction at one time, involves the use of dUTP and the DNA repair enzyme uracil-N-glycosylase (UNG) in PCR reactions. In this method UNG digests the dU-incorporated nonspecific products before thermal cycling commences, thereby reducing the amplification of these side products in the reaction (Kwok et al. (1992) 92nd Gen. Mtg. Am. Soc. Microbiol., 116 (Abstract No. D-120). However, this method is not widely used owing to the added expense of the additional reagents and the reduced yield of specific products which may result as a consequence of using dUTP in PCR.
A “hot start” method which allows the addition of all components of the PCR reaction at one time employs an antibody marketed by Clontech Laboratories, Palo Alto, California, USA, which binds to and inactivates Taq polymerase at ambient temperatures, but releases the active DNA polymerase once the high temperatures (above 70° C.) have been obtained. However, not all antibodies are suitable for this methodology as it was found that of the IgGs derived from 12 hybridoma clones whose supernatants had affinity for Taq DNA polymerase, only the IgGs from 4 of the clones inactivated Taq polymerase in solution. The remainder although having affinity for the polymerase did not block its activity. Furthermore, this method suffers from the drawback that the active polymerase is released from the inactivating antibody at a particular temperature which cannot be manipulated to suit the particular requirements of different PCR reactions.
SUMMARY OF THE INVENTION
Surprisingly, it has now been found that enzymes may be reversibly inactivated by attachment to an immobilizing moiety, and activated by their release from said moiety. In particular, it has been found that Taq polymerase may be inactivated when immobilized, but may be activated by disruption of the association
Lundeberg Joakim
Uhlen Mathias
Dynal AS
Fish & Richardson P.C.
Naff David M.
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