Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-25
2003-03-18
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200
Reexamination Certificate
active
06534271
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for cloning and copying genetic material on surfaces as well as copying biological material insofar as it can be classified in a broader sense in a ligand-receptor system.
2. Background Information
Methods of exponentially amplifying molecular matrices are already known through the work of G. von Kiedrowski et al. (Nature 1998, Vol. 346, 245-248; Federal Republic of Germany Patent No. 198 48 403). The amplification cycles are characterized by:
binding of molecular matrices to the surface of a solid phase by means of a reversible linker on the matrix;
addition of matrix fragments, with one of the fragments displays a linker unit, which may, if necessary, be protected;
synthesizing copies of the matrix;
removal of superfluous matrix fragments and ancillary reaction substances;
detachment of the copies from the matrix; and
application of synthesized matrix copies to free binding sites on the solid phase.
This represents an iterative, progressive amplification method, allowing an exponential increase in the amount of molecular matrices available, thus enabling a significant process of evolution to take place. To achieve this the process makes use of the surface of a solid carrier. Chemical attachment to immobilized matrices enables copies to be synthesized from precursor matrices, which are then released to become new matrices. This process can be repeated any number of times.
In addition, so-called “bridge” amplification technology is described in U.S. Pat. No. 5,641,658. This is an amplification model based on conventional PCR methods, but which is intended to achieve localized amplification. Bridge amplification technology has many uses, especially in analytical methods that can also be carried out with the commonly used PCR. The bridge technology facilitates the separation and detection stages of the amplified products. The characteristics of this technology are that it combines amplification, selection and detection in a single process. Advanced state of the art systems can be found on the homepage of MOSAIC Technologies, Inc. (USA), the company marketing bridge amplification technology (www.mostek.com).
The bridge system describes a method for amplifying nucleic acids on a solid phase, with both amplification primers being bound covalently to a single solid phase through their 5′ ends. Consequently this represents a further development of the well-known polymerase chain reaction, known as PCR for short. This takes place in a solid phase PCR instead of in a solution. The particular advantage of this method is its ability to amplify and analyze many different genetic elements simultaneously using a single sample. The applications for bridge amplification technology include genetic expression, genome research, clinical diagnostics and the examination of biological fluids, e.g. blood. A higher rate of amplification is achieved by eliminating ineffective primer artifacts (such as primer dimers). This enables simple, sensitive and cost-effective DNA detection methods to be developed, for example using fluorescence. Because bridge amplification technology ensures that all amplification products remain bound to the solid phase, contamination through prolongation remains low, and this in turn enhances the diagnostic value of the method compared with the usual PCR.
Whereas the method proposed by G. von Kiedrowski et al. referred to above demonstrates the benefits of the solid phase amplification of entire populations, the method described in U.S. Pat. No. 5,641,658 offers the advantage of amplifying a single matrix on a solid phase. However, the drawback of this bridge amplification method is linked with the problem of product inhibition, i.e. a newly produced copy may occur not only with the adjacent immobilized primer, but also with the original matrix strand, which is also adjacent. Another disadvantage is the lower linear limitation needed to achieve bridging as a double strand. Furthermore, there is no separation between the strands, with the result that, for diagnostic purposes, the hybridization signals are weakened due to hybridization with complementary strands.
Federal Republic of Germany Patent No. 694 09 646 T2 describes a method for amplifying a nucleic acid, in which the one primer is bound to a solid phase and the second primer to a particle that reacts with a magnetic field. These primers are incorporated in target nucleic acid sequences. Following an extension stage the nucleic acid strands are separated by the application of an electric current. The magnetic primer can be particle-bonded, existing as a form of solid phase. The avidin/biotin system is suitable for binding the primer to the solid phases. This method is also suitable for cloning.
U.S. Pat. No. 5,795,714 describes a method which, in one form, uses an array of oligonucleotides, which are connected to the surface of the solid phases by means of the reciprocal reaction between biotin and avidin. The method described consists of the hybridization of complementary strands, primer extension reactions, the hybridization of a second biotinylized primer to the primer extension products, and the extension of the second primer. Mention is made of the blotting of copies on a second surface which is coated with avidin.
The disadvantage of the methods referred to above is that multiple replication in the sense of exponential propagation is not possible, and that the translocation of the copies by means of an electromagnetic field cannot be achieved without loss of site information. Based on this state of the art, and avoiding the shortcomings referred to above, it is therefore the task of this invention to provide a method of cloning and copying onto surfaces which permits the propagation of biological material while retaining site information.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages of current amplifications methods and enables biological systems including, nucleic acids, ligands and receptors, to be propagated and separated from one another by means of an electric field for immobilization and fixation on one or more solid surfaces, while retaining site information.
The present invention considers a biological system to be basically an interaction between nucleic acids of any kind and/or with peptides/proteins/polymerases/enzymes (DNA/RNA/PNA/pRNA/2′-5′ nucleotides and RNA/DNA mirror mers (see PCT/EP97/04726)), in exactly the same way as antigen/antibody complexes or, in general terms, ligand/receptor systems.
For a basic understanding of the invention it is necessary to appreciate that complementary nucleic acids themselves represent nothing more than a special form of a complementary ligand/receptor system in a traditional sense. For purposes of the present application, the term “ligand” will refer to one molecule of the biological binding pair and the term “receptor” will refer to the opposite molecule of the biological binding pair. Two complementary strands of nucleic acid are biological binding pairs. One of the strands is designated the ligand and the other strand is designated the receptor. Biological binding pairs may also comprise antigen and antibodies, drugs and drug receptor sites and enzymes and enzyme substrates.
The invention utilizes the fact that, because of their charge, nucleic acids and many other biologically relevant molecules can be moved within an electrical field when such a field is applied. In the present case a stationary, bound molecule is separated from a corresponding molecule in this way, in that the non-stationary, bound molecule is either separated from the other by synthesization of that molecule or following an “identification reaction”, with the aid of an electrical field. Because the molecules tend to migrate along the line of the electrical field, they retain site information while migrating. This is also what mainly distinguishes the method referred to in this invention from the previously known state of the art, because in
Fürste Jens Peter
Klein Thomas
Klussmann Sven
Von Kiedrowski Günter
Horlick Kenneth R.
Nils H. Ljungman & Associates
Noxxon Pharma AG
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