Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-11-08
2003-04-08
Sisson, B. L. (Department: 1653)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C436S094000, C536S023100, C536S025300
Reexamination Certificate
active
06544775
ABSTRACT:
The invention relates to a device and to a process for the automatic synthesis of macromolecules, in particular oligonucleotides, and to the use of such a device.
Synthetic oligonucleotide sequences are routinely produced in machine-assisted processes. Nucleoside amidophosphites, nucleoside H phosphonates or other monomer or oligomer building blocks of biological or abiologically modified nucleic acids which are described in the literature are in this case used as synthons (Sonveaux, E. (1986)
Bioorg. Chem.
14, 274-325; Uhlmann, E. and Peyman, A. (1990)
Chem. Rev.
90: 544-584). The synthesis operations are as a rule carried out with solid phases. Typically, the automatic instruments used for the oligonucleotide synthesis operations are designed in such a way that the substrate materials are used in granulated form. With these as a solid bed in a column, reagents, solvents and other liquid phases flow through them. In a similar way, membranes are used as solid phases which can be washed thoroughly. The liquid phases are generally metered with time control using a process computer in a commercially available synthesis machine. The chain propagation takes place in steps. For each addition of a monomer, a multi-stage reaction cycle is carried out. The yield obtained in a chain propagation cycle is generally measured on separation of the protective group by spectroscopic or other analytical methods.
Examples of granulated substrate materials in common use include silica gel, controlled pore glass, polystyrene, composite materials, etc. in the form of irregularly shaped articles or spheres of varying diameter. For macroporous non-swellable substrate materials, concentrations of the order of 0.8-0.9 &mgr;mol nucleoside/m
2
external and internal surface area have been measured, irrespective of the material (Kotschi, U. (1988)
Dissertation, University of Ulm, Germany
). Routinely, batches in the region of about 0.2 &mgr;mol to 1 &mgr;mol of growing chains are run in machines.
The simultaneous synthesis of a plurality of oligonucleotide sequences can, on the one hand, on purely preparative basis be carried out by increasing the number of columns filled with polymer substrate which are to be operated, and on the other hand substrate-filled cartridges may be incorporated, for example in a stackable arrangement, in parallel in synthesis cycles of, the same type (Seliger, H. et al. (1989)
Bioengineering
6: 144-147).
Recently, surface-functionalized two-dimensional materials have also frequently been used as substrates for parallel oligonucleotide synthesis operations. These include glass plates (Maskos, U. and Southern E. M. (1992)
Nucleic Acids Res.
20: 1679-1684), silicon wafers (Pease, A. C. et al.
Proc. Natl. Acad. Sci. USA
91: 5022-5026), as well as surface-functionalized polypropylene sheets (Matson, R. S. et al. (1994)
Analytical Biochemistry
217: 306-310). The aim with all these substrate methods involves fitting the largest possible number of “synthesis” points on a given substrate area, in order thereby to pursue analytical/diagnostic goals.
The number of oligomer sequences to be synthesized in parallel is limited, in the case of conventional commercially available synthesis machines, on the one hand by the equipment outlay and on the other hand by the arrangement of the substrate elements in the stack. Permanent utilization of the available instrument capacity is therefore not possible because of idle times, since the individual cartridges always need to be manually taken from the synthesis machine and re-connected.
In the two-dimensional substrates used for analytical/diagnostic goals, the amounts of oligonucleotides synthesized per “pixel” are extremely small (in the lower picomol range), that is to say normally unsuitable for preparative purposes. Furthermore, the purity of the oligonucleotide material grafted onto a “synthesis point” is limited by the resolution of the “pixels”, that is to say it is generally very difficult to prevent one oligonucleotide synthesis “spreading” to neighbouring synthesis points.
In contrast to these works, the inventors of the present application even earlier prepared polypropylene sheets by chemical surface functionalization in such a way as to allow nucleoside concentrations of 5-7 nmol/cm
2
(50-70 &mgr;mol/m
2
). Such two-dimensional substrates which are loaded with extremely high concentrations, although exclusively on the surface, make it possible to carry out oligonucleotide synthesis operations on surface segments in the cm
2
range and, this being the case, to produce a sufficient quantity of nucleotide sequences for biochemical/preparative studies (Seliger, H. et al. (1995)
Reactive
&
functional polymers
26:119-126). The extraordinarily good accessibility of the surface concentration for dissolved reagents has been demonstrated by the synthesis of an oligonucleotide comprising 200 bases in a single synthesis operation (Bader, R. et al. (1997)
Nucleotides
&
Nucleosides
16: 829-833).
German patent document DE 42 06 488 A2 describes a device for carrying out simultaneous or sequential chemical reactions, which consists of four rods which are placed on top of one another and whose contact surfaces form an air-tight seal.
European patent application EP 0 385 433 A2 describes a device for continuous polymer synthesis, in which a tape-like substrate sheet is passed through a plurality of dipping baths.
WO 96/15450 describes a microelectronic semiconductor array which is subdivided in grid fashion into individual reaction fields. Reagents can be applied to the individual fields.
An article by J. Weiler et al. (1996,
Analytical Biochemistry
243: 218-227) describes a device for oligonucleotide synthesis and its application in DNA sequencing. A polypropylene sheet is brought into contact with elongate reaction chambers. After a first reaction step, the sheet is rotated through 90° and a further coupling reaction is carried out.
An article by R. S. Matson et al. (1995,
Analytical Biochemistry
224, 110-116) describes oligonucleotide synthesis on polypropylene substrates and its application in the location of genetic defects.
In the dissertation by Raoul Bader, University of Ulm, Germany, 1996, a manually actuable device was described, with the aid of which oligonucleotides can be synthesized on a functionalized tape-like substrate sheet with the use of a conventional synthesis machine (the most important results of this dissertation were described in brief by R. Bader et al. in
Nucleosides and Nucleotides,
16, 835-842 (1997)). The device described there and in Bader's dissertation has a synthesis module which can be sealed from the outside and comprises reaction chambers and fluid lines for filling and emptying the reaction chambers with and of reaction media, it being possible to introduce the tape-like substrate material into the synthesis module and bring it into contact with the reaction chambers by pressing two elements of the synthesis module together.
When the two module elements are again released from one another, the sheet tape can be transported forwards through a particular distance which corresponds to the separation between two synthesis chambers. By transporting the substrate sheet, for example a polypropylene strip, a linear sequence of synthesis fields can be produced on the strip, which contain either sequentially independent or sequentially overlapping oligonucleotides. With this manual device, it was possible to demonstrate the principal function of the synthesis process proposed by Bader et al. The known device is, however, not suitable for the automatic synthesis of macromolecules on a tape-like substrate material, and in particular the accuracy with which the substrate sheet is positioned in the synthesis module is poor with manual adjustment.
The object of the present invention is therefore to provide a device which allows automatic synthesis of macromolecules on a two-dimensional substrate material, in particular on a tape-like two-dimensional substrate material, and which avoids dead times
Bader Raoul
Brugger Hermann
Hofer Eberhard P.
Rembe Christian
Seliger Hartmut
Bacon & Thomas
Merckle GmbH
Sisson B. L.
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