Optical waveguides – Optical fiber bundle
Reexamination Certificate
2002-07-23
2004-11-16
Healy, Brian (Department: 2874)
Optical waveguides
Optical fiber bundle
C606S002000, C606S003000, C606S010000, C606S011000, C606S012000, C606S013000, C606S014000, C606S015000, C606S016000
Reexamination Certificate
active
06819843
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention concerns a device and a method for the photolithographic exposure of biological substances.
DNA chips are ultra-small, generally planar surfaces, on which a large number of different oligomers (short, single-stranded DNA molecules) are introduced in a spatially organized manner. Such chips are used, for example, for the parallel recognition of numerous DNA sequences in a prepared tissue specimen. For this purpose, the chip surface is wetted with a solution of single-stranded DNA segments from the tissue specimen. Complementary DNA segments from the solution are deposited on the corresponding oligomers introduced on the chip surface (hybridization). After this, a determination is made of which places on the chip a hybridization has occurred by means of suitable methods, such as, e.g., fluorescent labeling. If one knows where on the chip the respective oligomers are introduced, conclusions can be made relative to the DNA sequences in the tissue specimen. For this purpose, usually a dense rectangular grid is defined on the chip support surface. One type of oligomer is introduced at each grid point in the form of a small spot. The maximum possible number of different DNA sequences on the chip is consequently equal to the number of grid points. Since one wishes to introduce as many types of oligomers as possible on one chip, but the chips must be as small as possible at the same time in order to be able to effectively hybridize, it is an important objective in the production of DNA chips to achieve as high a grid density as possible.
Different methods for DNA chip production are known in the prior art:
1) All oligomers are synthesized individually in a conventional manner in the test tube and then are pipetted onto the provided grid points on the support, typically by an automatic micropipetting device. This method is very time-consuming and expensive, since each oligomer must be prepared or purchased individually and must be introduced by hand into the pipetting device. The grid density is limited by the high angular imprecision of the typical piezoelectric micropipettes that are presently available.
2) The oligomers are synthesized directly on the chip by means of an automatic pipetting device. The oligomer chain provided on each grid point is built up base by base (nucleobases). The chemical method is basically the same as in conventional oligomer synthesis in the test tube. The difference is that all oligomers are produced simultaneously directly at the provided determination site by a single automatic device. The separate operating steps of oligomer synthesis and micropipetting of method 1) are thus combined into one uniform operating step. This in-situ synthesis normally proceeds as follows: The automatic pipetting device sequentially drops the first nucleobase provided for each grid point onto a prepared substrate. This is mechanically not very time-consuming or expensive, since there are only 4 different nucleobases (C, T, G, A). For example, 4 micropipettes coupled to one another can be used for this purpose. After applying the first nucleoside building block at each grid point, the substrate is washed and after a “capping step”, the protecting groups at the 5′—OH functions are removed, in order to make possible the reaction with the respective subsequent nucleoside building block. After this, the second nucleobase is pipetted onto each grid point. The substrate is then washed again and deprotected. In this way, the necessary oligomer chains are constructed step by step on each grid point. This method is not particularly rapid, since each nucleobase must be newly pipetted one after the other onto each grid point. As in the case of method 1), the grid density is limited by the imprecision of the micropipettes. The imprecision is even worse here, since each grid point must be contacted several times sequentially in a way that is as identical as possible.
3) The oligomers are synthesized directly on the support as in 2), but the targeted binding of the correct nucleobases to the correct grid points is done by means of a completely parallel, photolithographic technique instead of sequential, target-precise pipetting steps. The method is based on the fact that the 5′—OH protecting groups of oligonucleotides can be removed in a targeted manner by light of a specific wavelength. By suitable local irradiation patterns, oligonucleotide ends can thus be made capable of reaction at precisely those grid points at which one wishes to introduce a new nucleoside in the next step. By complete wetting of the chip surface with a nucleotide building-block solution, a nucleotide base is thus bound only to the sites that have been previously exposed, and all unexposed sites remain unchanged. The local exposure patterns are produced by positioning a photomicrographic black-white mask between the substrate and the light source. The mask covers all of the grid points, which are not to be made capable of reaction. The elongation of the oligomer chains by one nucleobase at all grid points is then conducted as follows: Those grid points which must be extended by the first of the 4 possible types of nucleobases (e.g., C) are precisely exposed by means of a first mask. Then the chip is wetted with a solution of the corresponding nucleotide base, whereupon only the exposed points are elongated by this base. Since the newly bound bases all have a protecting group, they do not further react in the following steps until their protecting groups are cleaved by another exposure. The chip is washed after this reaction step. Now, those grid sites, which must be elongated by the second of the 4 possible types of nucleobases (e.g., T) are precisely exposed by means of a second mask. Then the chip is again wetted with a solution of the corresponding nucleotide building block and the exposed sites in this way are elongated by this base. The procedure is the same for the remaining two bases (e.g., G and A). For the elongation of all oligomers by one nucleobase, one consequently requires four exposure steps and 4 photomasks. This method is very efficient due to the high parallel operation, and it is also suitable for obtaining very high grid densities, due to the high precision that can be obtained with photolithography. Of course, the method is very time-consuming and thus expensive, since a large number of photomasks must first be created for the production of a specific type of chip. Also, rigid requirements are placed on the positioning accuracy of the masks during exposure in the case of high grid densities, and these requirements can be fulfilled efficiently only by using very expensive apparatus.
4) The same method is applied as in 3), but instead of the large number of photographic masks, only a single, transmissive, liquid crystal display, which is controlled electronically and serves as a dynamic mask, is used. This method is simple and inexpensive, since photographic masks need not be produced and there is thus no positioning problem. One possible problem of this method is the limited optical contrast of the liquid crystal displays that are currently available (maximum 1:100). The light intensity ratio between exposed and covered points is thus reduced, which can have as a consequence a reduction in yield in the case of oligomer synthesis.
These methods of the prior art have a number of disadvantages. Of the above-described production methods for DNA chips, the photolithographic method with dynamic liquid crystal masks is the only one that permits a simple, inexpensive and reliable production of chips with high grid density. The deficient contrast of liquid crystal displays, however, has as a consequence a reduction in the quality of the oligomer points, which in the final analysis reduces the detection sensitivity of the chip.
SUMMARY OF THE INVENTION
The object of the present invention is thus to create a device, which overcomes the disadvantages of the prior art. Another object of the invention is the creation of another method for the photolithogr
Braun Aron
Heuermann Arno
Epigenomics AG
Healy Brian
Kriegsman & Kriegsman
Petkovsek Daniel
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