Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-02-21
2003-05-06
Jones, W. Gary (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C436S046000, C435S288500, C422S105000, C422S105000, C156S503000, C156S158000, C156S499000
Reexamination Certificate
active
06559296
ABSTRACT:
This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 09-234145, filed Aug. 29, 1997.
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for detecting a target DNA, a target mRNA, etc. by using a DNA probe.
In recent years, a human genome project, i.e., an attempt to analyze the base sequence in all the human genes, is being carried out worldwide. The human genome analysis including the determination of the base sequence is a very complex and laborious work. It is said, however, that the human genome project will be completed at the beginning of the 21st century. Development of many novel technologies as well as improvement and automation of the analytical equipment greatly contributes to the promotion of the human genome project. A DNA chip technology is one of the newly developed analytical technologies.
The DNA chip is a chip prepared by spotting many kinds of DNA probes at predetermined positions on a substrate, e.g., a silicon wafer, by utilizing the lithographic technology used in the field of semiconductor devices. The DNA probe is formed of an oligonucleotide having a predetermined sequence of 4 kinds of bases constituting DNA, i.e., adenine (A), guanine (G), cytosine (C) and thymine (T). The sequence is complementary to the base sequence of the target DNA or mRNA. For example, if the sequence of AGCTT (5′→3′) is used as a DNA probe, a DNA having a base sequence of AAGCT, which is complementary to the sequence of AGCTT noted above, is hybridized with the DNA probe so as to be selectively caught. Incidentally, the actual constituting unit of the DNA probe is nucleotide having the above-noted base portion (base portion+deoxyribose portion+phosphoric acid ester portion). For simplifying the description, however, nucleotide is represented by the base alone in the following description.
A method of immobilizing a DNA probe on the substrate is exemplified in, for example, “Science 251:767-773” published in February 1991. In this method, a DNA probe is formed on a flat substrate by utilizing a photochemical reaction. Let us describe briefly how to form a DNA probe having a 4-base length on a silicon substrate by this method with reference to
FIGS. 1A
to
1
F.
In the first step, an amino group is formed on a silicon substrate S by treatment with silane, followed by allowing a photo-protective group X to be coupled with each amino group. Then, a desired position is selectively irradiated with an ultraviolet light by using a first mask M
1
. As a result, a protective group X at a desired position is removed so as to expose the amino group, as shown in FIG.
1
A. Then, an optional DNA base (shown by K here) accompanied by the photolabile protecting group is reacted with the exposed amino group, as shown in FIG.
1
B. As a result, a portion in which the base K accompanied by the photolabile protecting group X is coupled with the amino group and another portion in which the photolabile protecting group X alone is coupled with the amino group are formed on the substrate, as shown in FIG.
1
C. Further, the portion in which the photolabile protecting group X alone is coupled with the amino group is selectively irradiated with light through a second mask M
2
so as to selectively remove the photolabile protecting group X in the irradiated portion. Then, an optional DNA base (shown by L here) accompanied by the photolabile protecting group X is coupled with the exposed amino group, as shown in FIG.
1
D. As a result, bases K and L accompanied by the photolabile protecting group X are immobilized to the substrate surface with an amino group (not shown) interposed therebetween, as shown in FIG.
1
E. Further, similar operations are performed by using an optional DNA base (shown by M here) and another optional DNA base (shown by N here). As a result, a DNA probe having a length of two bases is immobilized to the substrate surface. Still further, similar operations are repeated to stack bases in a three dimensional direction so as to form DNA probes each having a length of four bases, said DNA probes having different base sequences depending on the process units, as shown in FIG.
1
F. For forming a base sequence having a length of, for example, eight bases, photolithography using 32 masks and the photoreaction are repeated 32 times so as to form DNA probes having all the desired base sequences. It is theoretically possible to form efficiently DNA probes each having an optional base length and an optional base sequence on a single substrate by utilizing the above-described technology.
An improved method of manufacturing a DNA chip is disclosed in International Laid-open Application WO 93/096668 (Japanese translation version No. 7-506561). In this method, a DNA probe is immobilized on a silicon substrate having an amino group formed thereon by using a flow type channel block. The flow type channel block used in this method is a pattern plate having a plurality of slender channels. In the case of using the pattern plate, it is possible to immobilize the DNA probe along each of these channels. In this case, the base sequence of the DNA probe differs depending on the channel. However, the base sequence is the same over the entire length in a single channel. In this preferred mode of the conventional technology, an amino group is attached first to the substrate, followed by combining the substrate with a block having a plurality of channels arranged in parallel. Then, a process solution containing a base, which is a constituting unit of the selected DNA probe, is allowed to flow through a predetermined channel so as to immobilize the first base of the aimed DNA probe. Further, the substrate and the channel block are rotated relative to each other by a predetermined angle, e.g., 90°, followed by combining again the substrate and the channel block and subsequently immobilizing a base corresponding to the second base along the channel. A DNA probe having a desired base sequence can be prepared by successively repeating the above-noted steps. Also, a large number of DNA chips referred to previously can be manufactured in a single operation by combining the method described above with the photochemical reaction. It should also be noted that screening can be performed by using the DNA chip prepared by the above-noted method in combination with the channel block described above.
In order to prepare the conventional DNA chip, it is necessary to carry out mutual reactions among a large number of reagents on a flat substrate in immobilizing the probe. Also, when the DNA chip is used for measurement, it is necessary to apply many times a liquid material to a surface of a flat DNA chip in order to carry out reactions with a sample and to wash the DNA chip surface. In order to apply the above treatment to a flat substrate (or DNA chip), the substrate (or the DNA chip) must be dipped in a reactant solution filling a container. Alternatively, a flow path must be formed on the DNA chip surface by using an additional tool such as the channel block noted above, followed by applying the treatment with the liquid. However, the dipping method gives rise to a problem that each kind of the treating liquid must be used in an excessive amount in the immobilizing step and in the sample measuring step. On the other hand, in the method using a flow path, only a limited region of the surface of the DNA chip having a DNA probe immobilized thereon is used, resulting in failure to utilize sufficiently the formed DNA chip. Further, the DNA chip is an open system in which the probe-formed surface is exposed to the outside, giving rise to the defects that the surface tends to be contaminated and that the DNA chip cannot be handled conveniently.
The conventional DNA chip technology gives rise to an additional problem besides the problems given above. Specifically, the conventional DNA chip technology is certainly effective for performing the sequencing of DNA having an unknown base sequence, but is not adapted for the d
Chakrabarti Arun K.
Frishauf Holtz Goodman & Chick P.C.
Jones W. Gary
Olympus Optical Co,. Ltd.
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