Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
2001-04-05
2003-04-29
Whisenant, Ethan (Department: 1655)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C435S006120, C435S091100, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06555383
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a test piece that is employed in deoxyribonucleic acid (DNA) analysis and immunological analysis and to a method and apparatus for measuring the quantity of an organism-originated substance using the test piece.
2. Description of the Related Art
It is now considered possible that the human genome project for determining and analyzing all the base sequences of a human genome as huge as about 3000 Mbp will be completed sooner than the original scheduled date and determined by 2003, and the focus of the human genome project is now shifting from systematic base sequence determination to systematic function analysis.
The specific content of generic information comes down to what protein is synthesized and under what condition. With respect to the former, i.e., what protein is synthesized, methods of analysis, such as Western blotting, Northern blotting, and Southern blotting, are hitherto well known. These methods can analyze what a specific protein, DNA, and ribonucleic acid (RNA) sampled are, but are not necessarily suitable for analyzing all proteins, DNA, and RNA, extracted from a cell, at the same time.
On the other hand, regarding the latter, i.e., under what conditions protein is synthesized, the conventional methods of analysis cannot perform sufficient analysis, because protein is controlled at a transfer level. The main reason for this is that control data of both the base sequence in DNA and the corresponding contents are insufficient.
However, with the latest advancements in techniques for fixing arbitrary oligonucleotide with high density on the surface of a 1-centimeter-square carrier called a DNA chip or a DNA micro array chip, it is expected that the analysis of gene expression information will increasingly advance. The DNA chip is formed by dividing a silicon chip into a plurality of sections using photolithography and directly synthesizing single-stranded DNA having a specific base sequence on each section. As to the DNA micro array chip, a DNA macro array chip having a spot size of about 300&mgr; or more previously blotted on the membrane is reduced to a spot size of about 200&mgr; or less and blotted on a slide glass. The DNA chip or the DNA micro array chip is connected to a signal reader and a computer system, and it can be known which probe DNA is hybridized by the DNA disposed on the chip or the micro array chip. Depending on the DNA type disposed on the DNA chip or the DNA micro array chip and the disposition, it is possible to employ the chip or the array chip in various analyses such as DNA mutation analysis, DNA polymorphism analysis, DNA base sequence analysis, and DNA expression analysis.
The analysis employing the DNA micro array chip, however, still has quite a number of problems because discussions about array chip generation and a detector thereof have only just started. For instance, the micro array chip is made by blotting complementary DNA (cDNA) by means of a spotter, and as a method of generating the micro array chip, there is a contact printing method and a non-contact printing method. In the contact printing method, cDNA
43
is disposed on a slide glass
42
by a pin
41
in direct contact with the slide glass
42
, as shown in FIG.
4
A. In the non-contact printing method, cDNA
43
is blotted on the slide glass
42
by a syringe
44
in non-contact with the slide glass
42
, as shown in FIG.
4
B. In both the printing methods, however, there is a difference in quantity between blotted spots. Even in the best case, there is a quantity difference of 5 to 10% CV for the contact printing method and a quantity difference of 3 to 5% CV for the non-contact printing method. Further, there are sometimes defect spots and spoiled spots. For this reason, DNA varies in quantity between spots a and b on a DNA micro array chip
151
, as shown in FIG.
5
. DNA also varies in quantity between spot a on the DNA micro array chip
51
and spot a on a DNA micro array chip
152
generated in the same way. Because of this difference in quantity, there is a problem that the quantitative analysis of different DNAs generated from a single cell and the quantitative comparison of DNAs in the same cell differing in quantity of expression at a different time will actually include a considerable error.
To solve this problem, an improvement in the spotter was initially considered, but improvements to enhance the reproducibility of the quantity of a sample to be spotted are considered to have limits.
SUMMARY OF THE INVENTION
The present invention has been made in view of the aforementioned circumstances. Accordingly, it is the object of the present invention to provide a test piece, such as a DNA micro array chip, which is capable of performing accurate quantitative analysis even when there is a difference in quantity between blotted spots, without relying on an improvement in a spotter which enhances the reproducibility of the blotted spots.
To achieve the aforementioned object of the present invention and in accordance with one aspect of the present invention, there is provided a test piece for analyzing an organism-originated substance labeled with a second labeling substance. The test piece comprises a carrier on which a plurality of known specific binding substances differing from one another are disposed at a plurality of predetermined positions. The specific binding substances are labeled with a first labeling substance.
Since a plurality of known specific binding substances differing from one another are disposed at a plurality of predetermined positions on a carrier and labeled with a labeling substance, the quantity of a specific binding substance disposed on the test piece can be specified regardless of a difference in quantity between the specific binding substances that is caused when they are disposed on the carrier.
In accordance with another aspect of the present invention, there is provided a quantitative method comprising the steps of:
detecting a level of a first labeling signal emitted by a first labeling substance, which labels a plurality of known different specific binding substances respectively disposed at a plurality of predetermined positions on a carrier of a test piece, for each of the plurality of predetermined positions;
binding an organism-originated substance, labeled with a second labeling substance differing from the first labeling substance, to the specific binding substance and detecting a level of a second labeling signal emitted from the second labeling substance for each of the plurality of predetermined positions; and
measuring a quantity of the organism-originated substance bound to the specific binding substance, based on the detected level of the first labeling signal and the detected level of the second labeling signal.
In accordance with still another aspect of the present invention, there is provided a quantitative apparatus comprising:
first detection means for detecting a level of a first labeling signal emitted by a first labeling substance, which labels a plurality of known different specific binding substances respectively disposed at a plurality of predetermined positions on a carrier of a test piece, for each of the plurality of predetermined positions;
second detection means for detecting a level of a second labeling signal emitted by a second labeling substance, which differs from the first labeling substance and labels an organism-originated substance bound to the specific binding substance, for each of the plurality of predetermined positions; and
analyzing means for measuring a level of the organism-originated substance bound to the specific binding substance, based on the detected level of the first labeling signal and the detected level of the second labeling signal.
According to the quantitative method and the quantitative apparatus of the present invention, a level of a first labeling signal emitted by a first labeling substance, which labels a plurality of known different specific binding substances respectively disposed at a pl
Fuji Photo Film Co. , Ltd.
Lu Frank
Sughrue & Mion, PLLC
Whisenant Ethan
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