Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-26
2001-02-06
Brusca, John S. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06183970
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a probe chip for assaying various detection items about objects to be detected, such as DNA, RNA and proteins, at a time (i.e., a many item-detecting sensor), and in particular to a polynucleotide probe chip for assaying DNA and a polynucleotide detection method using the same.
The genome project rapidly approaches the stage of functional genomics. DNA analysis has been clarifying the mechanism of livings and living phenomena. And the DNA analysis has been used for diagnosis of the many diseases. It is effective for understanding living phenomena and examining the effect of genes that the situation of expression of the genes is examined. As a high-powered means, a DNA probe array or a DNA chip wherein a great deal of DNA probes are divided depending on each kind thereof and fixed on the surface of a solid starts to be used. As a method for preparing this DNA chip, there are known a method of using photochemical reaction and lithography, which is widely used in the semiconductor industry, to synthesize oligomers having a designed sequence, one base by one base, on many cells divided up to areas (prior art 1: Science 251, pp. 767-773 (1991)); a method of planting a DNA probe one by one in every area (prior art 2: Proc. Natl. Acad. Sci. USA 93, pp. 4913-4918 (1996)); and the like. In order to increase the amount of probes fixed on a chip, a method is contrived wherein an acrylamide gel film is formed on a chip and probes are fixed on this gel (prior art 2). As a method for fixing DNA probes used in a DNA probe chip, there are known a method of using the bonding of biotin and avidin or fixing DNA probes on the surface of gold (Au) through a SH residue (prior art 3: Biophysical Journal 71, pp. 1079-1086 (1996)); a method of fixing DNA probes on the surface of glass (prior art 4: Analytical Biochemistry 247, pp. 96-101 (1997)); a method of fixing DNA probes on an element matrix of acrylamide gel applied on the surface of glass (prior art 2), and the like.
SUMMARY OF THE INVENTION
Not only DNA or its derivative but also RNA or its derivative may be held on the surface of a chip. Therefore, a chip which holds any polynucleotide on its surface is referred to as a polynucleotide probe chip, hereinafter. In both of the prior arts 1 and 2, there remains problems that their methods for producing DNA chips require much labor and time and costs for the production are high, in particular a problem that the production of a DNA chip composed of minutes parts having closely formed probe arrays requires far more labor and time. That is, usual users cannot form the chip easily. In examination using a DNA chip, there generally arises a serious problem that kinetics of hybridization is slow since a target DNA in a solution needs to be associated with probes held on the surface of a narrow chip by bringing a large amount of the solution into contact with the probes. In the case that the target DNA is long for the probes, it is necessary that the DNA approaches the held probes along such a sequence direction that the DNA becomes complementary to the probes. Therefore, kinetics of hybridization is especially slow. Since the area of the surface of the chip is limited, the amount of the held probes is such an amount as represented by fmol and the amount is restrictive. Thus, there remains a problem that when a large amount of DNAs having highly similar sequences are present, the detection of a target DNA of a very small amount is frequently disturbed. In the case that the amount of probes is small, falsely positive hybridization, i.e., a phenomenon that the probes on the surface of a chip are occupied by DNA having highly similar sequences arise easily. Among the above-mentioned problems, the assay using the DNA chip in the prior arts 1 and 2 has the problem that much time is required and high sensitivity is not attained. The prior art 2 has the problem that the diffusion of a sample DNA into gel in a chip results in a rate-determining step of hybridization, and also has the problem that it is a skilled job and difficult for usual users to make a polynucleotide probe chip holding probes uniformly and having reproducibility by treating gels formed on the surface of the chips chemically in such a manner that the probes can bond to the gels. In order to detect simultaneously fluorescence from respective areas in chips in the prior art, it is necessary to apply a laser beam widened by an expander from the same face at the side of a camera. High output is also necessary since the density of the laser beam applied to the respective areas drops. The laser beam which is reflected on the chip surface and then directly projected onto a detector results in a background. Thus, detection with high sensitivity is difficult. In the case of using chips in the prior art, therefore, it is general to use a method of scanning a laser beam by means of a laser scanning microscopy. As a result, there arises a problem that much time is required for assay. Furthermore, it is difficult to separate DNA captured in each of areas in chips in the prior art, dependently on each of the areas, and collect the DNA, dependently on each of sizes of DNA.
In order to overcome the above-mentioned problems, an object of the present invention is to provide a method for producing a polynucleotide probe chip which makes it possible to form desired polynucleotide probes in a close state easily and is low in production costs. Another object of the present invention is to provide a low-priced polynucleotide probe chip which causes an improvement in the kinetics of hybridization on the surface of the chip, makes high-sensitivity assay for a short time possible, and makes falsely positive hybridization less; and a method for detecting polynucleotide(s) and a polynucleotide detecting device which make it possible to separate DNA captured in each of areas in a chip, dependently on each of the areas, and collect the DNA, dependently on each of sizes of the DNA.
The polynucleotide probe chip of the present invention wherein plural areas holding different polynucleotide probes are arranged has the following features.
(A) Gels hold the polynucleotide probes in the respective areas. A polynucleotide sample is migrated in the gels in the areas by electrophoresis to hybridize the polynucleotide probes of the gels with sample polynucleotides. Thus, the possibility that the polynucleotide probe chips collide with the sample polynucleotides are raised, so that the kinetics of hybridization is made higher and the amount of the probes held by the gels can be increased.
(B) In actual use, sample polynucleotides labelled with a fluorophore are added to the polynucleotide probe chip wherein the areas holding the different polynucleotide probes are arranged, and then the polynucleotide sample is migrated in the gels of the respective gels by electrophoresis to hybridize the polynucleotide probes in the gels with specific polynucleotides. The specific polynucleotides captured in the respective areas can be detected by detecting the fluorophore-labelled polynucleotides captured in the gels of the respective areas of the polynucleotide probe chip.
The sample polynucleotides are not labelled in advance. First, the sample polynucleotide samples are added to the polynucleotide probe chip, and then they are migrated in the gels in the areas by electrophoresis, to hybridize the polynucleotide probes in the gels with specific polynucleotides. Next, dNTP labelled with a fluorophore by extension reaction using DNA polymerase or ddNTP labelled with a fluorophore is introduced to the polynucleotide probes hybridized with the polynucleotides in the areas of the gels in the polynucleotide probe chip, so that the polynucleotides are labelled. In this way, the specific polynucleotides captured in the respective areas can be detected.
In order to detect a DNA (cDNA) fragment having an unknown base sequence as a sample, for example, in mRNA-expression-profile assay, there is used a polynucleotide probe chip holding polynucleot
Irie Takashi
Kajiyama Tomoharu
Kambara Hideki
Matsunaga Hiroko
Okano Kazunori
Brusca John S.
Hitachi , Ltd.
Kim Young
Mattingly Stanger & Malur, P.C.
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