U.S. patent application number 10/508946 was filed with the patent office on 2006-04-20 for gel having biosubstance fixed thereto and microarray utilizing the gel.
Invention is credited to Chiho Itou, Chiaki Nagahama.
Application Number | 20060084060 10/508946 |
Document ID | / |
Family ID | 28672124 |
Filed Date | 2006-04-20 |
United States Patent
Application |
20060084060 |
Kind Code |
A1 |
Nagahama; Chiaki ; et
al. |
April 20, 2006 |
Gel having biosubstance fixed thereto and microarray utilizing the
gel
Abstract
The present invention provides a biological
substance-immobilized gel which comprises a gel containing 2%-7% by
mass of N,N-dimethylacrylamide and a biological substance
immobilized on and/or in the gel.
Inventors: |
Nagahama; Chiaki; (Kanagawa,
JP) ; Itou; Chiho; (Hiroshima, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Family ID: |
28672124 |
Appl. No.: |
10/508946 |
Filed: |
April 3, 2003 |
PCT Filed: |
April 3, 2003 |
PCT NO: |
PCT/JP03/04274 |
371 Date: |
July 11, 2005 |
Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/6.16 |
Current CPC
Class: |
B01J 2219/00576
20130101; B01J 2219/00644 20130101; B01J 2219/00659 20130101; B01J
19/0046 20130101; B01J 2219/0052 20130101; B01J 2219/00524
20130101; B01J 2219/00585 20130101; B01J 2219/00673 20130101; B01J
2219/00317 20130101; B01J 2219/00722 20130101; Y10T 428/1352
20150115; B01J 2219/00639 20130101; B01J 2219/00641 20130101; B01J
2219/00596 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 3, 2002 |
JP |
2002-101675 |
Claims
1. A biological substance-immobilized gel which comprises a gel
containing 2%-7% by mass of N,N-dimethylacrylamide and a biological
substance immobilized on and/or in the gel.
2. A biological substance-immobilized gel which comprises a gel
having the following composition and a biological substance
immobilized on and/or in the gel: TABLE-US-00010 (a)
N,N-dimethylacrylamide 2% to 7% by mass (b) cross-linking agent
0.1% to 1.5% by mass.
3. The biological substance-immobilized gel according to claim 1 or
2, wherein the biological substance is a nucleic acid.
4. The biological substance-immobilized gel according to claim 2 or
3, wherein the cross-linking agent is a multifunctional monomer
having at least two ethylenically unsaturated bonds.
5. The biological substance-immobilized gel according to claim 4,
wherein the cross-linking agent is methylenebisacrylamide.
6. A method for preparing a biological substance-immobilized gel,
which comprises immobilizing a biological substance on and/or in a
gel containing 2%-7% by mass of N,N-dimethylacrylamide.
7. The method according to claim 6, wherein the gel is obtained by
reacting 2%-7% by mass of N,N-dimethylacrylamide in the presence of
0.1%-1.5% by mass of a cross-linking agent.
8. A gel-filled hollow tube which comprises a hollow tube whose
hollow space is filled with the biological substance-immobilized
gel according to any one of claims 1 to 5.
9. The gel-filled hollow tube according to claim 8, wherein the
hollow tube is a hollow fiber.
10. A method for manufacturing a biological substance-immobilized
gel microarray, which comprises allowing a plurality of gel-filled
hollow tubes according to claim 8 or 9 to be tied in a bundle and
cutting the resulting tube bundle in a direction intersecting with
the longitudinal direction of the tubes.
11. A method for manufacturing a biological substance-immobilized
gel microarray, which comprises the following steps: (1) allowing a
plurality of hollow tubes to be tied in a bundle; (2) filling the
biological substance-immobilized gel according to any one of claims
1 to 5 into the hollow space of each tube in the resulting tube
bundle; and (3) cutting the tube bundle in a direction intersecting
with the longitudinal direction of the tubes.
12. A biological substance-immobilized gel microarray which
comprises the biological substance-immobilized gel according to any
one of claims 1 to 5, wherein the gel is arranged in multiple
compartments.
13. The biological substance-immobilized gel microarray according
to claim 12, wherein the surface area of each compartment is
10.sup.-6 m.sup.2 or less.
14. The biological substance-immobilized gel microarray according
to claim 12 or 13, wherein the compartments are formed by slots or
through holes.
15. A biological substance-immobilized gel microarray which is
obtained by allowing a plurality of gel-filled hollow tubes
according to claim 8 or 9 to be tied in a bundle and cutting the
tube bundle in a direction intersecting with the longitudinal
direction of the tubes.
16. The biological substance-immobilized gel microarray according
to claim 15, wherein the hollow tubes are hollow fibers.
17. A method for detecting a target to be measured, which comprises
reacting an analyte with the microarray according to any one of
claims 12 to 16 and detecting the target in the analyte.
18. The method according to claim 17, wherein the target to be
measured is a nucleic acid.
19. The method according to claim 18, wherein the nucleic acid is
100 nucleotides or less in length.
Description
TECHNICAL FIELD
[0001] The present invention relates to a biological
substance-immobilized gel and a biological substance-immobilized
gel microarray using the same. The microarray is used for analysis
of gene expression, etc.
BACKGROUND ART
[0002] The decoding of human genome is now progressing and helping
clarify causal relations between various diseases or diatheses and
specific gene sequences. For example, such a gene analysis is
intended to use for predicting the onset of diseases, side effects
of drugs, etc.
[0003] A means conventionally used for gene analysis is gel-based
electrophoresis. In recent years, capillary gel electrophoresis has
been developed with the aim of separating and analyzing trace
amounts of biological samples in a short time. Capillary gel
electrophoresis uses glass capillaries filled with a hydrogel such
as acrylamide.
[0004] In addition, microarrays carrying multiple capture probes
for DNA or protein detection (i.e., probes capable of capturing
target DNA or protein molecules and the like through hybridization
or binding to the DNA or protein molecules to be detected) are
employed as useful tools for detecting mutations and expression
levels of many genes all at once. Such microarrays are also known
to have a large number of variations which are constructed using a
gel. Among them, microarrays known to use a gel for immobilization
of capture probes include, for example, those having multiple slots
or holes on a substrate (e.g., a resin board), in which the slots
or holes are filled with a DNA-containing gel (see JP 2000-60554
A), as well as those having gel spots containing DNA or other
substances on a flat substrate (see U.S. Pat. No. 5,770,721). Also,
some of the inventors of the present invention have developed a
microarray that is obtained by creating a hollow fiber alignment
comprising hollow fibers whose hollow space is filled with a
capture probe-containing gel and then cutting the alignment in a
direction intersecting with its fiber axis. This microarray has
been filed for patent application (see JP 2000-270877 A, JP
2000-270878 A and JP 2000-270879 A).
[0005] These capture probe-immobilized microarrays may be used for
hybridization with an analyte to detect specific nucleotide
sequences. Detection of hybrids is accomplished by using a known
means capable of specifically recognizing the hybrids, as
exemplified by fluorescence detection.
[0006] However, there has been a problem that when the microarrays
after hybridization are measured for fluorescence intensity in each
of their compartments where capture probes are immobilized, the
fluorescence intensity is higher in the outer regions of the
compartments, but lower in the center regions of the
compartments.
DISCLOSURE OF THE INVENTION
[0007] The object of the present invention is to obtain gel
composition which ensures a uniform distribution of fluorescence
intensity in each compartment and provides a higher value for total
fluorescence intensity summed over the entire area of each
compartment, i.e., higher hybridization efficiency in the detection
of a microarray after hybridization.
[0008] As a result of extensive and intensive efforts made to
overcome the problem stated above, the inventors of the present
invention have found that when capture probes are immobilized on
and/or in a gel satisfying the following properties, it is possible
to ensure a uniform distribution and increased level of
fluorescence intensity in each compartment, i.e., higher
hybridization efficiency. This finding led to the completion of the
present invention.
[0009] Namely, the present invention provides a biological
substance-immobilized gel which comprises a gel containing 2%-7% by
mass of N,N-dimethylacrylamide and a biological substance
immobilized on and/or in the gel. The present invention also
provides a biological substance-immobilized gel which comprises a
gel having the following composition and a biological substance
immobilized on and/or in the gel: TABLE-US-00001 (a)
N,N-dimethylacrylamide 2% to 7% by mass (b) cross-linking agent
0.1% to 1.5% by mass.
[0010] In the above biological substance-immobilized gel, examples
of a biological substance include nucleic acids. On the other hand,
examples of a cross-linking agent include multifunctional monomers
having at least two ethylenically unsaturated bonds, as exemplified
by methylenebisacrylamide.
[0011] The present invention further provides a method for
preparing a biological substance-immobilized gel, which comprises
immobilizing a biological substance on and/or in a gel containing
2%-7% by mass of N,N-dimethylacrylamide. In the present invention,
the gel is preferably obtained by reacting 2%-7% by mass of
N,N-dimethylacrylamide in the presence of 0.1%-1.5% by mass of a
cross-linking agent.
[0012] The present invention further provides a gel-filled hollow
tube which comprises a hollow tube whose hollow space is filled
with the biological substance-immobilized gel mentioned above.
Examples of a hollow tube include hollow fibers.
[0013] The present invention further provides a method for
manufacturing a biological substance-immobilized gel microarray,
which comprises allowing a plurality of the above gel-filled hollow
tubes to be tied in a bundle and cutting the tube bundle in a
direction intersecting with the longitudinal direction of the
tubes.
[0014] The present invention further provides a method for
manufacturing a biological substance-immobilized gel microarray,
which comprises the following steps: [0015] (a) allowing a
plurality of hollow tubes to be tied in a bundle; [0016] (b)
filling the above biological substance-immobilized gel into the
hollow space of each tube in the resulting tube bundle; and [0017]
(c) cutting the tube bundle in a direction intersecting with the
longitudinal direction of the tubes.
[0018] The present invention further provides a biological
substance-immobilized gel microarray which comprises the above
biological substance-immobilized gel arranged in multiple
compartments. In this case, the surface area of each compartment is
preferably 10.sup.-6 m.sup.2 or less. It is also possible to employ
a biological substance-immobilized gel microarray whose
compartments are formed by slots or through holes.
[0019] The present invention further provides a biological
substance-immobilized gel microarray which is obtained by allowing
a plurality of the above gel-filled hollow tubes (e.g., hollow
fibers) to be tied in a bundle and cutting the tube bundle in a
direction intersecting with the longitudinal direction of the
tubes.
[0020] The present invention further provides a method for
detecting a target to be measured (e.g., nucleic acids such as
DNA), which comprises reacting an analyte with the microarray
mentioned above and detecting the target in the analyte. In a case
where a target to be measured in this detection is DNA, it is
preferably 100 nucleotides or less in length.
[0021] The present invention will be described in more detail
below.
[0022] The present invention is directed to a gel comprising a
biological substance immobilized thereon and/or therein (i.e., a
biological substance-immobilized gel), whose composition includes
N,N-dimethylacrylamide (2% to 7% by mass).
[0023] As used herein, the term "biological substance" is intended
to mean a biological material which may be used as a capture probe.
Examples include deoxyribonucleic acids (DNA), ribonucleic acids
(RNA), proteins and lipids. These biological substances may be
commercially available or derived from living cells, etc.
[0024] For example, DNA extraction from living cells may be
accomplished, e.g., by the method of Blin et al. [Nucleic. Acids.
Res. 3. 2303 (1976)], while RNA extraction may be accomplished,
e.g., by the method of Favaloro et al. [Methods. Enzymol. 65. 718
(1980)].
[0025] DNA used for this purpose is linear or circular plasmid DNA
or chromosomal DNA. It is also possible to use DNA fragments
cleaved with restriction enzymes or by chemical treatments, DNA
molecules synthesized in vitro by enzymatic or other processes, or
oligonucleotides chemically synthesized, etc.
[0026] Biological substances prepared by the methods stated above
or other techniques are immobilized on and/or in a gelatinous
material (hereinafter referred to as a gel). As used herein, the
term "immobilized" is used to mean that a biological substance is
retained on and/or in a gel.
[0027] The composition of such a gel includes
N,N-dimethylacrylamide in an amount of 2% to 7% by mass of the gel,
but the following composition is preferred: TABLE-US-00002 (a)
N,N-dimethylacrylamide 2% to 7% by mass (b) cross-linking agent
0.1% to 1.5% by mass.
[0028] More preferably, the lower limit of the amount of
N,N-dimethylacrylamide is 2.5% to 5.0% by mass.
[0029] Preferred cross-linking agents are multifunctional monomers
having at least two ethylenically unsaturated bonds. The amount of
such a cross-linking agent is preferably 0.1% to 1.5% by mass of
the gel, and more preferably 0.3% to 0.7% by mass of the gel. Any
cross-linking agent can be used without particular limitations as
long as it is among the multifunctional monomers stated above.
Examples include methylenebisacrylamide, divinylbenzene, and
polyethylene glycol di(meth)acrylate.
[0030] To prepare such a gel, for example, N,N-dimethylacrylamide
and a cross-linking agent may be mixed and copolymerized in an
aqueous medium, or alternatively, N,N-dimethylacrylamide may be
polymerized to give a prepolymer, which in turn may be mixed and
copolymerized with a cross-linking agent.
[0031] To immobilize biological substances on and/or in the above
gel, for example, biological substances modified to have a terminal
vinyl group may be added during polymerization and copolymerized
with components of the gel (see WO 02/62817), or a
hydrazine-treated gel may be prepared and reacted with biological
substances having an amino group (see JP 6-507486 A).
[0032] The biological substance-immobilized gel prepared in the
present invention preferably has a water permeability of
1.0.times.10.sup.-5 m.sup.3m/m.sup.2/hr/MPa or more. The water
permeability of the gel is calculated from the amount of water
permeating through the gel. A water permeation experiment is
performed as follows and the measured value is defined as the water
permeability.
[0033] A gel disk of 1 mm thickness and 20 mm diameter is prepared
and overlaid on a support filter (Millipore SMWPO4700). The gel
disk is then placed in a filtration holder (ADVANTEC UHP-43K) and
the holder is filled with water. Nitrogen pressure is then applied
to the filtration holder and a PE tube of 2 mm diameter is
connected to the filtrate outlet. The amount of water permeating
through the gel disk is estimated from the time required for the
front-end of the filtrate to move a given distance (40 cm) through
the tube, followed by calculation of the water permeability.
[0034] In addition, the gel preferably has a shape retention rate
of 0.4 or more, more preferably 0.6 or more. The shape retention
rate of the gel is defined as the value measured as follows.
[0035] A gel is prepared in a cylindrical container of 13 mm
diameter and 4 cm length. The gel is removed from the container,
allowed to stand at 25.degree. C. for 24 hours in an airtight
container, and then measured for its height The shape retention
rate is then calculated by the following equation: Shape retention
rate=height (mm) of the gel after 24 hours/13 mm (initial diameter
of the gel)
[0036] The thus prepared biological substance-immobilized gel may
be used as a tool for gene analysis as a gel carrying capture
probes.
[0037] For example, the above gel may be filled into the hollow
space of a hollow tube to prepare a gel-filled hollow tube, which
in turn can be used as an analysis tool for genes, etc. It should
be noted that the hollow space may be filled with the gel in the
same manner as in the production of capillary columns used for
capillary gel electrophoresis.
[0038] The gel of the present invention may also be used as a
component of a microarray. For example, when the above gel carrying
capture probes immobilized thereon and/or therein (hereinafter
referred to as an immobilized gel) is arranged on a flat substrate,
it is possible to manufacture a microarray in which the immobilized
gel is arranged in multiple compartments on the flat substrate (see
JP 6-507486 A and U.S. Pat. No. 5,770,721). A flat substrate having
multiple slots or through holes may also be used for this purpose.
In this case, a biological substance-containing monomer solution
before or immediately after initiation of polymerization may be
introduced into each compartment formed by a slot or a through
hole, followed by polymerization and cross-linking within each
compartment to give a microarray in which a biological
substance-immobilized gel is arranged on the substrate (i.e., a
biological substance-immobilized gel microarray) (see JP 2000-60554
A).
[0039] The type of biological substance to be retained in each
compartment may vary from compartment to compartment.
Alternatively, multiple immobilized gels of the same type may be
grouped together and arranged on a microarray. Likewise, a gel
carrying, e.g., a pigment instead of a biological substance may be
retained in a compartment(s) to determine the coordinates of
compartments.
[0040] The surface area of each compartment is usually 10.sup.-6
m.sup.2 or less. The lower limit is not restricted in any way as
long as biological substances can be detected.
[0041] In the present invention, examples of hollow tubes include
glass tubes, stainless steel tubes, and hollow fibers. In terms of
processability and ease of handling, hollow fibers are preferred
for use. Examples of fibers available for use in the present
inventions include chemical fibers such as synthetic fibers,
semi-synthetic fibers, regenerated fibers and inorganic fibers, as
well as natural fibers (JP 2000-270878 A). Representative examples
of synthetic fibers include various types of polyamide-type fibers
such as Nylon 6, Nylon 66 and aromatic polyamide fibers, various
types of polyester-type fibers such as polyethylene terephthalate,
polybutyrene terephthalate, polylactic acid and polyglycolic acid
fibers, various types of acrylic-type fibers such as
polyacrylonitrile fibers, various types of polyolefin-type fibers
such as polyethylene and polypropylene fibers, various types of
polyvinyl alcohol-type fibers, various types of polyvinylidene
chloride-type fibers, polyvinyl chloride-type fibers, various types
of polyurethane-type fibers, phenol-type fibers, fluoro-type fibers
such as polyvinylidene fluoride and poly(tetrafluoroethylene),
polyalkylene parahydroxybenzoate-type fibers, as well as fibers
formed using (meth)acrylic-type resins such as
polymethylmethacrylate.
[0042] Representative examples of semi-synthetic fibers include
various types of cellulose-type derivative-type fibers originated
from diacetate, triacetate, chitin, chitosan and the like, as well
as various types of protein-type fibers called promix.
Representative examples of regenerated fibers include various types
of regenerated cellulose fibers (e.g., rayon, cupra, polynosic)
which are obtained by viscose or cuprammonium process or by organic
solvent process.
[0043] Representative examples of inorganic fibers include glass
fibers and carbon fibers. Representative examples of natural fibers
include vegetable fibers such as cotton, linen, ramie and jute,
animal fibers such as sheep wool and silk, as well as mineral
fibers such as asbestos.
[0044] Hollow fibers other than natural fibers may be produced in a
known manner using special nozzles. The melt spinning technique is
preferred for polyamides, polyesters, polyolefins and the like,
which can use a horseshoe- or C-shaped nozzle, a double-tubed
nozzle, etc.
[0045] The solvent spinning technique is preferred for spinning
synthetic polymers that are not melt-spinnable and polymers that
are used in semi-synthetic fibers or regenerated fibers. As in the
case of melt spinning, a double-tubed nozzle is also used in this
case to give hollow fibers having a continuous hollow space by
spinning the fibers while filling an appropriate liquid as a core
material into the hollow space.
[0046] The hollow tubes thus prepared may each be used as a base
unit for supporting the biological substance-immobilized gel of the
present invention. In the case of using hollow tubes, microarrays
(biological substance-immobilized gel microarrays) may be
manufactured, for example, by allowing a plurality of the above
hollow tubes to be tied in a bundle, filling the biological
substance-immobilized gel into the hollow space of each hollow tube
in the resulting tube bundle, and then cutting the tube bundle in a
direction intersecting with the longitudinal direction of the tubes
in such a manner as to give cross-sectional slices (see WO
00/53736). In the present invention, individual hollow tubes may be
filled with the gel before being tied in a bundle.
[0047] In this case, these hollow tubes may be regularly arranged
and bonded with an adhesive or the like to give, e.g., a tube
alignment in which the hollow tubes are regularly arranged in both
vertical and horizontal directions. The term "regularly" is used to
mean that tubes are arranged in an orderly manner such that the
number of hollow tubes contained in a fame of certain size can be
the same.
[0048] Such a tube alignment may be produced as follows, by way of
example. Namely, two perforated plates with a regular arrangement
of holes are provided, and hollow tubes are threaded through the
holes in both plates such that the positions of holes in both
perforated plates are matched with each other. The space between
these perforated plates is then adjusted. It should be noted that
the step of threading hollow tubes through the holes and the step
of adjusting the space between perforated plates may be conducted
in reverse order. Then, tension is applied to the hollow tubes and,
under this condition, spaces between the hollow tubes (spaces
within the tube bundle) are filled with a resin so as to bond the
bundle of the tubes, thereby obtaining a tube alignment (JP
2001-239594 A).
[0049] The tube alignment may be of any shape in cross section. For
example, hollow tubes may be regularly arranged to form a square or
rectangular cross section, or alternatively, hollow tubes may be
concentrically arranged to form a circular cross section.
[0050] In the present invention, the above tube alignment is cut in
a direction intersecting with, preferably perpendicular to, the
longitudinal direction (i.e., the axial direction of the hollow
tubes) to obtain slices. An example of a cutting method involves
cutting slices from the tube alignment using a microtome. The
thickness of slices can be arbitrarily adjusted, but it usually
ranges from 1 to 5,000 .mu.m, preferably 10 to 2,000 .mu.m.
[0051] The slices thus prepared may each be used as a microarray
for supporting the biological substance-immobilized gel.
[0052] Biological substances immobilized on and/or in the gel in
the microarray serve as capture probes for nucleic acids or
proteins which hybridize or bind to the biological substances (such
nucleic acids or proteins being called targets to be measured).
Thus, the microarray of the present invention can be used as a kit
for detecting a target(s) to be measured (e.g., nucleic acids or
proteins).
[0053] An analyte containing biological substances to be detected
(e.g., nucleic acids such as DNA) is prepared, added to the
microarray and then reacted with biological substances immobilized
on and/or in the gel of the microarray. For example, DNA targets to
be measured are fluorescently labeled and then hybridized with DNA
in the microarray. Subsequently, the microarray is washed to remove
unreacted DNA, followed by detection of fluorescence intensity. The
fluorescence intensity may be detected using any device (e.g., a
commercially available DNA detector). According to the present
invention, the inventive "biological substance-immobilized gel
which comprises a gel containing 2%-7% by mass of
N,N-dimethylacrylamide and a biological substance immobilized on
and/or in the gel" has good reactivity and ensures uniform
fluorescence intensity per compartment of a microarray, thus
providing highly sensitive detection results.
BRIEF DESCRIPTION OF DRAWINGS
[0054] FIG. 1 presents photographs showing the results of DNA
detection using the microarray of the present invention.
[0055] FIG. 2 presents photographs showing the results of DNA
detection using the microarray of the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0056] The present invention will be further described in more
detail in the following Examples, which are not intended to limit
the scope of the invention.
EXAMPLE 1
(1) Production of Polymethylmethacrylate (PMMA) Hollow Fibers
[0057] An acrylic resin with a mass molecular weight of about
90,000, which was composed of methyl methacrylate (MMA) and methyl
acrylate (MA) in a monomer ratio of 82:18, was used as a source
material and melt-extruded using an extruder through a spinning
nozzle having a circular outlet, thereby obtaining hollow fibers
with an outer diameter of 0.3 mm, an inner diameter of 0.2 mm and a
length of 600 mm.
(2) Production of a Hollow Fiber Alignment
[0058] Two perforated plates of 0.1 mm thickness were placed one
upon another, each of which had 9 holes (diameter: 0.32 mm;
center-to-center distance: 0.42 mm) arranged in a 3 by 3 array, and
9 hollow fibers prepared above were then threaded through the
respective holes in these perforated plates. The space between
these two perforated plates was set to 50 mm and the hollow fibers
were fixed under tension at two points, 50 mm and 100 mm from one
end.
[0059] A resin raw material was then poured into the space between
these two perforated plates. As a resin, a polyurethane resin
adhesive (Nipporan 4276/Coronate 4403, Nippon Polyurethane Industry
Co., Ltd.) was used, which was supplemented with carbon black in an
amount of 2.5% by mass, based on the total weight of this adhesive.
The plates were allowed to stand at room temperature for 1 week to
cure the resin. The perforated plates were then removed to give a
hollow fiber alignment.
(3) Preparation of an Oligonucleotide having a Terminal Vinyl Group
(Vinyl-Terminated Oligonucleotide
[0060] Oligonucleotide synthesis was carried out using an automated
DNA/RNA synthesizer (PE Biosystems Model 394). In the final step of
synthesis, an amino group [NH.sub.2(CH.sub.2).sub.6--] was
introduced at the 5'-terminus to synthesize oligonucleotide A (SEQ
ID NO: 1) shown below. The same procedure was repeated to
synthesize oligonucleotide B (SEQ ID NO: 2), except that no amino
group was introduced at the 5'-terminus. Amino group introduction
at the 5'-terminus was accomplished by using AminoLink II.TM.
(Applied Biosystem).
[0061] These oligonucleotides were deprotected and purified in a
standard manner before use. TABLE-US-00003 [Oligonucleotide A (SEQ
ID NO: 1)] caaccaacca caactacata cacatac [Oligonucleotide B (SEQ ID
NO: 2)] gtcatttaga caactctgca agcgt
[0062] Subsequently, oligonucleotide A (500 nmol/ml, 5 .mu.l) and
glycidyl methacrylate (0.5 .mu.l) were mixed and reacted at
70.degree. C. for 2 hours. After completion. of the reaction, water
was added to a total volume of 25 l to give an oligonucleotide (100
nmol/ml) having a terminal methacrylate group (GMA-denatured
oligonucleotide A).
(4) PCR Reaction of a Vinyl-Terminated Oligonucleotide
[0063] Saccharomyces cerevisiac JCM7255 was grown in 100 ml YPD
medium (20 g/L glucose, 10 g/L yeast extract, 20 g/L polypeptone,
pH 6.0) at 30.degree. C. for 1 day, followed by collection of the
bacterial cells. The chromosomal DNA was prepared in a routine
manner from the collected bacterial cells and used as a PCR
template.
[0064] The GMA-denatured oligonucleotide A and oligonucleotide B
were diluted with sterilized water to 50 .mu.M and 5 .mu.M,
respectively. These oligonucleotides were used as primers to
perform polymerase chain reaction (hereinafter referred to as PCR)
with the template prepared above.
[0065] PCR conditions were as described in the specification of
Ex-Taq (Takara Shuzo Co., Ltd.) and PCR was performed using a
TaKaRa PCR Thermal Cycler PERSONAL. The reaction was repeated for
30 cycles with 100 .mu.l under temperature conditions of 93.degree.
C. for 30 seconds, 65.degree. C. for 30 seconds and 72.degree. C.
for 2 minutes. A vinyl-terminated nucleic acid (capture probe A;
SEQ ID NO: 3) was amplified by PCR.
(a) Preparation of a Monomer Solution and a Polymerization
Initiator Solution
[0066] Polymerization solutions 1 and 2 having the compositions
shown in Table 1 were prepared. Monomer solution A and a
polymerization initiator solution were prepared as follows.
[Monomer Solution A]
[0067] Dimethylacrylamide (0.45 g) and methylenebisacrylamide (0.05
g) were dissolved in a 50/50 (by mass) mixture of glycerine and
pure water to give a total volume of 10 ml.
[Polymerization Initiator Solution]
[0068] 2,2'-Azobis(2-imidazolin-2-yl)propane) dihydrochloride (1 g)
was dissolved in pure water to give a total volume of 10 ml.
TABLE-US-00004 TABLE 1 Polymerization Polymerization solution 1
solution 2 Monomer solution A 1000 .mu.l 1000 .mu.l Polymerization
initiator solution 10 .mu.l 10 .mu.l Capture probe A (100 nmol/ml)
5 .mu.l 0
(6) Preparation of Slices
[0069] Polymerization solution 1 was filled into the hollow space
of three hollow fibers in the center row of the hollow fiber
alignment obtained in (2) above, while polymerization solution 2
was filled into the hollow space of the other hollow fibers.
Polymerization solutions 1 and 2 were filled. The alignment was
transferred to an airtight glass container, inside of which was
saturated with water vapor, and then allowed to stand at 55.degree.
C. for 1 hour to perform polymerization.
[0070] After polymerization, the hollow fiber alignment was
repeatedly cut using a microtome in a direction perpendicular to
the longitudinal direction of the hollow fibers, thereby obtaining
slices of about 500 .mu.m thickness.
(7) Hybridization
[0071] A hybridization solution was prepared, which was
supplemented with 200 fmol/ml oligonucleotide C (SEQ ID NO: 4)
complementary to a part of the nucleotide sequence of capture probe
A (nucleotides 241 to 339 of SEQ ID NO: 3).
[0072] Oligonucleotide C was synthesized in the same manner as
shown in (3) above using an automated DNA synthesizer, and Cy5 was
introduced at the 5'-terminus. After completion of the synthesis,
the oligonucleotide was deprotected and purified in a standard
manner before use. TABLE-US-00005 [Oligonucleotide C (SEQ ID NO:
4)] gccaacaatg gaatgttgat tgggcccaaa ccaccttcct ttcttgggat
attggtccat gccaaaaggg agtattcgga gtcagtggag gogaaaaga
<Composition of Hybridization Solution>
[0073] 5.times.SSC (0.75 mol/L sodium chloride, 0.075 mol/l sodium
citrate, pH 7.0) 0.02% SDS (sodium lauryl sulfate)
[0074] The slice obtained in (6) and the above hybridization
solution (1 ml) were poured into a HybriPack, followed by
heat-sealing the top end of the pack. Hybridization was performed
at 65.degree. C. for 20 hours.
(8) Washing
[0075] The slice was removed from the HybriPack and washed under
the conditions shown in Table 2 in the order listed. The volume of
a washing solution was 10 ml. TABLE-US-00006 TABLE 2 Composition of
washing solution Washing temperature Washing time 2 .times. SSC
0.2% SDS 25.degree. C. 20 minutes 0.2 .times. SSC 0.2% SDS
25.degree. C. 20 minutes 0.2 .times. SSC 0.2% SDS 55.degree. C. 20
minutes 0.2 .times. SSC 0.2% SDS 55.degree. C. 20 minutes 0.2
.times. SSC 0.2% SDS 25.degree. C. 20 minutes
(9) Detection
[0076] The washed slice was placed on a non-fluorescent slide glass
and a few drops of sterilized water were put onto the slice. The
slide glass was then covered with a cover glass and mounted on a
DNA chip detector (GeneTac V, Genomic Solutions K. K.), followed by
detection using a Cy5 laser. The image size was set to 10 .mu.m per
pixel.
(10) Fluorescence Intensity Measurement
[0077] The sum of fluorescence intensity obtained from 80 pixels
around the center of each compartment was calculated as the
intensity per compartment. FIG. 1 shows the fluorescence intensity
obtained, along with an image of the washed hollow fiber and its
surrounding area. The center of each compartment was determined as
appropriate. As a result, the distribution of fluorescence
intensity in the hybridized compartments was uniform.
COMPARATIVE EXAMPLE 1
[0078] The same procedure as used in Example 1 was repeated, except
that monomer solution A was replaced by monomer solution B.
[Monomer Solution B]
[0079] Acrylamide (0.475 g) and methylenebisacrylamide (0.025 g)
were dissolved in a 50/50 (by mass) mixture of glycerine and pure
water to give a total volume of 10 ml.
[0080] FIG. 1 shows the fluorescence intensity obtained, along with
an image of the washed hollow fiber and its surrounding area.
[0081] The distribution of fluorescence intensity in the hybridized
hollow space was uniform, but the fluorescence intensity decreased
as compared to Example 1.
COMPARATIVE EXAMPLE 2
[0082] The same procedure as used in Example 1 was repeated, except
that monomer solution A was replaced by monomer solution C.
[Monomer Solution C]
[0083] Acrylamide (0.76 g) and methylenebisacrylamide (0.04 g) were
dissolved in a 50/50 (by mass) mixture of glycerine and pure water
to give a total volume of 10 ml. FIG. 1 shows the fluorescence
intensity obtained, along with an image of the washed hollow fiber
and its surrounding area
[0084] The fluorescence intensity was lower than in Example 1, and
the fluorescence intensity in the hollow space was high in the
peripheral region, but low in the center region.
EXAMPLE 2
[0085] The same procedure as used in Example 1 was repeated to
prepare slices, except that monomer solution A and capture probe A
were replaced by monomer solution D and capture probe B (SEQ ID NO:
5), respectively. Capture probe B was constructed to have a
terminal methacrylate group by introducing an amino group at the
5'-terminus and then reacting the same with glycidyl
methacrylate.
[Monomer Solution D]
[0086] Dimethylacrylamide (0.27 g) and methylenebisacrylamide (0.03
g) were dissolved in a 50/50 (by mass) mixture of glycerine and
pure water to give a total volume of 10 ml. TABLE-US-00007 [Capture
probe B (SEQ ID NO: 5)] aaatacgcct gcaggcggag atcttccagg cccgcctcaa
gggctggttc gagccaatag tggaagacat
[0087] Hybridization and washing were performed as follows.
(1) Hybridization
[0088] A hybridization solution was prepared, which was
supplemented with 1 pmol/ml oligonucleotide E (SEQ ID NO: 6)
including, as a part thereof, a complementary sequence to the
nucleotide sequence of capture probe B (nucleotides 16 to 85 of SEQ
ID NO: 6).
[0089] Oligonucleotide E was synthesized using an automated DNA
synthesizer, and Cy5 was introduced at the 5'-terminus. After
completion of the synthesis, the oligonucleotide was deprotected
and purified in a standard manner before use. TABLE-US-00008
[Oligonucleotide E (SEQ ID NO: 6)] gcccactggc gatgcatgtc ttccactatt
ggctcgaacc agcccttgag gcgggcctgg aagatctccg cctgcaggcg tatttgctgg
gtctgttcc
[Composition of Hybridization Solution]
[0090] 6.times.SSC (0.75 mol/L sodium chloride, 0.075 mol/I sodium
citrate, pH 7.0) 0.02% SDS (sodium lauryl sulfate)
[0091] The resulting slice and the above hybridization solution (1
ml) were poured into a HybriPack, followed by heat-sealing the top
end of the pack. Hybridization was performed at 37.degree. C. for
16 hours.
(2) Washing
[0092] The slice was removed from the HybriPack and washed under
the conditions shown in Table 3 in the order listed. The washing
temperature was 45.degree. C. The volume of a washing solution was
10 ml. TABLE-US-00009 TABLE 3 0.2 .times. SSC 0.1% SDS 20 minutes
0.2 .times. SSC 0.1% SDS 20 minutes 0.2 .times. SSC 20 minutes
(3) Detection
[0093] The washed slice was placed on a non-fluorescent slide glass
and a few drops of sterilized water were put onto the slice. The
slide glass was then covered with a cover glass and mounted on a
DNA chip detector (GeneTac IV, Genomic Solutions K. K.), followed
by detection using a Cy5 laser. The image size was set to 10 .mu.m
per pixel.
(4) Fluorescence Intensity Measurement
[0094] The fluorescence intensity averaged over 200 pixels around
the center of each compartment was calculated as the intensity per
compartment.
[0095] FIG. 2 shows the fluorescence intensity obtained, along with
an image of the washed hollow fiber and its surrounding area. The
center of each compartment was determined as appropriate. As a
result, the distribution of fluorescence intensity in the
hybridized compartments was uniform.
EXAMPLE 3
[0096] The same procedure as used in Example 2 was repeated, except
that monomer solution D was replaced by monomer solution A. FIG. 2
shows the fluorescence intensity per compartment, along with an
image of the washed hollow fiber and its surrounding area. The
center of each compartment was determined as appropriate. As a
result, the distribution of fluorescence intensity in the
hybridized compartments was uniform.
COMPARATIVE EXAMPLE 3
[0097] The same procedure as used in Example 2 was repeated, except
that monomer solution A was replaced by monomer solution E.
[Monomer Solution E]
[0098] N,N-Dimethylacrylamide (0.72 g) and methylenebisacrylamide
(0.08 g) were dissolved in a 50/50 (by mass) mixture of glycerine
and pure water to give a total volume of 10ml.
[0099] FIG. 2 shows the fluorescence intensity obtained, along with
an image of the washed hollow fiber and its surrounding area. The
fluorescence intensity was lower than in Example 2, and the
fluorescence intensity in the hollow space was high in the
peripheral region, but low in the center region.
COMPARATIVE EXAMPLE 4
[0100] The same procedure as used in Example 2 was repeated, except
that monomer solution A was replaced by monomer solution F.
[Monomer Solution F]
[0101] N,N-Dimethylacrylamide (0.18 g) and methylenebisacrylamide
(0.02 g) were dissolved in a 50/50 (by mass) mixture of glycerine
and pure water to give a total volume of 10 ml.
[0102] The sliced chip did not hold any gel and its hollow spaces
were not filled (FIG. 2).
INDUSTRIAL APPLICABILITY
[0103] The present invention provides a biological
substance-immobilized gel. The gel of the present invention is
useful for detection of genes such as DNA because its use ensures
uniform fluorescence intensity throughout the compartment and
achieves higher hybridization efficiency.
Sequence Listing Free Text
[0104] SEQ ID NO: 1: synthetic DNA [0105] SEQ ID NO: 2: synthetic
DNA [0106] SEQ ID NO: 3: synthetic DNA [0107] SEQ ID NO: 4:
synthetic DNA [0108] SEQ ID NO: 5: synthetic DNA [0109] SEQ ID NO:
6: synthetic DNA
Sequence CWU 1
1
6 1 27 DNA Artificial Sequence synthetic DNA 1 caaccaacca
caactacata cacatac 27 2 25 DNA Artificial Sequence synthetic DNA 2
gtcatttaga caactctgca agcgt 25 3 651 DNA Artificial Sequence
synthetic DNA 3 caaccaacca caactacata cacatacata cacaatggtc
gctcaagttc aaaagcaagc 60 tccaactttt aagaaaactg ccgtcgtcga
cggtgtcttt gacgaagtct ccttggacaa 120 atacaagggt aagtacgttg
tcctagcctt tattccattg gccttcactt tcgtctgtcc 180 aaccgaaatc
attgctttct cagaagctgc taagaaattc gaagaacaag gcgctcaagt 240
tcttttcgcc tccactgact ccgaatactc ccttttggca tggaccaata tcccaagaaa
300 ggaaggtggt ttgggcccaa tcaacattcc attgttggct gacaccaacc
actctttgtc 360 cagagactat ggtgtcttga tcgaagaaga aggtgtcgcc
ttgagaggtt tgttcatcat 420 cgacccaaag ggtgtcatta gacacatcac
cattaacgat ttgccagtcg gtagaaacgt 480 tgacgaagcc ttgagattgg
ttgaagcctt ccaatggacc gacaagaacg gtactgtctt 540 gccatgtaac
tggactccag gtgctgctac catcaagcca accgttgaag actccaagga 600
atacttcgaa gctgccaaca aataagacgc ttgcagagtt gtctaaatga c 651 4 99
DNA Artificial Sequence synthetic DNA 4 gccaacaatg gaatgttgat
tgggcccaaa ccaccttcct ttcttgggat attggtccat 60 gccaaaaggg
agtattcgga gtcagtggag gcgaaaaga 99 5 70 DNA Artificial Sequence
synthetic DNA 5 aaatacgcct gcaggcggag atcttccagg cccgcctcaa
gggctggttc gagccaatag 60 tggaagacat 70 6 99 DNA Artificial Sequence
synthetic DNA 6 gcccactggc gatgcatgtc ttccactatt ggctcgaacc
agcccttgag gcgggcctgg 60 aagatctccg cctgcaggcg tatttgctgg gtctgttcc
99
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