U.S. patent application number 11/109774 was filed with the patent office on 2005-09-15 for hybridization probe and target nucleic acid detecting kit, target nucleic acid detecting apparatus and target nucleic acid detecting method using the same.
This patent application is currently assigned to FUJI PHOTO FILM CO., LTD.. Invention is credited to Kinoshita, Takatoshi, Washizu, Shintaro.
Application Number | 20050202495 11/109774 |
Document ID | / |
Family ID | 18941707 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050202495 |
Kind Code |
A1 |
Kinoshita, Takatoshi ; et
al. |
September 15, 2005 |
Hybridization probe and target nucleic acid detecting kit, target
nucleic acid detecting apparatus and target nucleic acid detecting
method using the same
Abstract
The present invention relates to a hybridization probe having a
rod-shaped body and nucleic acid which is bonded to the rod-shape
material and specifically bonds to a target nucleic acid and also
relates to a target nucleic acid detecting kit, a target nucleic
acid detecting apparatus and a target nucleic acid detecting method
using the same.
Inventors: |
Kinoshita, Takatoshi;
(Aichi, JP) ; Washizu, Shintaro; (Shizuoka,
JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
FUJI PHOTO FILM CO., LTD.
|
Family ID: |
18941707 |
Appl. No.: |
11/109774 |
Filed: |
April 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11109774 |
Apr 20, 2005 |
|
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|
10103830 |
Mar 25, 2002 |
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Current U.S.
Class: |
435/6.13 ;
435/287.2; 435/6.16 |
Current CPC
Class: |
C12Q 1/6832
20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 23, 2001 |
JP |
2001-86306 |
Claims
1-14. (canceled)
15. A target nucleic acid detecting apparatus for detecting a
target nucleic acid, comprising: a hybridization probe; means for
adding a sample to the hybridization probe; and means for measuring
changes in a wavelength by the colored interference light of the
hybridization probe hybridized aligned in a film-like shape to the
target nucleic acid; wherein the hybridization probe comprises a
rod-shaped body and a nucleic acid bonded to the rod-shaped body in
a length of 810 nm or shorter and specifically bonds to a target
nucleic acid and reflects the incident light as colored
interference light when aligned in a film-like shape.
16. A target nucleic acid detecting apparatus according to claim
15, wherein the hybridization probe is amphiphilic and the means
for adding is an adding means in which the hybridization probe is
added to an aqueous phase together with an oil phase so that the
hybridization probe contacts the sample.
17. A target nucleic acid detecting apparatus comprising: a
biosensor which comprises one of quartz oscillator and surface
acoustic wave (SAW) element and a hybridization probe aligned to
the one of quartz oscillator and surface acoustic wave (SAW)
element in a film-like shape; an oscillation circuit in which
changes in mass or changes in viscoelasticity when a target nucleic
acid is hybridized to the biosensor are oscillated as frequency;
and a frequency counter in which a frequency oscillated from the
oscillation circuit is measured; wherein the hybridization probe
comprises a rod-shaped body and a nucleic acid bonded to the
rod-shaped body in a length of 810 nm or shorter and specifically
bonds to a target nucleic acid and reflects the incident light as
colored interference light when aligned in a film-like shape and
the hybridization probe is amphiphilic.
18. The target nucleic acid detecting apparatus according to claim
17, wherein the hybridization probe is aligned in a monomolecular
film-like shape to one of the quartz oscillator and the surface
acoustic wave element.
19. A target nucleic acid detecting apparatus according to claim
17, wherein the hybridization probe is aligned in a bimolecular
film-like shape to one of the quartz oscillator and the surface
acoustic wave element.
20. A target nucleic acid detecting method apparatus for detecting
a target nucleic acid, comprising: a step for contacting a sample
to a hybridization probe; a step for measuring changes in a
wavelength by the colored interference light of the hybridization
probe hybridized to the target nucleic acid; wherein the
hybridization probe comprises a rod-shaped body and a nucleic acid
bonded to the rod-shaped body in a length of 810 nm or shorter and
specifically bonds to a target nucleic acid and reflects the
incident light as colored interference light when aligned in a
film-like shape and the hybridization probe is amphiphilic.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a highly sensitive
hybridization probe and to a target nucleic acid detecting kit, a
target nucleic acid detecting apparatus and a target nucleic acid
detecting method using the same.
[0003] 2. Description of the Related Art
[0004] For a method in which a target gene sequence is detected in
a nucleic acid polymer or a method in which difference or homology
of plural nucleic acid polymers is judged, there has been known a
hybridization method in which a single-stranded polymer (DNA or
RNA) which is complementary to a partial sequence of the target
nucleic acid polymer is used as a probe.
[0005] In that hybridization method, a single-stranded target
nucleic acid polymer is fixed to a nitrocellulose film or a Nylon
film, an aqueous solution of probe nucleic acid labeled with
radioisotope or enzyme is added onto the film and, since only
hybridized probe nucleic acid polymer remains on the film after
being washed with water when the probe nucleic acid polymer is
hybridized to the target nucleic acid polymer, radioactivity from
the labeled radioisotope of the probe nucleic acid or
chemiluminescence or color of precipitate produced by the enzyme is
detected whereby it is possible to judge whether a target base
sequence is present in the target nucleic acid polymer.
[0006] However, in the handling of radioisotopes, a special license
or equipment is necessary and that is not for general use. In
addition, there is a problem that labeling of a single-stranded
probe nucleic acid polymer with enzyme, luminous substance, and the
like is expensive, labor-intensive and time-consuming.
[0007] In the Japanese Patent Laid-Open No. 210198/1991 for
example, there is a description for a method in which a hybrid is
formed from DNA which is modified by antigen having a base sequence
complementary to the target DNA and then it is made to react with
an enzymatically modified antibody to detect the product.
[0008] However, there are problems in the method that its
quantitative property is poor due to inactivation of the enzymatic
activity and that much time is needed for the operation of
preparing the enzymatically modified antibody, and the like, the
treatment and the measurement.
SUMMARY OF THE INVENTION
[0009] Under such circumstances, an object of the present invention
is to solve various problems in the past and to achieve the
following object.
[0010] Thus, an object of the present invention is to provide a
hybridization probe by which the reaction for the formation of DNA
hybrid may easily and directly be measured and the formation of DNA
hybrid may be tested easily and highly precisely and also to
provide a target nucleic acid detecting kit, a target nucleic acid
detecting apparatus and a target nucleic acid detecting method
using the same.
[0011] The hybridization probe of the present invention has a
rod-shaped body and nucleic acid which is bonded to the rod-shaped
body and specifically bonds to a target nucleic acid. As a result
thereof, it is now possible to manifest the reaction for the
formation of DNA hybrid easily and directly and to test the
formation of DNA hybrid easily and highly precisely.
[0012] The target nucleic acid detecting kit of the present
invention contains a hybridization probe having a rod-shaped body
with a length of 810 nm or shorter and nucleic acid which is bonded
to the rod-shaped body and specifically bonds to a target nucleic
acid and reflects an incident light as colored interference light
when aligned in a film-like shape; and any of dish, plate and
tube.
[0013] The hybridization probe aligned in a film-like shape
reflects the incident light as colored interference light on the
basis of a multi-layer thin film interference theory which is a
basic principle of color formation of the scaly powder of the wings
of a Morpho butterfly. When the change in wavelength based on the
reflection of the incident light as colored interference light
brought out by changes in length or refractive index at the time
the film-like hybridization probe hybridizes with a target nucleic
acid is measured, the target nucleic acid in the sample may be
detected quickly by a simple operation in a reliable manner.
[0014] The first embodiment of the target nucleic acid detecting
apparatus of the present invention is equipped with a hybridization
probe having a rod-shaped body of a length of 810 nm or shorter and
nucleic acid which is bonded to the rod-shaped body and bonds to a
target nucleic acid and reflecting the incident light as colored
interference light when aligned in a film-like shape; a sample
adding means n which the hybridization probe is contacted to a
sample; and a colored wavelength measuring means in which changes
in wavelength by reflection of an incident light as colored
interference light of the film-like hybridization probe which is
hybridized to the target nucleic acid are measured.
[0015] The hybridization probe aligned in a film-like shape
reflects incident light as colored interference light on the basis
of a multi-layer thin film interference theory which is a basic
principle of color formation of the scaly powder of the wings of a
Morpho butterfly. When the change in wavelength based on the
reflection of the incident light as colored interference light
brought out by changes in length or refractive index at the time
the film-like hybridization probe hybridizes with a target nucleic
acid is measured, the target nucleic acid in the sample may be
detected.
[0016] The second embodiment of the target nucleic acid detecting
apparatus of the present invention is equipped with a biosensor
having a rod-shaped body and nucleic acid which is bonded to a
hybridization probe having a rod-shaped body and specifically bonds
to a target nucleic acid in which an amphiphilic biosensor is
aligned on quartz oscillator or surface acoustic wave element in a
film-like shape; an oscillation circuit whereby changes in mass or
changes in viscoelasticity when a target nucleic acid is hybridized
to the biosensor are oscillated as frequency; and a frequency
counter whereby frequency of the oscillation oscillated from the
oscillation circuit is measured.
[0017] As a result, changes in mass or changes in viscoelasticity
when the hybridization probe of the biosensor hybridized the target
nucleic acid may be detected as a frequency with a high sensitivity
and within a short time.
[0018] The target nucleic acid detecting method according to the
present invention comprises a contacting step in which a
hybridization probe having a rod-shaped body of a length of 810 nm
or shorter, having nucleic acid which is bonded to the rod-shaped
body and specifically bonds to a target nucleic acid, reflecting
the incident light as colored interference light when aligned in a
film-like shape and being amphiphilic with a sample and a
wavelength measuring step in which changes in wavelength based on
reflection of an incident light as colored interference light of
film-like hybridization probe hybridized to the target nucleic acid
are measured.
[0019] The hybridization probe aligned in a film-like shape
reflects the incident light as colored interference light on the
basis of a multi-layer thin film interference theory which is a
basic principle of color formation of the scaly powder of the wings
of a Morpho butterfly. When the change in wavelength based on the
reflection of the incident light as colored interference light
brought out by changes in length or refractive index at the time
the film-like hybridization probe hybridizes with a target nucleic
acid is measured, the target nucleic acid in the sample may be
detected efficiently by a simple operation in a reliable
manner.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic view of a hybridization probe relating
to one example of the present invention.
[0021] FIG. 2 is a view for explaining a principle of light
reflection of the incident light as colored interference light.
[0022] FIG. 3 is a typical view to explain the principle of light
reflection of the incident light as colored interference light.
[0023] FIG. 4 is a schematic view for showing a formation of a
monomolecular film by a functional molecule of the present
invention.
[0024] FIG. 5 is a schematic view for showing an example of an
amphiphilic functional molecule aligned on water (aqueous
phase).
[0025] FIG. 6 is a schematic view for showing an example of an
amphiphilic functional molecule vertically aligned on water
(aqueous phase).
[0026] FIGS. 7A and 7B is an example view of a quartz oscillator in
which FIG. 7A is a plan view and FIG. 7B is a front view.
[0027] FIG. 8 is a schematic view which shows an example of a
nucleic acid detecting apparatus.
[0028] FIG. 9 is a schematic plan view showing a surface acoustic
wave (SAW) element.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] Hereinafter, the present invention will be described in more
detail.
[0030] As shown in FIG. 1 as an example, the hybridization probe 10
of the present invention has a rod-shaped body 1 and nucleic acid 2
which is bonded to the rod-shaped body 1 and specifically bonds to
a target nucleic acid. Incidentally, the exemplary hybridization
probe 10 of FIG. 1 is an amphiphilic polypeptide of an
.alpha.-helix structure in which the part 1a of the rod-shaped body
is hydrophobic while the part lb thereof is hydrophilic.
[0031] <Rod-Shaped Body>
[0032] The rod-shaped body is not particularly limited provided
that it is rod-shaped, and may be appropriately selected in
accordance with the object. The rod-shaped body may be either a
rod-shaped inorganic substance or rod-shaped organic substance, but
a rod-shaped organic substance is preferable.
[0033] Examples of rod-shaped organic substances are biopolymers,
polysaccharides, and the like.
[0034] Suitable examples of biopolymers are fibrous proteins,
.alpha.-helix polypeptides, nucleic acids (DNA, RNA), and the like.
Examples of fibrous proteins are fibrous proteins having
.alpha.-helix structures such as .alpha.-keratin, myosin,
epidermin, fibrinogen, tropomyosin, silk fibroin, and the like.
Suitable examples of polysaccharides are amylose and the like.
[0035] Among rod-shaped organic substances, spiral organic
molecules whose molecules have a spiral structure are preferable
from the standpoints of stable maintenance of the rod shape and
internal intercalatability of other substances in accordance with
an object. Among the aforementioned substances, those with spiral
organic molecules include .alpha.-helix polypeptides, DNA, amylose,
and the like.
[0036] {.alpha.-Helix Polypeptides}
[0037] .alpha.-helix polypeptides are referred to one of secondary
structures of polypeptides. The polypeptide rotates one time (forms
one spiral) for each amino acid 3.6 residue, and a hydrogen bond,
which is substantially parallel to the axis of the helix, is formed
between a carbonyl group (--CO--) and an imide group (--NH--) of
each fourth amino acid, and this structure is repeated in units of
seven amino acids. In this way, the .alpha.-helix polypeptide has a
structure which is stable energy-wise.
[0038] The direction of the spiral of the .alpha.-helix polypeptide
is not particularly limited, and may be either wound right or wound
left. Note that, in nature, only structures whose direction of
spiral is wound right exist from the standpoint of stability.
[0039] The amino acids which form the .alpha.-helix polypeptide are
not particularly limited provided that an .alpha.-helix structure
can be formed, and can be appropriately selected in accordance with
the object. However, amino acids which facilitate formation of the
.alpha.-helix structure are preferable. Suitable examples of such
amino acids are aspartic acid (Asp), glutamic acid (Glu), arginine
(Arg), lysine (Lys), histidine (His), asparagine (Asn), glutamine
(Gln), serine (Ser), threonine (Thr), alanine (Ala), valine (Val),
leucine (Leu), isoleucine (Ile), cysteine (Cys), methionine (Met),
tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), and the
like. A single one of these amino acids may be used alone, or two
or more may be used in combination.
[0040] By appropriately selecting the amino acid, the property of
the .alpha.-helix polypeptide can be changed to any of hydrophilic,
hydrophobic, and amphiphilic. In the case in which the
.alpha.-helix polypeptide is to be made to be hydrophilic, suitable
examples of the amino acid are serine (Ser), threonine (Thr),
aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), lysine
(Lys), asparagine (Asn), glutamine (Gln), and the like. In the case
in which the .alpha.-helix polypeptide is to be made to be
hydrophobic, suitable examples of the amino acid are phenylalanine
(Phe), tryptophan (Trp), isoleucine (Ile), tyrosine (Tyr),
methionine (Met), leucine (Leu), valine (Val), and the like.
[0041] In the .alpha.-helix polypeptide, the carboxyl group, which
does not form a peptide bond and which is in the amino acid which
forms the .alpha.-helix, can be made to be hydrophobic by
esterification. On the other hand, an esterified carboxyl group can
be made to be hydrophilic by hydrolysis.
[0042] The amino acid may be any of a L-amino acid, a D-amino acid,
a derivative in which the side chain portion of a L-amino acid or a
D-amino acid is modified, and the like.
[0043] The number of bonds (the degree of polymerization) of the
amino acid in the .alpha.-helix polypeptide is not particularly
limited and may be appropriately selected in accordance with the
object. However, 10 to 5000 is preferable.
[0044] If the number of bonds (the degree of polymerization) is
less than 10, it may not be possible for the polyamino acid to form
a stable .alpha.-helix. If the number of bonds (the degree of
polymerization) exceeds 5000, vertical orientation may be difficult
to achieve.
[0045] Suitable specific examples of the .alpha.-helix polypeptide
are polyglutamic acid derivatives such as poly(.gamma.-methyl
L-glutamate), poly(.gamma.-ethyl L-glutamate), poly(.gamma.-benzyl
L-glutamate), poly(n-hexyl L-glutamate), and the like; polyaspartic
acid derivatives such as poly(.beta.-benzyl L-aspartate) and the
like; polypeptides such as poly(L-leucine), poly(L-alanine),
poly(L-methionine), poly(L-phenylalanine),
poly(L-lysine)-poly(.gamma.-methyl L-glutamate), and the like.
[0046] The .alpha.-helix polypeptide may be a commercially
available .alpha.-helix polypeptide, or may be appropriately
synthesized or prepared in accordance with methods disclosed in
known publications and the like.
[0047] As one example of synthesizing the .alpha.-helix
polypeptide, the synthesis of block copolypeptide
[poly(L-lysine).sub.25-poly(.gamma.-meth- yl
L-glutamate).sub.60]PLLZ.sub.25-PMLG.sub.60 is as follows. As is
shown by the following formula, block copolypeptide
[poly(L-lysine).sub.25-poly- (.gamma.-methyl
L-glutamate).sub.60]PLLZ.sub.25-PMLG.sub.60 can be synthesized by
polymerizing N.sup..epsilon.-carbobenzoxy L-lysine
N.sup..alpha.-carboxy acid anhydride (LLZ-NCA) by using
n-hexylamine as an initiator, and then polymerizing .gamma.-methyl
L-glutamate N-carboxy acid anhydride (MLG-NCA). 1
[0048] Synthesis of the .alpha.-helix polypeptide is not limited to
the above-described method, and the .alpha.-helix polypeptide can
be synthesized by a genetic engineering method. Specifically, the
.alpha.-helix polypeptide can be manufactured by transforming a
host cell by a expression vector in which is integrated a DNA which
encodes the target polypeptide, and culturing the transformant, and
the like.
[0049] Examples of the expression vector include a plasmid vector,
a phage vector, a plasmid and phage chimeric vector, and the
like.
[0050] Examples of the host cell include prokaryotic microorganisms
such as E. coli, Bacillus subtilis, and the like; eukaryotic
microorganisms such as yeast and the like; zooblasts, and the
like.
[0051] The .alpha.-helix polypeptide may be prepared by removing
the .alpha.-helix structural portion from a natural fibrous protein
such as .alpha.-keratin, myosin, epidermin, fibrinogen,
tropomyosin, silk fibroin, and the like.
[0052] {DNA}
[0053] The DNA may be a single-stranded DNA. However, the DNA is
preferably a double-stranded DNA from the standpoints that the
rod-shape can be stably maintained, other substances can be
intercalated into the interior, and the like.
[0054] A double-stranded DNA has a double helix structure in which
two polynucleotide chains, which are in the form of right-wound
spirals, are formed so as to be positioned around a single central
axis in a state in which they extend in respectively opposite
directions.
[0055] The polynucleotide chains are formed by four types of
nucleic acid bases which are adenine (A), thiamine (T), guanine
(G), and cytosine (C). The nucleic acid bases in the polynucleotide
chain exist in the form of projecting inwardly within a plane which
is orthogonal to the central axis, and form so-called Watson-Crick
base pairs. Thiamine specifically hydrogen bonds with adenine, and
cytosine specifically hydrogen bonds with guanine. As a result, in
a double-stranded DNA, the two polypeptide chains are bonded
complementarily.
[0056] The DNA can be prepared by known method such as PCR
(Polymerase Chain Reaction), LCR (Ligase Chain Reaction), 3SR
(Self-Sustained Sequence Replication), SDA (Strand Displacement
Amplification), and the like. Among these, the PCR method is
suitable.
[0057] Further, the DNA can be prepared by being directly removed
enzymatically from a natural gene by a restriction enzyme. Or, the
DNA can be prepared by a genetic cloning method, or by a chemical
synthesis method.
[0058] In the case of a genetic cloning method, a large amount of
the DNA can be prepared by, for example, integrating a structure,
in which a normal nucleic acid has been amplified, into a vector
which is selected from plasmid vectors, phage vectors, plasmid and
phage chimeric vectors, and the like, and then introducing the
vector into an arbitrary host in which propagation is possible and
which is selected from prokaryotic microorganisms such as E. coli,
Bacillus subtilis, and the like; eukaryotic microorganisms such as
yeast and the like; zooblasts, and the like.
[0059] Examples of chemical synthesis methods include liquid phase
methods or solid phase synthesis methods using an insoluble
carrier, such as a triester method, a phosphorous acid method, and
the like. In the case of a chemical synthesis method, the
double-stranded DNA can be prepared by using a known automatic
synthesizing device and the like to prepare a large amount of
single-stranded DNA, and thereafter, carrying out annealing.
[0060] {Amylose}
[0061] Amylose is a polysaccharide having a spiral structure in
which D-glucose, which forms starch which is a homopolysaccharide
of higher plants for storage, is joined in a straight chain by
.alpha.-1,4 bonds.
[0062] The molecular weight of the amylose is preferably around
several thousand to 150,000 in number average molecular weight.
[0063] The amylose may be a commercially available amylose, or may
be appropriately prepared in accordance with known methods.
[0064] Amylopectin may be contained in a portion of the
amylose.
[0065] The length of the rod-shaped body is not particularly
limited, and may be appropriately selected in accordance with the
object. However, from the standpoint of causing light reflection of
the incident light as colored interference light which will be
described later, a length of 810 nm or less is preferable, and 10
nm to 810 nm is more preferable.
[0066] The diameter of the rod-shaped body is not particularly
limited, and is about 0.8 to 2.0 nm in the case of the
.alpha.-helix polypeptide.
[0067] The entire rod-shaped body may be hydrophobic or
hydrophilic. Or, the rod-shaped body may be amphiphilic such that a
portion thereof is hydrophobic or hydrophilic, and the other
portion thereof exhibits the opposite property of the one portion.
In the case of an amphiphilic rod-shaped body, the numbers of the
lipophilic (hydrophobic) portions and hydrophilic portions are not
particularly limited, and may be appropriately selected in
accordance with the object. Further, in this case, the portions
which are lipophilic (hydrophobic) and the portions which are
hydrophilic may be positioned alternately, or either type of
portion may be positioned only at one end portion of the rod-shaped
body.
[0068] In the case of the amphiphilic rod-shaped body, there is no
particular limitation for the numbers of the moiety showing
hydrophobicity and the moiety showing hydrophilicity but that may
be appropriately selected according to the object. In that case,
the moiety showing hydrophobicity and the moiety showing
hydrophilicity may be alternately positioned. Any of the moieties
may be positioned only at one end of the rod-shaped body.
[0069] <Target Nucleic Acid>
[0070] For the target nucleic acid, there is no particular
limitation but may be appropriately selected depending on the
object although it is preferred to be a nucleic acid selected from
a part of a base sequence which is present in prokaryotes only, a
part of a base sequence which is present in eukaryotes (except
human being) only and a part of a base sequence which is present in
human being only. It is not necessary that the target nucleic acid
is a nucleic acid which is a final target in the detection for each
of the objects but may be a nucleic acid which co-exists with the
nucleic acid of the final target for detection.
[0071] For the target nucleic acid, there may be specifically
exemplified cancer-related gene, gene related to genetic diseases,
virogene, bacterial gene, gene showing polymorphism called a risk
factor for diseases, and the like.
[0072] For the cancer-related gene, examples include K-ras gene,
N-ras gene, p53 gene, BRCA1 gene, BRCA2 gene, src gene, ros gene,
APC gene, and the like.
[0073] For the gene related to genetic diseases, examples include
genes of various inborn error metabolisms such as phenylketonuria,
alkaptonuria, cystinuria Huntington's chorea, Down syndrome,
Duehnne muscular dystrophy, hemophilia, and the like.
[0074] For the virogene and bacterial gene, examples include genes
of hepatitis C virus, hepatitis B virus, influenza virus, measles
virus, HIV virus, mycoplasma, rickettsia, streptococcus,
salmonella, and the like.
[0075] The gene showing polymorphism means a gene which has a base
sequence being not always directly related to the cause of the
disease, and the like and being different for each body. Examples
include PS1 (presenlin 1) gene, PS 2 (presenilin 2) gene, APP
(.beta.-amyloid precursor protein) gene, lipoprotein gene, gene
related to HLA (human leukocyte antigen) and blood type, gene
believed to be related to onset of hypertension, diabetes and the
like, and the like.
[0076] Usually, those genes are present on chromosomes of the host
but, in some cases, they are coded by mitochondria gene.
[0077] For the sample to be examined containing the target antigen
as such, examples include pathogenic organism such as bacteria and
virus; blood, saliva, disease tissue pieces, and the like separated
from living organism; and excrement such as feces and urine.
Further, when diagnosis before birth is carried out, cells of fetus
existing in amniotic fluid and a part of divided ovules may be also
used as a sample to be examined. Furthermore, such a sample to be
examined may be used either directly or, if necessary, after
concentrating as a precipitate by a centrifugal operation or the
like and then subjected to a cytocidal treatment such as, for
example, enzymatic treatment, thermal treatment, surfactant
treatment, ultrasonic treatment or a combination thereof. In that
case, the cytocidal treatment is carried out with an object of
manifesting the DNA derived from the aimed tissue.
[0078] Incidentally, protein which is essential for cell division
and has been known to specifically bond to DNA such as tubulin,
chitin, and the like which is not nucleic acid is also included in
the target substance of the present invention.
[0079] <Nucleic Acid Specifically Bonding to Target Nucleic
Acid>
[0080] The nucleic acid which specifically bonds to the nucleic
acid is RNA or a single-stranded DNA having a base sequence
complementary to the target nucleic acid. Such a nucleic acid is
able to be prepared by PCR method, chemical synthetic method, and
the like as same as above.
[0081] There is no limitation for the length of nucleic acid as
long as it is able to hybridize to a target nucleic acid in a
stable manner under a usual hybridizing condition. Still it is
preferred not to be unnecessarily long and is preferably to be
10-50 bases, more preferably 10-30 bases and, still more
preferably, 15-25 bases.
[0082] When the resulting nucleic acid which specifically bonds to
a target nucleic acid is bonded to the rod-shaped body, a
hybridization probe of the present invention is prepared.
[0083] The bonding method may be appropriately selected depending
on the nucleic acid the rod-shaped body and there may be used known
methods such as a method in which covalent bond such as ester bond,
amide bond, and the like is utilized; a method in which protein is
labeled with avidin and bonded to biotinated nucleic acid; a method
in which protein is labeled with streptoavidin and bonded to
biotinated nucleic acid; and the like.
[0084] For the covalent bond method, examples include peptide
method, diazo method, alkylation method, cyan bromide activation
method, bonding method by a cross-linking reagent, immobilization
method utilizing Ugi reaction, immobilization method utilizing a
thiol-disulfide exchange reaction, Schiff base formation method,
chelate bonding method, tosyl chloride method, biochemically
specific bonding method, and the like. For more stable bonding such
as covalent bond, there is preferably carried out utilizing a
reaction of thiol group with maleimide group, a reaction of pyridyl
disulfide group with thiol group, a reaction of pyridyl disulfide
group with thiol group, a reaction of amino group with aldehyde
group, and the like and there may be applied a method which is
appropriately selected from known methods, methods which may be
easily carried out by the persons skilled in the art and methods
which are modified therefrom. Among them, there may be used a
chemically bonding agent and a cross-linking agent which are able
to form more stable bond.
[0085] For such chemically bonding agent and cross-linking agent,
examples include carbodiimide, isocyanate, diazo compound,
benzoquinone, aldehyde, periodic acid, maleimide compound, pyridyl
disulfide compound, and the like. For the preferred reagent,
examples include glutaraldehyde, hexamethylene diisocyanate,
hexamethylene diisothiocyanate, N,N'-polymethylenebisiodoacetamide,
N,N'-ethylenebismaleiimide, ethylene glycol bissuccinimidyl
succinate, bisdiaobenzidine, 1-ethyl-3-(3-diethylamiopropyl)
carbodiimide, succinimidyl 3-(2-pyridylthio) propionate (SPDP),
N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), N-sulfosuccinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl)
aminobenzoate, N-succinimidyl 4-(1-maleimidophenyl) butyrate,
iminothiolane, S-acetylmercaptosuccinic acid anhydride,
methyl-3-(4'-dithiopyridyl)propionimidate, methyl-4-mercaptobutyryl
imidate, methyl-3mercaptopropionimidate, N-succinimidyl-S-acetyl
mercaptoacetate, and the like.
[0086] <Hybridization Probe>
[0087] As shown in FIG. 1, the hybridization probe 10 of the
present invention has a rod-shaped body 1 and nucleic acid 2 which
is bonded to the rod-shaped body 1 and specifically bonds to a
target nucleic acid. In the hybridization probe 10, when the target
nucleic acid is bonded to the nucleic acid moiety, properties of
the hybridization probe such as refractive index and transmittance
of light, mass, viscoelasticity, and the like change and,
therefore, when the change is detected, it may be utilized for the
detection of the target nucleic acid.
[0088] The above method for the detection may be appropriately
selected depending on the object and, for example, various methods
such as that color change is observed by naked eye, that wavelength
change is detected by spectrophotometer, that oscillation of
frequency of quartz oscillator, surface acoustic wave (SAW) element
or the like is detected by a frequency counter, and the like may be
carried out.
[0089] This hybridization probe 10 may be used as it is and, in
that case, when it is used by aligned in single or plural layer(s)
on the surface of a solvent containing the target nucleic acid or
at the boundary between the solvent and an immisible liquid,
changes in wavelength may be easily detected and, therefore, that
is preferred.
[0090] It is also able to be formed in a film-like state such as
monomolecular film or two layered monomolecular films on a
substrate which is vertically aligned by, for example, a
Langmuir-Brodgett (HYBRIDIZATION PROBE) technique and the above is
preferred in such respects that changes in wavelength may be apt to
be detected, that quartz oscillator, surface acoustic wave (SAW)
element or the like may be fixed, that handling is easy, and the
like.
[0091] For the hybridization probe of the present invention, the
one in which to reflect an incident light as colored interference
light is preferred from a viewpoint of recognition and
discrimination.
[0092] The reflection of the incident light as colored interference
light is a color formation resulted on the basis of a multi-layer
thin film interference theory which is a basic principle of color
formation of the scaly powder of the wings of a Morpho butterfly
and is a color formation on the film as a result of reflection of
light of specific wavelength corresponding to the thickness of the
film and the refractivity thereof when stimulation from outside
such as electric field, magnetic field, heat, light (for example,
natural light, infrared light and ultraviolet light), and the like
is applied to the film. The color tone may be freely controlled
like the surface skin of chameleon by the stimulation from
outside.
[0093] Principle of the light reflection of the incident light as
colored interference light will be described herein after.
[0094] As shown in FIG. 2 and FIG. 3, when light is irradiated on
the film of the rod-shaped body, wavelength (.lambda.) of the
interference light by the film is emphasized under the condition as
shown in the following (1) and enfeebled under the condition as
shown in the following (2). 1 = 2 tl m n 2 - sin 2 ( 1 ) = 4 tl 2 m
- 1 n 2 - sin 2 ( 2 )
[0095] In the formulae (1) and (2), the .lambda. means wavelength
(nm) of the interference light, the .alpha. means angle of
incidence (degree) of the light to the film, the t means thickness
(nm) of a single film, the 1 means numbers of the film, the n means
a refractive index of the film and the m means an integer of 1 or
more.
[0096] The light reflection of the incident light as colored
interference light may be achieved by aligning the hybridization
probe in a film-like shape.
[0097] Thickness of the single film is preferably 810 nm or less
and, more preferably, it is from 10 nm to 810 nm.
[0098] When the thickness is changed suitably, a color (wavelength)
of the light interfered by the light reflection of the incident
light may be changed.
[0099] The film may be either a monomolecular film or a layered
film comprising the monomolecular film.
[0100] The monomolecular film or the layered film comprising the
same may be formed by, for example, a Langmuir-Brodgett method (LB
method) and, in that case, a known LB film forming apparatus (such
as NL-LB 400 NK-MWC manufactured by Nippon Laser & Electronics
Laboratories) may be used.
[0101] Formation of the monomolecular film may be carried out, for
example, in such a state that the above mentioned rod-shaped body
which is lipophilic (hydrophobic) or amphiphilic is floated on
water surface (on an aqueous phase) or in such a state that the
rod-shaped body which is lipophilic (hydrophobic) or amphiphilic is
floated on oil surface (on an oil phase) or, in other words, the
rod-shaped body 1 is aligned as shown in FIG. 4 so as to form on a
substrate 50 using an extrusion material 60. When such an operation
is repeatedly carried out, the layered film where the monomolecular
films are layered in any number may be formed on the substrate 50.
Incidentally, it is preferred that the monomolecular film or the
layered film is fixed on the substrate 50 since the reflection of
the incident light as colored interference light by the
monomolecular film or layered film is expressed in a stable
manner.
[0102] In that case, there is no particular limitation for the
substrate 50 and, according to the object, its material, shape,
size, and the like may be appropriately selected although it is
preferred that its surface is appropriately subjected to a surface
treatment previously with an object that the rod-shaped body 1 is
easily aligned thereto. When the rod-shaped body 1 (such as
.alpha.-helix polypeptide) is hydrophilic for example, it is
preferred that a surface treatment such as hydrophilizing treatment
using octadecyl trimethylsiloxane and the like is previously
carried out.
[0103] With regard to the state where the rod-shaped body is
floated on an oil phase or an aqueous phase in the formation of the
monomolecular film of the amphiphilic rod-shaped body, the
lipophilic areas (hydrophobic areas) 1a of the rod-shaped body 1
are aligned in an adjacent state each other on the aqueous phase or
oil phase while the hydrophilic areas 1b are aligned in an adjacent
state each other as shown in FIG. 5.
[0104] The above is an example of a layered membrane or a layered
film comprising the same where the rod-shaped body is aligned in
the plane direction of the monomolecular film (in a horizontal
state) while a monomolecular film where the rod-shaped body is
aligned in the thickness direction of the monomolecular film (in a
vertical state) may be manufactured, for example, as follows.
First, as shown in FIG. 6, water (aqueous phase) is made alkaline
of around pH 12 under such a state that the amphiphilic rod-shaped
body 1 (.alpha.-helix polypeptide) is floated on the water surface
(aqueous phase) (i.e., in a horizontal state). As a result, in the
hydrophilic area lb in the rod-shaped body 1 (.alpha.-helix
polypeptide), the .alpha.-helix structure thereof is disentangled
to give a random structure. At that time, the lipophilic area
(hydrophobic area) 1a of the rod-shaped body 1 (.alpha.-helix
polypeptide) maintains its .alpha.-helix structure. Then, pH of the
water (aqueous phase) is made acidic to about 5 thereby the
hydrophilic area lb in the rod-shaped body 1 (.alpha.-helix
polypeptide) forms an .alpha.-helix structure again. When the
pushing material attached to the rod-shaped body 1 (.alpha.-helix
polypeptide) is pushed by the pressure of air from its side to the
rod-shaped body 1 (.alpha.-helix polypeptide), the rod-shaped body
1 maintains vertical against water (aqueous phase) while its
hydrophilic area 1b forms an .alpha.-helix structure in the
direction substantially orthogonal to the water surface in the
aqueous phase. When the aligned rod-shaped body 1 (.alpha.-helix
polypeptide) is pushed out onto the substrate 50 using a pushing
material 60 as mentioned above by referring to FIG. 4, it is
possible to form a monomolecular film on the substrate 50. When
such operation is repeatedly carried out, the layered film having
prescribed number of monomolecular film may be formed on the
substrate 50.
[0105] With regard to the hybridization probe which is able to give
single-layered film or multi-layered film which reflects incident
light as colored interference light, there may be exemplified an
hybridization probe which is amphiphilic and an amphiphilic
hybridization probe wherein the rod-shaped body is .alpha.-helix
polypeptide is preferred.
[0106] In the hybridization using the hybridization probe of the
present invention, there have been commonly used SSC (20.times.SSC:
3M sodium chloride, 0.3M sodium citrate), SSPE (20.times.SSPE: 3.6M
sodium chloride, 0.2M sodium phosphate, 2 mM EDTA), and the like
and, in the present invention, those solutions may also be used by
diluting to an appropriate concentration. If necessary, it is
preferred to carry out in a hybridization solution containing an
organic solvent such as dimethyl sulfoxide (DMSO), dimethyl
formamide (DMF), and the like, formamide, salt, protein,
stabilizer, buffer, and the like.
[0107] Concentration of the formamide is 0-60% by mass, preferably
10-50% by mass and, more preferably, 20-30% by mass. For the salt,
examples include inorganic salts such as sodium chloride, potassium
chloride, and the like and organic salts such as sodium citrate,
sodium oxalate, and the like and the salt concentration is 0-2.0 M
or, preferably, 0.15-1.0 M. For the protein, examples include serum
albumin, and the like. For the stabilizer, examples include phycol,
and the like. For the buffer, examples include a phosphate buffer,
and the like and its preferred concentration is 1-100 mM.
[0108] The hybridization using the hybridization probe may be
carried out in such a manner that the hybridization probe and a
sample nucleic acid (single-stranded DNA or single-stranded RNA)
are added to the hybridization solution to an extent of equimolar
concentration, heated at 60-90.degree. C. for 1-60 minute(s) and
then gradually cooled down to 040.degree. C. during 1-24
hour(s).
[0109] <Target Nucleic Acid Detecting Kit>
[0110] The target nucleic acid detecting kit of the present
invention contains a hybridization probe having a rod-shaped body
with a length of 810 nm or shorter and nucleic acid which is bonded
to the rod-shaped body and specifically bonds to a target nucleic
acid and causing a light reflection of the incident light as
colored interference light when aligned in a film-like shape; and
any of dish, plate and tube.
[0111] The target nucleic acid detecting kit may contain a solvent
containing the hybridization probe in an amount suitable for the
size of the dish, and the like in a container which is different
from the container. For example, an aqueous sample is added to the
container and the oily or amphiphilic hybridization probe is added
to the sample so that the hybridization probe is aligned on the
sample in a film-like shape in which a target nucleic acid may be
detected by the changes in wavelength based on the light reflection
of an incident light as colored interference light of the film-like
probe.
[0112] If necessary, the detecting kit may be combined with
cytocidal reagent for a pretreatment of the sample, washing liquid
for washing the amplified reaction product, oil for preventing the
evaporation of water from the reaction solution, and the like.
[0113] In the target nucleic acid detecting kit of the present
invention, the hybridization probe aligned in a film-like shape
reflects the incident light as colored interference light on the
basis of the multi-layered thin film interference theory which is a
basic principle for color formation of the scaly powder of the
wings of a Morpho butterfly. Accordingly, When the change in
wavelength based on the reflection of the incident light as colored
interference light brought out by changes in length or refractive
index at the time the film-like hybridization probe hybridizes with
a target nucleic acid is measured, it is now possible to detect the
target nucleic acid in the sample by a simple operation in a
reliable manner.
[0114] <Target Nucleic Acid Detecting Apparatus>
[0115] The target nucleic acid detecting apparatus according to the
first embodiment of the present invention is equipped with a
hybridization probe having a rod-shaped body of a length of 810 nm
or shorter, nucleic acid which is bonded to the rod-shaped body and
bonds to a target nucleic acid and reflecting the incident light as
colored interference light when aligned in a film-like shape; a
sample adding means in which the hybridization probe is contacted
to a sample; and a colored wavelength measuring means in which
changes in wavelength based on the reflection of the incident light
as colored interference light brought out by changes in length or
refractive index at the time the film-like hybridization probe
hybridizes with a target nucleic acid is measured.
[0116] For the sample, there is no particular limitation as long as
it is a thing which is an object of the test whether or not it
contains the target nucleic acid and examples include the sample to
be tested, a nucleic acid library containing a target nucleic acid,
and the like.
[0117] For an appropriate embodiment of the target nucleic acid
detecting apparatus, it is preferred that the hybridization probe
is further amphiphilic and that the sample adding means is a sample
adding means in which the hybridization probe is added to an
aqueous sample together with an oil phase so that the hybridization
probe is contacted to the sample.
[0118] For the sample adding means, there is no particular
limitation as long as it is a means for adding a predetermined
amount of hybridization probe to the sample or is a means for
adding a predetermined amount of sample to the hybridization probe.
It is however preferred that the amount of the hybridization probe
is set in such an amount that the light reflection of an incident
light as colored interference light may apt to be detected by
aligning in a film-like shape.
[0119] The fact that the hybridization probe is amphiphilic is
preferred because of such a view that the probe is vertically
aligned at the interface between an oil phase and an aqueous phase
in which changes in wavelength due to the light reflection of an
incident light as colored interference light are easily
measured.
[0120] In accordance with the target nucleic acid detecting
apparatus according to the first embodiment, when the nucleic acid
having a base sequence complementary to the target nucleic acid of
the hybridization probe is hybridized to the target nucleic acid,
length or refractive index of the probe aligned in a film-like
shape changes and, when the change in wavelength based on a colored
interference light brought about by the incident light reflection
of the probe is measured by a colored wavelength measuring means
such as a spectrophotometer, it is now possible to specifically
test whether the target nucleic acid is present. In addition, when
a calibration curve is previously prepared using a sample DNA in a
known amount, concentration of DNA in the sample to be detected or
quantified may be detected or quantified.
[0121] The target nucleic acid detecting apparatus according to the
second embodiment of the present invention comprises a biosensor
the hybridization probe of the present invention is aligned on
quartz oscillator or surface acoustic wave (SAW) element in a
film-like shape, an oscillation circuit whereby changes in mass or
changes in viscoelasticity when a target nucleic acid is hybridized
to the biosensor are oscillated as frequency and a frequency
counter whereby frequency of the oscillation oscillated from the
oscillation circuit is measured.
[0122] In that case, it is preferred that the hybridization probe
is aligned in a monomolecular film-like shape to the quartz
oscillator or surface acoustic wave (SAW) element or is aligned in
a bimolecular film-like shape thereto. For the frequency counter,
there is no particular limitation as long as it can precisely
measure the frequency from the quartz oscillator or the surface
acoustic wave (SAW) element.
[0123] In the quartz oscillator, metal electrodes are vapor
deposited on the surface and the back of a thin quartz plate. An
example of the quartz oscillator 20 is shown in FIGS. 7A and 7B.
FIG. 7A is a plane view while FIG. 7B is a front view. An electrode
12 is vapor deposited on the surface of the quartz plate 21 while
another electrode 14 is vapor deposited on the back thereof. The
electrodes extend to the left side from the electrodes 12, 14 and
the left ends thereof are connected to clip-type lead wires (not
shown) followed by connecting to an alternating current source (not
shown). When alternating current is applied between the electrodes
12, 14, there is generated oscillation of a predetermined period in
the quartz plate 21 due to a back piezoelectric effect.
[0124] Although not shown in the drawing, an hybridization probe
film is aligned on the surface of the quartz oscillator 20. The
antibody of this hybridization probe film is bonded to the target
antigen and mass of the surface of the quartz oscillator 20 changes
to an extent of the mass of the bonded target antigen whereby a
resonance frequency changes.
[0125] Between the changes in the resonance frequency and changes
in the mass of the hybridization probe film coated on the surface
of the quartz oscillator 20 which oscillates in parallel to the
plane vertical to the thickness direction, there is a relation as
shown in the following formula (3) whereby changes in the mass may
be detected from changes in the resonance frequency. For example,
in the case of an oscillator of resonance frequency of 9 MHz (area:
about 0.5 cm.sup.2), a reduction in frequency of 400 Hz is resulted
by an increase in mass of 1 .mu.g.
.DELTA.F=-2.3.times.10.sup.6 (F.sup.2.times..DELTA.W/A) (3)
[0126] In the formula, F means resonance frequency (MHz) of the
quartz oscillator, .DELTA.F means changes (Hz) in the resonance
frequency by changes in mass, .DELTA.W means changes in mass (g) of
the film and A means a surface area (cm.sup.2) of the film.
[0127] An example of the nucleic acid detecting apparatus is shown
in FIG. 8. The quartz oscillator 20 (hybridization probe 10 is
aligned on the surface in a film-like shape) is attached to an arm
for attaching the quartz oscillator and dipped in a solution in a
thermostat heat block 23. The thermostat heat block 23 maintains
the temperature of the solution constant. The solution is stirred
by a stirrer 24. In a sample injection 25, a sample to be measured
is injected into a solution. In the oscillation circuit 26,
alternating current field is applied to the electrodes 12, 14 of
the quartz oscillator 20 to oscillate the quartz oscillator 20.
Oscillation frequency of the oscillation circuit 24 is counted by a
counter 27, analyzed by a computer 28 and mass of the target
antigen in the sample is indicated.
[0128] As such, when a nucleic acid having a base sequence
complementary to the target nucleic acid of the hybridization probe
is hybridized to the target nucleic acid, mass of the probe changes
and the quartz oscillator catches the change in mass and converts
to frequency. Accordingly, when the changes in frequency are
measured by the frequency counter, it is now possible to
specifically test whether the target nucleic acid is present.
[0129] When a calibration curve is previously prepared using a
sample DNA in a known amount, it is also possible to detect or
quantity the concentration of DNA in the sample to be detected or
quantified.
[0130] The surface acoustic wave (SAW) element is an element where
a pair of comb-shaped electrodes is set on the surface of the solid
and electric signal is converted to a surface acoustic wave (sonic
wave transmitting the solid surface, ultrasonic wave), transmitted
to the encountering electrode and outputted as electric signal
again whereby signal of specific frequency corresponding to the
stimulation may be taken out. Ferroelectric substance such as
lithium tantalite and lithium niobate, quartz, zinc oxide thin
film, and the like are used as the material therefor.
[0131] The SAW is elastic wave which transmits along the surface of
the medium and exponentially decreases in the inner area of the
medium. In the SAW, the transmitted energy is concentrated on the
surface of the medium whereby the changes in the medium surface may
be sensitively detected and, as a result of the changes in the mass
of the surface, the SAW transmitting velocity changes as same as in
the case of quartz oscillator. Usually, SAW transmitting velocity
is measured as the changes in oscillation frequency using an
oscillation circuit. Changes in the oscillation frequency are given
by the following formula.
.DELTA.f=(k.sub.1+k.sub.2)f.sup.2h.sub..rho.-k.sub.2f.sup.2h[(4.mu./V.sub.-
r.sup.2)(.lambda.+.mu./.lambda.+2.mu.)]
[0132] In the formula, k.sub.1 and k.sub.2 mean constants, h means
thickness of the fixed film, .rho. means density of the film,
.lambda. and .mu. mean Lame constants of the film and V.sub.r means
a SAW transmitting velocity.
[0133] FIG. 9 is a schematic plane view which shows an example of
constitution of main parts of a surface acoustic wave (SAW)
element. In FIG. 9, in the SAW element sensor 30, there are formed
gold electrode 38 and comb-shaped electrodes 36 at both ends
thereof on the SAW element having a resonance frequency of 90 MHz
made of an ST cut quartz and there is formed a film (not shown)
comprising the hybridization probe in the surface wave transmitting
region 37 as shown by dotted lines. The sensor is connected to a
frequency counter 39 from each comb-shaped electrode 36 via a
high-frequency amplifier 35 whereby the mass of the object to be
captured in the sample is indicated.
[0134] When a nucleic acid having a base sequence complementary to
the target nucleic acid of the hybridization probe is hybridized to
the target nucleic acid, mass or viscoelasticity of the probe
changes and the surface acoustic wave (SAW) catches the change in
mass or viscoelasticity and converts to frequency. Accordingly,
when the changes in the frequency are measured by the frequency
counter, it is now possible to specifically test whether the target
nucleic acid is present.
[0135] When a calibration curve is previously prepared using a
sample antigen of a known amount, the antigen concentration to be
detected or quantified in the sample may be detected or
quantified.
[0136] For a method of chemical bonding/fixing of the hybridization
probe on the electrode of the quartz oscillator or surface acoustic
wave (SAW) element constituting the biosensor, there is no
particular limitation but may be appropriately selected depending
on the object. For example, that may be carried out by means of a
chemical bond such as a covalent bond.
[0137] The above-mentioned covalent bond method is not particularly
limited, however, the same method which is used for bonding of
nucleic acid to rod-shaped body in the hybridization probe may be
appropriately selected and used.
[0138] To be specific, examples include a method in which a
substance in which thiol group is introduced into the end of the
hybridization probe is synthesized, then quartz oscillator or
surface acoustic wave (SAW) element is dipped into the above
solution and is made to react therewith and, after that, the
biosensor is taken out from the solution and dried. With regard to
the thiol group, S-trimethyl-3-mercaptopropyloxy-.beta.-cy-
anoethyl-N,N-diiso-propylaminophosphoramidide or the like is
covered and introduction of the thiol group into the end of the
probe may be carried out by a phosphoramide method.
[0139] <Target Nucleic Acid Detecting Method>
[0140] The target nucleic acid detecting method according to the
present invention comprises a contacting step in which a
hybridization probe having a rod-shaped body of a length of 810 nm
or shorter, having nucleic acid which is bonded to the rod-shaped
body and specifically bonds to a target nucleic acid, reflects the
incident light as colored interference light when aligned in a
film-like shape and being amphiphilic with a sample; and a
wavelength measuring step in which changes in wavelength caused by
the light reflection of the incident light as colored interference
light of the film-like hybridization probe hybridized to the target
nucleic acid.
[0141] For the contacting step, there is no particular limitation
but may be carried out by the same condition and method as in the
case of common hybridization.
[0142] For the colored wavelength measuring step, there is no
particular limitation so long as it is a method in which the
changes in the wavelength based on the light reflection of an
incident light as colored interference light brought about by
changes in length or refractive index at the time the hybridization
probe aligned in a film-like shape hybridizes with the target
nucleic acid may be measured and examples include a method in which
the changes in wavelength are measured using a
spectrophotometer.
[0143] In accordance with the target nucleic acid detecting method
of the present invention, the change in wavelength based on the
reflection of the incident light as colored interference light
brought out by changes in length or refractive index at the time
the film-like hybridization probe hybridizes with a target nucleic
acid is measured, it is now possible to quickly and surely detect
the presence of the target nucleic acid by a simple operation.
EXAMPLES
[0144] As hereunder, the present invention will be more
specifically illustrated by the following examples although the
present invention is not limited to such examples.
Example 1
[0145] DNA chain having a K-ras mutation sequence having a sequence
which is different only in one base from human normal chromosome
K-ras gene was prepared using a pair of primers having a one-base
mismatch from the following human normal chromosome K-ras gene.
1 k-ras-5m (mutation base is underlined)
5'-TATAAACTTGTGGTAGTTGGACCT-3' SEQ ID NO: 1 k-ras-3
5'-TATCGTCAAGGCACTCTTGCC-3' SEQ ID NO: 2
[0146] A gene amplifying reaction was carried out by a PCR method
in a reaction solution containing 100 .mu.l of 10 mM
Tris-hydrochloride buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2,
0.001% gelatin and 2 units of Ampli taq.TM. DNA polymerase in the
presence of 200 .mu.M dNTP using Bio-k-ras-5 m (100 ng) into which
biotin was introduced and Bio-k-ras-3 (100 ng) into which biotin
was introduced as a pair of primers and using 100 ng of human
normal chromosome K-ras gene as a template. In the PCR method, a
Thermal Cycler PJ 2000 (manufactured by Perkin-Elmer) was used and
a cycle of 94.degree. C. for 30 seconds and 56.degree. C. for 30
seconds was repeated for 35 times. The resulting reaction solution
was analyzed by an agarose gel electrophoresis and its size and
amplifying efficiency were measured.
[0147] Next, polymerization of N.sup..epsilon.-carbobenzoxy
L-lysine N.sup..alpha.-carboxylic acid anhydride (LLZ-NCA) was
carried out using n-hexylamine as an initiator and then
polymerization of .gamma.-methyl L-glutamate N-carboxylic acid
anhydride (MLG-NCA) was carried out to prepare a block
copolypeptide PLLZ.sub.2000-PMLG.sub.600 in which degree of
polymerization of a PLLZ moiety was 2000 and that of a PMLG moiety
was 600. After that, the PMLG segment was partially hydrolyzed to
give L-glutamic acid (LGA) in which .alpha.-helix copolypepide
PLLZ.sub.250-P(MLG.sub.420/LGA.sub.180) was prepared.
[0148] Avidin was introduced into this .alpha.-helix copolypeptide
followed by bonding to biotin of the DNA chain having the K-ras
mutation sequence in which a hybridization probe was prepared.
[0149] Then, the hybridization probe was kept in a state of being
floated (horizontal state) on a water surface (aqueous phase) and
pH of the water (aqueous phase) is made alkaline of around 12 in
which the .alpha.-helix structure in the hydrophilic area of the
probe is disentangled to give a random structure. At that time, the
hydrophobic area of the probe still maintains its .alpha.-helix
structure. Then, pH of the water (aqueous phase) is made acidic of
around 5 in which the hydrophilic area in the probe forms an
.alpha.-helix structure again. When the pushing material attached
to the probe is pushed by the pressure of air from its side to the
probe at that time, the probe is still in a state of being vertical
to the water (aqueous phase) while its hydrophilic area forms an
.alpha.-helix structure in the direction Substantially orthogonal
against the surface of water in the aqueous phase. When the probe
in an aligned state is pushed out onto the substrate (plate) using
a pushing material as mentioned above, it is possible to form a
monomolecular film in which the probe is vertically stood on the
substrate (plate). Incidentally, this operation was carried out
using an apparatus for forming a HYBRIDIZATION PROBE membrane
(NL-LB 400NK-NWC; manufactured by Nippon Laser & Electronics
Laboratories). Thickness of this monomolecular film was calculated
to be about 16 nm.
[0150] The resulting substrate in which the hybridization probe was
vertically stood in a state of a monomolecular film was aligned in
a solvent containing K-ras mutation gene prepared from a cDNA
library and changes in wavelength by the light reflection of an
incident light as colored interference light were measured in
which, as compared with the case in which addition was conducted to
a solvent containing no K-ras mutation gene, significant changes in
wavelength were observed.
Example 2
[0151] Monomolecular film in which a hybridization probe was
vertically stood on a substrate (plate) in Example 1 was used as a
structure unit comprising two layered monomolecular films to
prepare a substrate in which the hybridization probe was vertically
stood in a form of two layered monomolecular films. The substrate
was aligned in a solvent containing K-ras mutation gene prepared
from a cDNA library and the changes in wavelength based on the
light reflection of an incident light as colored interference light
were measured by a spectrophotometer in which, as compared with the
case in which addition was conducted to a solvent containing no
K-ras mutation gene, significant changes in wavelength were
observed.
Example 3
[0152] A product in which a gold electrode having an area of 0.2
cm.sup.2 and a gold-plated lead wire were attached to a quartz
oscillator (AT cut; area: 0.5 cm.sup.2; basic frequency: 9 MHz) was
used as a quartz oscillator electrode.
[0153] The quartz oscillation electrode was dipped at room
temperature for 1 hour in a 1% by volume aqueous solution of
aminopropyl triethoxysilane (manufactured by Chisso) and washed by
irradiating with ultrasonic wave of 20 kHz in pure water for 30
minutes to remove an excessive aminopropyl triethoxysilane. After
that, the quartz oscillation electrode was subjected to a thermal
treatment for 20 minutes at the temperature of 110.degree. C.
whereby a covalent bond was formed between aminopropyl
triethoxysilane and quartz oscillator surface.
[0154] Then this quartz oscillator was dipped for 1 hour in a 1% by
volume aqueous solution of glutaraldehyde to form a covalent bond
between glutaraldehyde and aminopropyl triethoxysilane and, after
that, the quartz oscillator was washed by irradiating with
ultrasonic wave of 20 kHz for 30 minutes in pure water to remove an
excessive glutaraldehyde.
[0155] The quartz oscillator electrode was dipped for 2 hours in
100 ml of a phosphate buffer of pH 7.2 containing the hybridization
probe prepared in Example 1. As a result thereof, the hybridization
probe was fixed to the quartz oscillator via glutaraldehyde. The
unreacted physical checkup medicament was removed by washing with a
phosphate buffer of pH 7.2.
[0156] After that, the quartz oscillator prepared as such was
attached to the target nucleic acid detecting apparatus shown in
FIG. 8, then a predetermined amount of solvent containing K-ras
mutation gene prepared from a cDNA library was added to a phosphate
buffer and changes in frequency during 10 minutes were checked.
Within one minute, the changes in the oscillation frequency almost
reached saturation. The thing in which a solvent containing K-ras
mutation gene was added showed a clear reduction in oscillation
frequency as compared to those without.
[0157] When the adding amount of K-ras mutation gene increased, it
was found that the oscillation frequency decreased in a certain
rate.
Example 4
[0158] A target nucleic acid detecting apparatus was assembled in
the same manner as in Example 3 except that, in place of the quartz
oscillator in Example 3, there was used a surface acoustic wave
(SAW) element of ST cut as shown in FIG. 9 in which oscillation
frequency was 10.3 MHz.
[0159] To a phosphate buffer was added a predetermined amount of a
solvent containing K-ras mutation gene prepared from a cDNA library
and changes in oscillation frequency during 10 minutes were
checked. Within one minute, the changes in the oscillation
frequency almost reached saturation. In the sample in which a
solvent containing K-ras mutation gene was added showed a clear
reduction in oscillation frequency as compared to those without
added.
[0160] When the adding amount of K-ras mutation gene increased, it
was found that the oscillation frequency decreased in certain
rate.
[0161] In accordance with the present invention, the formation
reaction of DNA hybrid may be directly manifested in aqueous phase
or gaseous phase without special technique within a short time and,
at the same time, formation of DNA hybrid may be tested easily and
highly precisely. In addition, the hybrid formation may be
quantitatively tested if necessary.
Sequence CWU 1
1
2 1 24 DNA Artificial Sequence DNA Synthesizer 1 tataaacttg
tggtagttgg acct 24 2 21 DNA Artificial Sequence DNA Synthesizer 2
tatcgtcaag gcactcttgc c 21
* * * * *