US 20020068282A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2002/0068282 A1
(43) Pub. Date:
Okamoto et al.
(54) DETECTION/QUANTIFICATION OF
TARGETED NUCLEOTIDE CHAINS, AND
DETECTION/QUANTIFICATION OF
MULTI-STRANDED NUCLEOTIDE CHAINS
BY FLUORESCENCE
(52)
(57)
Jun. 6, 2002
Us. 01. ................................................................ .. 435/6
ABSTRACT
A method for dry detection/quanti?cation of targeted nucle
otide chains, comprising the steps of:
(76) Inventors: Tadashi Okamoto, Yokohama-shi (JP);
Nobuko Yamamoto, Isehara-shi (JP);
(1) realizing a state in Which a hybrid (C) of a certain
Tomohiro Suzuki, Sagamihara-shi (JP)
derived from a sample solution and subjected to
detection or quanti?cation, and a probe nucleotide
chain (B), Which has a base sequence complemen
tary to a speci?c site of the base sequence of the
targeted nucleotide chain, is formed on a solid-phase
Correspondence Address:
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK, NY 10112 (US)
amount of targeted nucleotide chain (A), Which is
substrate by mutually reacting the tWo types of
(21) Appl. No.:
09/764,050
nucleotide chains With each other, and in Which there
exists a ?uorescence dye (D), Which acts on the
(22) Filed:
Jan. 19, 2001
hybrid (C), thereby emits ?uorescence or increases
its ?uorescence intensity, and is capable of continu
(30)
Foreign Application Priority Data
Jul. 26, 1999
Jul. 26, 1999
(JP) ......................................... .. 11-210701
(JP) ......................................... .. 11-210702
Publication Classi?cation
ing to emit ?uorescence even in the dried state While
acting on the hybrid;
(2) drying the hybrid (C) and the ?uorescence dye (D)
on the substrate; and
(3) measuring the ?uorescence emitted from the ?uo
rescence dye (D), as a measuring means, after the
(51)
Int. Cl? ..................................................... .. C12Q 1/68
drying operation.
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DETECTION/QUANTIFICATION OF TARGETED
NUCLEOTIDE CHAINS, AND
DETECTION/QUANTIFICATION OF
MULTI-STRANDED NUCLEOTIDE CHAINS BY
FLUORESCENCE
BACKGROUND OF THE INVENTION
[0001]
1. Field of the Invention
[0002] The present invention relates to detection/quanti
?cation of targeted nucleotide chains With speci?c base
sequences therein and detection/quanti?cation of multi
stranded nucleotide chains such as double-stranded nucle
otide chains.
[0003] 2. Related Background Art
[0004] In order to detect speci?c genes of organisms, in
the detection of a double-stranded nucleotide hybrid of a
targeted nucleotide chain and a nucleotide probe for the
targeted nucleotide chain, a technology has been Widely
used of detecting/quantifying the double-stranded nucle
otide chain in gel or solution using ?uorescence dyes, such
as ethidium bromide (hereinafter referred to as EB), Which
act on double-stranded nucleotide chains and increase their
?uorescence intensity.
[0005] In recent years, While polymerase chain reaction,
Which replicates nucleotide chains With an enZyme, has been
very commonly used to detect a very slight amount of
nucleotide chains, a method has also been used Which uses
[0011] The method, What is called solid-phase hybridiZa
tion, is Widely knoWn Which forms a hybrid on a solid phase
by ?xing a targeted nucleotide chain, Which has a speci?c
base sequence, for use in detection of a speci?c gene or
?xing a probe nucleotide chain having a base sequence
complementary to a speci?c site of the base sequence of the
targeted nucleotide chain on a solid phase and by reacting
the same With the corresponding probe nucleotide chain or
targeted nucleotide chain to detect the hybrid thereof. And
there have been developed various detecting techniques.
[0012] The most typical technique is the Southern hybrid
iZation technique, Which forms a hybrid on, for example, a
nylon ?lter by transferring a nucleotide chain, Which is
developed according to their length utiliZing gel electro
phoresis, to the nylon ?lter, denaturing the same to be
single-stranded, and alloWing a probe nucleotide chain
labeled With a radioisotope to act on the same, so as to detect
the formed hybrid thereof by using autoradiography.
[0013] And in order to overcome the problems attendant to
the use of radioisotope, an improved technique has been put
to practical use Which uses chemiluminescence as a detect
ing means.
[0014] In recent years, as means for detecting a speci?c
gene more simply and easily, a technique has also been
developed Which forms a hybrid on a micro-plate by ?xing
a probe nucleotide chain on the micro-plate and alloWing a
mRNA targeted nucleotide chain to act on the same, so as to
detect the formed hybrid thereof With a dye, for example,
ethidium bromide (hereinafter referred to as EB), Which acts
2-methyl-4,6-bis(4-N,N-dimethylaminophenyl)pyrylium
speci?cally on double-stranded nucleotide chains and
salt, a dye emitting ?uorescence only after acting on double
thereby increase its ?uorescence intensity (published J apa
stranded nucleotide chains in solution, to detect the PCR
nese Laid-Open Application of PCT Patent Application No.
ampli?cation products (Nucleic Acid Research, 1995, Vol.
7-506482).
23, No. 8 1445-1446).
[0006] Further, quite recently, a fact has been knoWn that
there exist in organisms triple-stranded and quadruple
stranded nucleotide chains having special base sequences
Which act on double-stranded nucleotide chains or multi
and those nucleotide chains play an important part in the
replication of genes and the lifetime of cells in organisms.
[0015] As described above, there have been knoWn dyes
stranded nucleotide chains in solution and thereby increase
their ?uorescence intensity or emit ?uorescence for the ?rst
time; hoWever, What is knoWn about them is just the behav
ior in solution.
[0007] These nucleotide chains form triple-stranded
chains and quadruple-stranded chains and, in each chain, 3
[0016] Usually for the determination of the ?uorescence
or 4 bases in corresponding positions form a base trio or a
etc.) of ?uorescence dyes, measurements are made for about
1 to 4 mL of sample solution With a dye dissolved therein
base quartet in a plane.
[0008] Although there have been very feW studies on dyes
acting on these triple- and quadruple-stranded chains, in
particular on ?uorescence dyes, there is found in a collection
of lecture resumes II (665) for the 72th spring meeting of the
Chemistry Society of Japan (1997) a description that 2-me
thyl-4,6-bis(4-N,N-dimethylaminophenyl)pyrylium salt acts
characteristics (intensity, excitation/?uorescence spectrum,
placed in measuring glass cells, generally With a spectrof
luorometer. Micro-cells of about 200 pL are also commer
cially available. And, lately, devices are also commercially
available from several manufacturers Which make measure
ments automatically and continuously for sample solutions
placed in about 100 to 250 pL Wells up to 96 of a plastic
on triple- and quadruple-stranded DNAs and emit ?uores
micro-plate (for example, Cyto Flour, by Nippon Perceptive
cence in a solution.
Ltd.).
[0009] Further, besides of the above dye, there have been
several dyes, including YOYO1 from Molecular Probe Co.,
[0017]
on the market in recent years.
[0010] Among these dyes, EB is generally considered to
be an intercalator Which enters the space betWeen the tWo
base pairs of double-stranded nucleotide chains, and there is
found in a literature (Nucleic Acid Symposium Series No. 29
1993 83-84) a description that 2-methyl-4,6-bis(4-N,N-dim
ethylaminophenyl)pyrylium salt is also an intercalator. On
the other hand, YOYO1 is generally considered to be a
groove binding type ?uorescence dye Which enters the
groove of double-stranded nucleotide chains.
In any of these measurements, since measurements
are made for the sample solution in solution as described
above, the containers for use in the measurements are
restricted, further, When making measurements using a
micro-plate, if the sample is illuminated With excitation light
from the solution surface side, there occurs a problem of
causing diffused re?ection and scattering on the solution
surface, and the illumination has to be conducted from the
plate back side.
[0018]
Further, When the amount of the sample solution is
as small as 0.5 to 5 pL, the containers for use in the
measurements are limited. In addition, When trying to mea
Jun. 6, 2002
US 2002/0068282 A1
sure ?uorescence With a microscope, there arise not only a
slight amount of sample can be used Without caring about its
problem of excitation direction as described above, but also
a problem of permitting the sample to be dried and the
getting dried; the detecting/quantifying operations become
?uorescence of the same to be quenched, even in a typical
the direction in Which excitation light is illuminated is not
restricted.
?uorescence dye, FITC (?uorescein isothiocyanate).
SUMMARY OF THE INVENTION
[0019] In light of the above problems, the present inven
tors concentrated their energies on an intensive investigation
of the detection/quanti?cation of targeted nucleotide chains,
in particular, of multi-stranded nucleotide chains including
double-stranded nucleotide chains, and have ?nally made
relatively simpler and easier because of dry conditions; and
[0029]
substrate at the ?nal stage, nucleotide chains other than the
hybrid or the ?uorescence dye used can be removed by
Washing operation, as the need arises. This reduces the
background, resulting in higher detection/quanti?cation sen
sitivity.
the present invention.
[0020] According to one aspect of the present invention,
there is provided a method for dry detection/quanti?cation
of targeted nucleotide chains comprising the steps of:
[0021] (1) realiZing a state in Which a hybrid (C) of
a certain amount of targeted nucleotide chain (A),
Which is derived from a sample solution and sub
jected to detection or quanti?cation, and a probe
nucleotide chain (B), Which has a base sequence
complementary to a speci?c site of the base
sequences of the targeted nucleotide chain, is formed
on a solid-phase substrate by mutually reacting the
above tWo types of nucleotide chains With each
other, and in Which there exists a ?uorescence dye
(D), Which acts on the hybrid (C), thereby emits
?uorescence or increases its ?uorescence intensity,
and is capable of continuing to emit ?uorescence
even in the dried state While acting on the hybrid;
[0022] (2) drying the hybrid (C) and the ?uorescence
dye (D) on the substrate; and
[0023] (3) measuring the ?uorescence emitted from
the ?uorescence dye (D), as a measuring means, after
the drying operation.
Further, in an aspect in Which a hybrid of a targeted
nucleotide chain and a probe nucleotide chain is ?xed on a
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a graphical representation of detection/
quanti?cation of a model targeted nucleotide chain using
EB, P2 and YOYOl by the hybrid nucleotide chain solution
method (solution system);
[0031] FIG. 2 is a graphical representation of detection/
quanti?cation of a model targeted nucleotide chain using
EB, P2 and YOYOl by the hybrid nucleotide chain solution
method (dry method);
[0032] FIG. 3 is a graphical representation of detection/
quanti?cation of M13mp18 using EB, P2 and YOYOl by
the hybrid nucleotide chain solution method;
[0033] FIG. 4 is a graphical representation of detection/
quanti?cation of mRNA using P2 by the hybrid nucleotide
chain solution method;
[0034] FIG. 5 is a graphical representation of detection/
quanti?cation of a model targeted nucleotide chain using
EB, P2 and YOYOl by the probe nucleotide chain ?xing
method (solution system);
[0035] FIG. 6 is a graphical representation of detection/
quanti?cation of a model targeted nucleotide chain using
EB, P2 and YOYOl by the probe nucleotide chain ?xing
method (dry method);
[0036]
FIG. 7 is a graphical representation of detection/
[0024] According to another aspect of the present inven
tion, there is provided a method for detection/quanti?cation
of multi-stranded nucleotide chains comprising the steps of:
quanti?cation of M13mp18 using EB, P2 and YOYOl by
the probe nucleotide chain ?xing method;
[0025] (1) adding to a sample solution, Which is
[0037] FIG. 8 is a graphical representation of detection/
quanti?cation of mRNA using P2 by the probe nucleotide
subjected to detection/quanti?cation of a multi
stranded nucleotide chain, a ?uorescence dye having
chain ?xing method;
a ?uorescence characteristic of emitting ?uorescence
or increasing its ?uorescence intensity in the pres
[0038] FIG. 9 is a graphical representation of detection/
quanti?cation of a double-stranded nucleotide chain using
ence of a multi-stranded nucleotide chain and
EB, P2 and YOYOl (solution system);
capable of maintaining the ?uorescence characteris
tic even in the dried state;
[0026] (2) placing a knoWn amount of the sample
solution With the ?uorescence dye added thereto on
a clean observation substrate so as to dry the same;
and
[0027] (3) measuring the ?uorescence emitted from
the dried sample and detecting/quantifying the multi
stranded nucleotide chain in the sample solution
based on the obtained measured values.
[0028] The present invention enables the observation of
targeted nucleotide chains by ?uorescence even under dry
conditions. This brings the folloWing good results: the
restrictions on the containers used for detection/quanti?ca
tion of targeted nucleotide chains can be relaxed; even a very
[0039] FIG. 10 is a graphical representation of detection/
quanti?cation of a double-stranded nucleotide chain using
EB, P2 and YOYOl (dry method);
[0040] FIG. 11 is a graphical representation of detection/
quanti?cation of a triple-stranded nucleotide chain using P2;
and
[0041] FIG. 12 is a graphical representation of detection/
quanti?cation of a quadruple-stranded nucleotide chain
using P2.
DETAILED DESCRIPTION OF THE
PREFERRED EMBODIMENTS
[0042] The method for detection/quanti?cation of targeted
nucleotide chains of the present invention is dry detection/
quanti?cation of targeted nucleotide chains characteriZed in
that they comprise:
Jun. 6, 2002
US 2002/0068282 A1
handling the sample becomes easier, and the direction of
[0043] a step (1) of realizing a state in Which a hybrid
(C) of a certain amount of targeted nucleotide chain
(A), Which is derived from a sample solution and
subjected to detection or quanti?cation, and a probe
nucleotide chain (B), Which has a base sequence
complementary to a speci?c site of the base sequence
of the targeted nucleotide chain, is formed on a
excitation light illumination is not restricted because there
exists no liquid. In addition, the restrictions on the sample
container used are relaxed substantially, since the ?uores
cence is measured for the sample in the dried state.
solid-phase substrate by mutually reacting the above
solution used before the step (2) is relatively large, the
tWo types of nucleotide chains With each other, and
in Which there exists a ?uorescence dye (D), Which
acts on the hybrid (C), thereby emits ?uorescence or
increases its ?uorescence intensity, and is capable of
container is needed Which has concave portions correspond
ing to the amount; hoWever, if the amount of the same is
suf?ciently small, a planar substrate may be used.
continuing to emit ?uorescence even in the dried
state While acting on the hybrid;
[0057] HoWever, in the case of (a), When quantitative
[0044] a step (2) of drying the hybrid (C) and the
?uorescence dye (D) on the substrate; and
[0045] a step (3) of measuring the ?uorescence emit
ted from the ?uorescence dye (D), as a measuring
means, after the drying operation.
[0046] In the above step (1), the formation (1-1) of the
hybrid (C) and the action (1-2) of the ?uorescence dye (D)
on the hybrid (C) may be carried out:
[0047] (a) in state Where they are dissolved in the
sample solution; or
[0048] (b) in state Where they are ?xed on the sub
strate.
[0049] The latter (b) case includes a step (1-0) or (1-0‘) of
previously ?xing either one of the targeted nucleotide chain
(B) or the probe nucleotide chain (A) on the clean obser
vation solid-phase substrate, and after this step, the forma
tion (1-1) of the hybrid (C) is carried out by alloWing the
other solution to act on the ?xed solid, subsequently the
action (1-2) of the ?uorescence dye (D) on the hybrid (C) is
carried out.
[0050] At this time, as the need arises, the folloWing
Washing steps may be added:
[0051] a step (1-1‘) of removing the nucleotide chain
other than the hybrid (C) in the solution by Washing
operation after the formation (1-1) of the hybrid (C)
and before the action (1-2) of the ?uorescence dye
(D) on the hybrid (C);
[0052] a step (1-3) of removing the ?uorescence dye
[0056]
It goes Without saying that if the amount of each
evaluation is required, it becomes necessary to prepare
micro concave portions With Which the area of the dropped
liquid can be speci?ed.
[0058] Further, according to the present invention, the step
is not necessary, in principle, of removing the ?uorescence
dye (D) not having acted on the hybrid by Washing opera
tion, because the dye (D) used for acting on the targeted
nucleotide chains in the solution is such that it emits
?uorescence or increases its ?uorescence intensity only after
acting on the hybrid (C) of the targeted nucleotide chains
and the probe nucleotide chains.
[0059] HoWever, in the case of (b), because the hybrid (C)
to be detected at the ?nal stage is ?xed on the surface of the
solid-phase substrate, particularly When using the ?uores
cence dye of a type Which increases its ?uorescence inten
sity after acting on the hybrid, it becomes easier to remove
the dye not having acted on the hybrid by Washing operation
so as to reduce the background, as the need arises, and it also
becomes easier to remove the nucleotide chains other than
the hybrid formed on the solid-phase substrate by Washing
operation so as to reduce the background.
[0060] Further, according to the present invention, the
problem of the ?uorescence being quenched by drying the
sample solution can be avoided in principle, because the
?uorescence dye (D) is used Which is capable of continuing
to emit ?uorescence even in the dried state While acting on
the double-stranded nucleotide chains and the hybrid.
[0061] Any ?uorescence dyes may be used in the present
invention as long as they emit ?uorescence or increase their
(D) not having acted on the hybrid (C) after the
action (1-2) of the ?uorescence dye (D) on the hybrid
?uorescence intensity only after acting on the hybrid in a
solution and are capable of continuing to emit ?uorescence
(C); and
even in the dried state While acting on the hybrid.
[0053] a step (1-4) of removing the solvent including
the hybrid (C) and the ?uorescence dye (D) having
acted on the hybrid (C) by gas ?oW betWeen the
above steps (1) and
[0054] In the (b) case, though there are different cases
Where the probe nucleotide chain (B) is ?xed on the solid
[0062] The mechanism is not clear for the ?uorescence
dye used in the present invention to act on the nucleotide
chain hybrid and continue to emit ?uorescence even in the
dried state; hoWever, considering the fact that in the sole
solutions of either of FITC and rhodamine, Which have high
?uorescence intensity (from another vieWpoint, high quan
phase substrate and the targeted nucleotide chain (A) is ?xed
tum yield of ?uorescence) and are relatively stable in a
on the same, there is basically no difference in means for
solution, their ?uorescence is rapidly quenched once they
detecting the hybrid (C) of the targeted nucleotide chains
are left in the dried state, it is presumed that the micro
environment of the ?uorescence dye in the present invention
undergoes some change When the dye acts on the nucleotide
chain and becomes suitable for the ?uorescence emission.
and the probe nucleotide chains ?nally formed on the
solid-phase substrate surface.
[0055] According to the present invention, the hybrid (C)
of the targeted nucleotide chain and the probe nucleotide
[0063]
chain as Well as the ?uorescence dye (D) having acted on the
above hybrid ?nally exist on the substrate surface in the
form of a dried thin ?lm With a certain area; therefore,
?uorescence dye in the present invention acts on the nucle
From that vieWpoint, the modes in Which the
otide chain hybrid are desirably such that its dye molecules
are included in the nucleotide chain hybrid.
Jun. 6, 2002
US 2002/0068282 A1
[0064] The ?uorescence dye’s action modes described
above include, for example, intercalation in Which the dye
substrate is required to be optically transparent to the
Wavelength of both excitation light and ?uorescence emit
molecules enter the space betWeen the tWo base pairs of the
nucleotide chain hybrid, Which is a double-stranded nucle
ted.
otide chain, and groove binding in Which the dye molecules
enter the groove of the double-stranded nucleotide chain.
[0065] After the intensive investigation of the ?uores
cence intensity and safety of the dyes acting on the nucle
otide chain hybrid in these modes, the present inventors have
found that EB and 2-methyl-4,6-bis(4-N,N-dimethylami
nophenyl)pyrylium salt are desirable as intercalators and
YOYOl is desirable as a groove binding type dye. It goes
Without saying that these dyes are not intended to limit the
present invention.
[0066] Any nucleotide chains may be the targeted nucle
otide chains detectable/quanti?able by the present invention
as long as they hybridiZe With probe nucleotide chains. Both
DNA and RNA can be the targeted nucleotide chains of the
present invention.
[0067] HoWever, in the case of (a), if there exist in the
solution double-stranded nucleotide chains other than the
[0072] On the other hand, even When using a ?uorescence
microscope Which illuminates the substrate With excitation
light from its substance side, in vieW of the scattering of
excitation light, the substrate is desirably optically transpar
ent like the above case or desirably such that it can control
the re?ection of excitation light to some extent, like a black
mirror ?nished or black frosted substrate. Translucent sub
strates are not desirable, especially in vieW of the scattering
of excitation light.
[0073] As the method of attaching the targeted nucleotide
chain or probe nucleotide chain used in the present invention
on a substrate, any methods may be adopted as long as they
alloW the targeted nucleotide chain or probe nucleotide
chain to attach on the substrate, alloW the same to form a
hybrid With the probe nucleotide chain or targeted nucle
otide chain, and alloW the hybrid to be detected/quanti?ed
by the method of the present invention.
[0074]
In such a case, the nylon ?lters used in the afore
hybrid of the targeted nucleotide chains and the probe
mentioned Southern hybridiZation or the like can also be
nucleotide chains, the double-stranded nucleotide chains
other than the hybrid are also detected in principle; thus, the
double-stranded nucleotide chains other than the hybrid
used in principle; hoWever, in vieW of the ef?ciency in
hybridiZation or the aforementioned ?uorescence detecting
method, desirably a method is adopted to bond targeted
need to be discriminated and removed. In order to remove
nucleotide chain or probe nucleotide chain covalently on a
such operations, nucleotide chains previously separated into
substrate Which is advantageous to optical detection.
single strands or RNA Which is single-stranded from the
beginning are desirably used as the targeted nucleotides.
[0075]
[0068] Further, among RNAs, mRNA, of Which partially
double-stranded portion is small compared With tRNA and
rRNA, is more desirably used as the targeted nucleotide
chains.
[0069] On the other hand, in the case of the method (b),
since the nucleotide chains other than the hybrid can be
easily removed by Washing operation, as described above,
both single-stranded and double-stranded nucleotide chains
can be subjected to detection as they are by controlling
hybridiZation conditions. In terms of the capability of mak
In recent years, as substrates for use in such a
method, polystyrene micro-plates With amino group
attached thereon (brand name: Sumiron N) and With car
boxyl group attached thereon (brand name: Sumiron C) are
commercially available from Sumitomo Bakelite Co., Ltd.
[0076] The nucleotide chain can be attached on the sub
strate by the method Which activates the above amino group
and the hydroxyl group at 5‘ terminal of the nucleotide chain
With carbonyldiimidaZole so as to subject the same to
peptide bond, or by the method Which subject the above
carboxyl group and the nucleotide chain With primary amino
group attached thereto to attachment by the method
ing the detection easier, single-stranded nucleotide chains
described in published Japanese Laid-Open Application of
may be more desirably used as the targeted nucleotide
PCT Patent Application No. 7-506482. Alternatively, the
chains; hoWever, there is basically no difference betWeen the
tWo types nucleotide chains. Among single-stranded nucle
otide chains, mRNA, Which has small partially double
stranded portion and codes directly the amino acid sequence,
is more desirably used, just like the case of (a).
[0070]
In the method (b) of the present invention, as the
substrate on Which a hybrid of a targeted nucleotide chain
method can also be adopted Which introduces a maleimide
group to the above carboxyl group, folloWed by subjecting
the carboxyl group and the nucleotide chain With sulfhydryl
group attached thereto to attachment.
[0077] In addition, the method can also be adopted Which
applies an aminosilane coupling agent on a glass substrate,
folloWed by subjecting the amino group and the nucleotide
and a probe nucleotide chain is ?nally ?xed, any substrates
chain to attachment in the same manner as described above,
may be used as long as the hybrid can be attached thereto.
or Which applies a glycidylsilane coupling agent on a glass
The substrates include, for example, glass substrates formed
substrate, folloWed by subjecting the epoxy group and the
of synthetic quartZ, fused quartZ or others and resin sub
strates formed of acryl, polycarbonate, polystyrene or oth
amino group attached to the nucleotide chain or amino group
of the nucleotide chain base to attachment in such a manner
as not to interfere With hybridiZation.
ers.
[0071] In the use of the above substrates, according to the
method of the present invention, the restrictions are relaxed
Which are imposed on the excitation light illumination
direction When detecting ?uorescence; hoWever, When
using, for example, a ?uorescence microscope Which illu
[0078] The substrate have only to be in the dried state
Which enables the elimination of the obstacles to ?uores
cence measurements due to the existence of Water having a
?oW property or due to its containing moisture. The dry
minates the substrate, on Which the substances to be detected
conditions under Which Water does not interfere With ?uo
rescence measurements should be determined, for example,
are ?xed, With excitation light from its back side, the
based on the data obtained by preparing a sample equivalent
Jun. 6, 2002
US 2002/0068282 A1
to that of the actual measurements, conducting a pretest in
accordance With the same sample drying procedure as an
actual one, and making NMR measurements for the dried
[0087] Any ?uorescence dye may be used in the present
invention as long as it emits ?uorescence or increases its
sample.
?uorescence intensity only after acting on the multi-stranded
nucleotide chains in the solution and is capable of continu
ing to emit ?uorescence even in the dried state While acting
[0079] The concrete drying methods include, for example,
on the multi-stranded nucleotide chains.
the drying-up method using a vacuum pump as adopted in
the folloWing examples; hoWever, the methods are not
limited to such an example and any methods can be adopted
as long as they have the effect equivalent to that of the
drying-up method.
[0080] The detection/quanti?cation of multi-stranded
nucleotide chains of the present invention are characteriZed
in that they comprise the steps of:
[0081] (1) adding to a sample solution, Which is
subjected to detection/quanti?cation of multi
stranded nucleotide chains, a ?uorescence dye hav
ing a ?uorescence characteristic of emitting ?uores
cence or increasing its ?uorescence intensity in the
presence of the multi-stranded nucleotide chains and
capable of maintaining the ?uorescence characteris
tic even in the dried state;
[0082] (2) placing a knoWn amount of the sample
[0088] The mechanism is not clear for the ?uorescence
dye used in the present invention to act on the multi-stranded
nucleotide chains and continue to emit ?uorescence even in
the dried state; hoWever, considering the fact that in the sole
solutions of either of FITC and rhodamine, Which have high
?uorescence intensity (from another vieWpoint, high quan
tum yield of ?uorescence) and are relatively stable in a
solution, their ?uorescence is rapidly quenched once they
are left in the dried state, it is presumed that the micro
environment of the ?uorescence dye in the present invention
undergoes some change When the dye acts on the multi
stranded nucleotide chain and becomes suitable for the
?uorescence emission.
[0089]
From that vieWpoint, the modes in Which the
?uorescence dye in the present invention acts on the multi
stranded nucleotide chain are desirably such that its dye
molecules are included in the multi-stranded nucleotide
chain. The ?uorescence dye’s action modes described above
include, for example, intercalation in Which the dye mol
solution With the ?uorescence dye added thereto on
ecules enter the space betWeen the tWo base pairs of the
a clean observation substrate so as to dry the same;
multi-stranded nucleotide chains including double-stranded
nucleotide chains, and groove binding in Which the dye
and
[0083] (3) measuring the ?uorescence emitted from
the dried sample and detecting/quantifying the multi
stranded nucleotide chains in the sample solution
based on the obtained measured values.
[0084] According to the present invention, multi-stranded
molecules enter the groove of the multi-stranded nucleotide
chain.
[0090] After the intensive investigation of the ?uores
cence intensity and safety of the dyes acting on the multi
stranded nucleotide chains in these modes, the present
inventors have found that EB and 2-methyl-4,6-bis(4-N,N
nucleotide chains as Well as the ?uorescence dye having
dimethylaminophenyl)pyrylium salt are desirably used as
acted on the above multi-stranded nucleotide chains exist on
the substrate surface in the form of a dried thin ?lm With a
intercalators and YOYOl is desirably used as a groove
certain area at the ?nal stage; therefore, handling the sample
becomes easier, and the direction of excitation light illumi
nation is not restricted. In addition, the restrictions on the
sample container used are relaxed substantially, since the
?uorescence is measured for the sample in the dried state.
binding type dye in the dry detection/quanti?cation of
multi-stranded nucleotide chains by ?uorescence.
[0091] It goes Without saying that these dyes are not
intended to limit the present invention.
It goes Without saying that if the amount of the
[0092] The multi-stranded nucleotide chains detectable/
quanti?able by the present invention may include not only
the ordinary double-stranded DNA, but also the triple
solution dropped in the step (2) is relatively large, the
stranded DNA and quadruple-stranded DNA as described
[0085]
container is needed Which has concave portions correspond
ing to the amount; hoWever, if the amount of the same is
sufficiently small, a planar substrate may be used. HoWever,
When quantitative evaluation is required, it becomes neces
sary to prepare micro concave portions With Which the area
of the dropped liquid can be speci?ed.
[0086] Further, according to the present invention, the step
is not necessary, in principle, of removing the ?uorescence
dye not having acted on the multi-stranded nucleotide chains
by Washing operation, because the dye used for acting on the
above, in addition, natural type multi-stranded nucleotide
chains such as DNA-RNA hybrid and RNA-RNA hybrid,
multi-stranded nucleotide chains of Which backbone has a
non-natural type structure, such as those at least one strand
of Which is phosphorothionate-type or hydrogenphospho
nate-type or protein nucleotide chains of Which backbone
consists of nucleotide, and non-natural type nucleotide
chains of Which sacchari?ed portion or base portion is
modi?ed. And any nucleotide chains may be included as
long as they are capable of forming multi-stranded chains by
recogniZing their partners With hydrogen bond betWeen
multi-stranded nucleotide chains in the solution is such that
it emits ?uorescence or increases its ?uorescence intensity
only after acting on the multi-stranded nucleotide chains. In
bases.
addition, the problem of the ?uorescence’s being quenched
cence measurements due to the existence of Water having a
by drying the sample solution, as described above, can be
avoided in principle, because the ?uorescence dye is used
Which is capable of continuing to emit ?uorescence even in
the dried state While acting on the multi-stranded nucleotide
chains.
[0093] The substrate have only to be in the dried state
Which enables the elimination of the obstacles to ?uores
?oW property or due to its containing moisture. The dry
conditions under Which Water does not interfere With ?uo
rescence measurements should be determined, for example,
based on the data obtained by preparing a sample equivalent
to that of the actual measurements, conducting a pretest in
Jun. 6, 2002
US 2002/0068282 A1
accordance With the same sample drying procedure as an
actual one, and making NMR measurements for the dried
[0106] (7) Ten ILL (?nal concentration 1 IIM) of each
dye solution Was added to each of the above DNA
solutions.
sample.
[0094] The concrete drying methods include, for example,
the drying-up method using a vacuum pump as adopted in
the following examples; hoWever, the methods are not
limited to such an example and any methods can be adopted
as long as they have the effect equivalent to that of the
[0107] (8) 0.5 ILL of each solution (5) Was placed on
a properly cleaned and dried transparent acrylic
substrate 1 mm thick (from Asahi Chemical Industry
Co., Ltd., Deluglass A). For the solutions in the
above state and another solutions having been sub
jected to the same operation as above and the drying
drying-up method.
up by a vacuum pump, the ?uorescence Was
observed and the intensity of the same Was mea
[0095] In the folloWing, the present invention Will be
described in detail With reference to the folloWing examples.
EXAMPLE 1
[0096] Detection of Oligodeoxynucleotide With EB,
2-Methyl-4,6-bis(4-N,N-Dimethylaminophenyl) Pyrylium
Iodide (hereinafter referred to as P2) and YOYO1
[0097] (1) 20-mer oligodeoxynucleotide having com
pletely the same base sequence as the speci?c part of
that of M13mp18, Which is a single-stranded DNA,
Was obtained (from Kanto Chemical Co., Ltd.) as a
model targeted nucleotide chain. The base sequence
Was as folloWs:
[009s] 5‘ACTGGCCGTCGTTTTACAAC3‘ (SEQ ID
NO: 1)
[0099] A 100 IIM, in terms of the base concentration, stock
solution of the model targeted nucleotide chain Was prepared
by properly mixing the aqueous solution of the above
oligonucleotide and Water.
[0100] (2) 20-mer oligodeoxynucleotide having base
sequence complementary to that of the above model
targeted nucleotide chain Was obtained as a probe
nucleotide chain, and a 100 IIM, in terms of the base
sured.
[0108]
For the ?uorescence observation, used Was an
inverted-type ?uorescence microscope IMT2 (objective lens
of 10 magni?cations) from Olympus Optical Co., Ltd. And
as the ?lter cubes for the ?uorescence observation, a G
excitation ?lter Was used for each case using EB, a B
excitation ?lter for each case using YOYO1 and a ?lter
specially prepared (by Asahi Bunko, excitation: 580 nm,
?uorescence: 540 nm, dichroic mirror: 610 nm) for each
case using P2.
[0109]
For the measurements of ?uorescence intensity,
used Were a CCD equipped With an image intensi?er (from
Hamamatsu Photonics, ICCD C2400-87) and an image
processing apparatus (from Hamamatsu Photonics, Argus
50) Which Were connected to the above ?uorescence micro
scope via a relay lens (NFK 2.5><LD).
[0110]
The measurements of ?uorescence intensity Were
carried out in the area of 200x200 pm Which Was considered
to have the average brightness and be uniform to some
extent, and the average values on the pixels (2><2 pm) in the
area Were adopted as measured values.
[0111] The ampli?cation degree of the image intensi?er
Was 0.2 in terms of the indicated value. The ?uorescence
intensity values of the dyes cannot be directly compared
concentration, stock solution of the probe nucleotide
because the spectral characteristics of the ?lters are different
chain Was prepared in the same manner as described
in
The base sequence Was as folloWs:
from each other, the excitation light source (high pressure
mercury lamp) consists of bright lines, and because the
sensitivity of the ICCD camera depends on Wavelength.
[0101] 3‘TGACCGGCAGCAAAATGTTG5‘ (SEQ ID
NO: 2)
[0102] (3) Four mg of EB (from Sigma Ardrich
Japan) Was dissolved in 1 mL of DMSO and 9 mL of
Water Was added. Additional Water Was added to the
solution to bring about a 100-fold dilution as a 10 pM
stock solution.
[0103] (4) Five mg of P2 (synthesiZed by the present
inventors) Was dissolved in 1 mL of acetonitrile and
9 mL of Water Was added. Additional Water Was
added to the solution to bring about a 100-fold
dilution as a 10 IIM stock solution.
[0112] The measured values of the ?uorescence intensity
are shoWn in FIGS. 1 (solution system) and 2 (dry type). It
is apparent from FIGS. 1 and 2 that the detection/quanti
?cation of model targeted nucleotide chains can be per
formed even in the dry-up state With any one of EB, P2 and
YOYO1, though there are observed differences in ?uores
cence intensity difference and linearity. The comparison of
the three types of dyes shoWs that the degree of ?uorescence
quenching is high in EB and YOYO1, especially in EB, in
the dried up state. On the other hand, the degree of ?uores
cence quenching is relatively loW in P2 in the dried up state;
accordingly, P2 can be said to be suitable for the measuring
method of the present invention.
[0104] (5) Water Was added to YOYO1 (from
Molecular Probe Co., 1 mM/DMSO) to bring about
a 100-fold dilution as a 10 pM stock solution.
[0105] (6) Each of 0, 2, 5, 10 ILL of model targeted
nucleotide chain solutions (?nal concentrations of 0,
2.0, 5.0 and 10.0 IIM, respectively, at the stage Where
a dye solution has been added), 15 ILL of probe
nucleotide chain solution (a ?nal concentration of 15
IIM) and 5 ILL of 100 mM Tris-HCl buffer solution
(pH 7.5) Were mixed and Water Was added to a
volume of 90 ILL. Each solution Was annealed and
cooled to a ?nal temperature of 4° C.
EXAMPLE 2
[0113] Detection of Targeted DNA With EB, P2 and
YOYO1
[0114] (1) M13mp18 (from Takara ShuZo Co., Ltd.),
Which is a single-stranded DNA, Was obtained as a
targeted nucleotide chain. The targeted base
sequence Was the same as in Example 1, as shoWn
beloW:
[0115] 5‘ACTGGCCGTCGTTTTACAAC3‘ (SEQ ID
NO: 1)
Jun. 6, 2002
US 2002/0068282 A1
[0116] A 10 MM, in terms of the base concentration of the
targeted portion, a stock solution of the targeted nucleotide
chain Was prepared by properly mixing the aqueous solution
of the above DNA and Water. The reason that the concen
tration of the base of the targeted portion Was dilute com
pared With that of Example 1 is that, since M13mp18
consists of 7249 bases, the concentration as a nucleotide
chain had to be kept properly.
[0117] (2) Ten pM aqueous solution of oligodeoxy
nucleotide having the same base sequence as in
Example 1, as shoWn beloW, Was prepared as a probe
nucleotide chain stock solution.
[011s] 3‘TGACCGGCAGCAAAATGTTG5‘ (SEQ ID
NO: 2)
[0119] (3) Ten pM stock solutions of EB, P2 and
YOYOl Were prepared in the same manner as in
Example 1.
in the same manner as in Example 1.
[0132] (4) Each of 0, 2, 5, 10 ML of targeted nucle
otide chain solutions (?nal concentrations of 0, 0.2,
0.5 and 1.0 MM, respectively, at the stage Where a dye
solution has been added), 15 ML of probe nucleotide
chain solution (a ?nal concentration of 1.5 ILM) and
5 ML of 100 mM Tris-HCl buffer solution (pH 7.5)
Were mixed and Water Was added to a volume of 90
ILL. And the solution Was annealed and cooled to a
?nal temperature of 4° C.
[0133] (5) 1.0 ML (?nal concentration 0.1 ILM) of P2
solution Was added to each of the above DNA
solutions.
[0134] (6) The ?uorescence intensity of the sample
Was measured in the same manner as in Example 2
[0120] (4) Each of 0, 2, 5, 10 ML of targeted nucle
otide chain solutions (?nal concentrations of 0, 0.2,
0.5 and 1.0 MM, respectively, at the stage Where a dye
solution has been added), 15 ML of probe nucleotide
chain solution (a ?nal concentration of 1.5 ILM) and
5 ML of 100 mM Tris-HCl buffer solution (pH 7.5)
Were mixed and Water Was added to a volume of 99
ILL. Each solution Was annealed and cooled to a ?nal
temperature of 4° C.
[0121] (5) 1.0 ML (?nal concentration 0.1 ILM) of each
dye solution Was added to each of the above DNA
solutions.
[0122] (6) The ?uorescence intensity of the sample
Was measured in the same manner as in Example 1
in state Where the sample Was dried up.
[0123] The ampli?cation degree of the image intensi?er
Was 1.0 in terms of the indicated value, because the con
centration of the targeted DNA Was loW.
[0124]
[0131] (3) Ten IIM stock solution of P2 Was prepared
The measured results are shoWn in FIG. 3. It is
apparent from FIG. 3 that the targeted single-stranded
nucleotide chains can be detected/quanti?ed by the method
of the present invention.
EXAMPLE 3
[0125] Detection of Targeted mRNA With P2
[0126] (1) Human [32 adrenergic receptor mRNAWas
synthesiZed from human [32 adrenergic receptor
in state Where the sample Was dried up.
[0135] The measured results are shoWn in FIG. 4. It is
apparent from FIG. 4 that the targeted mRNA can be
detected/quanti?ed by the method of the present invention.
EXAMPLE 4
[0136] Detection of Oligodeoxynucleotide With EB, 2-me
thyl-4,6-bis(4-N,N-dimethylaminophenyl)pyrylium iodide
(hereinafter referred to as P2) and YOYOl (probe nucleotide
chain being ?xed)
[0137] (1) The same 20-mer oligodeoxynucleotide
(from Kanto Chemical Co., Ltd.) as used in Example
1 Was used as a model targeted nucleotide chain. The
base sequence Was as folloWs:
[013s] 5‘ACTGGCCGTCGTTTTACAAC3‘ (SEQ ID
NO: 1)
[0139] A 100 MM, in terms of the base concentration, stock
solution of the model targeted nucleotide chain Was prepared
by properly mixing the aqueous solution of the above
oligonucleotide and Water.
[0140] (2) 20-mer oligodeoxynucleotide having base
sequence complementary to that of the above model
targeted nucleotide chain Was obtained as a probe
nucleotide chain, and a 100 MM, in terms of the base
concentration, stock solution of the probe nucleotide
chain Was prepared in the same manner as described
cDNA using T7RNA polymerase by conventional
in
procedure and puri?ed after the D Nase treatment. A
10 MM, in terms of the base concentration of the
targeted portion, stock solution of a targeted nucle
In order to ?x the probe nucleotide chain on
oligodeoxynucleotide Was obtained and used at
Which 5‘ terminal an amino group Was attached using
a solid-phase substrate by the covalent bond, 20-mer
otide chain Was prepared by properly mixing the
hexamethylene as a linker. The base sequence Was as
aqueous solution of the above mRNA and Water.
folloWs:
[0127] The portion of the targeted base sequence of the
above mRNA Was as folloWs:
[012s] S‘TGCTGTGCGTCACGGCCAGCAT3‘ (SEQ
ID NO: 3)
[0129] (2) Ten pM aqueous solution of oligodeoxy
nucleotide (from Kanto Chemical Co., Ltd.) having
the folloWing base sequence Was prepared as a probe
nucleotide chain stock solution.
[0130] 3‘ACGACACGCAGTGCCGGTCGTAS‘ (SEQ
ID NO: 4)
[0141] 3‘TGACCGGCAGCAAAATGTTG-NH25‘
(SEQ ID NO: 2)
[0142] (3) Ten pM stock solutions of EB, P2 and
YOYOl Were prepared in the same manner as in
Example 1.
[0143] (4) The probe nucleotide chain of (2) Was
?xed on a 96-Well micro-plate With carboxyl groups
attached to its surface (from Sumitomo Bakelite Co.,
Ltd., Sumiron C) using 1-hyroxy-2,5-dioxo-3-pyrro
lidinesulfonic acid monosodium salt (from Sigma