Cu: a CuS04 solution and (+)-Sodium L-ascorbate
(hereinafter referred to as 'S.A.") were purchased from
Sigma-Aldrich.
Nucleic acid: Sonicated Salmon Sperm .DNA
(hereinafter referred to as "ssDNAN) purchased from
56
BioDynamics laboratory Inc. (Tokyo, Japan) was used. In
addition, as oligo-DNAs, Custom Oligo purchased from
Invitrogen Corporation was used.
Buffer: HEPPSO purchased from DOJINDO Laboratories
5 (Kumamoto, Japan) was used by adjusting the pH to 8.5
pursuant to the protocols provided by the manufacturer.
Fluorophotometer: NanoDrop 3300 (Thermo Fisher
Scientific, Inc., Waltham, MA, USA) or type F-4500
spectrofluorophotometer (Hitachi High-Technologies
10 Corporation) was used. In the NanoDrop 3300, a UV LED
light source was used to provide exciting light. A
fluorescent spectrum excited by the exciting light was
measured. Using a companion software, Relative
Fluorescence Units (RFU) at a wavelength where a
15 spectrum intensity became at maximum was acquired as a
peak RFU value. In the type F-4500
spectrofluorophotometer, a quartz capillary and a
dedicated adapter cell manufactured by Helix Biomedical
Accessories, Inc. were used. Unless otherwise noted
20 below, the NanoDrop 3300 was used.
Spectrophotometer: NanoDrop 1000
Spectrophotometer was used to measure an absorption
spectrum.
Sample preparation and fluorescence measurement:
25 50 mM of a HEPPSO buffer was mixed with sodium chloride
(250 mM), CuS04 (0 to 4 mM), S.A. (4, 50 mM), ssDNAs (1
mg/ml) or oligo-DNAs (50, 250, 500 pM) to provide 20 p1
of a sample. It is known that the S.A. has an action to
reduce Cu(I1) ions generated from CuS04 in the solution
to Cu (I) (see Non-Patent Document 31) .
[0090]

Figs. 1 and 2 are graphs each showing a
fluorescent spectrum and an RFU value obtained by
changing a concentration of CuS04 under the condition of
an S.A. concentration of 50 mM; (A) shows the
fluorescent spectrum and (B) shows a peak RFU value.
[0091] Figs. 3 and 4 are graphs each showing
fluorescent spectra obtained under the conditions of
CuS04 having a concentration of 0.4 mM and an S.A.
15 concentration of 4 mM. The oligo-DNAs having base
lengths of 20, 10, 6 and 3 had a concentration of 50,
50, 250, and 500 pM, respectively. Fig. 3 shows a
result of the oligo DNAs having the base sequences
described in SEQ ID NOS: 1 to 6. An abscissa axis
20 represents a wavelength, an ordinate axis in (A)
represents the RFU value in each wavelength, and an
ordinate axis in (B) represents a value provided by
dividing the RFU value in each wavelength by a maximum
RFU value. Fig. 4 shows a result (A) of the oligo-DNAs
25 having the base sequences described in SEQ ID NO: 2
(hereinafter described as T(20)), a result (B) of the
oligo-DNAs having the base sequences described in SEQ
ID NO: 10 (hereinafter described as T (6) ) , a result (C)
of the oligo-DNAs having the base sequences described
in SEQ ID NO: 12 (hereinafter described as T(3)), and a
5 result (D) of the oligo-DNAs having the base sequences
described in SEQ ID NO: 11 (hereinafter described as
T(3)). Each abscissa axis represents a wavelength, and
each ordinate axis represents an RFU value in each
wavelength.
10 [0092] As shown in the Figures, it was confirmed
that the patterns of the fluorescent spectra (a peak
wavelength and an intensity) were changed depending on
the base sequences of the nucleic acids.
[0093] Next, a change with elapsed time of
15 fluorescent spectra and absorption spectra obtained in
oligo-DNAs T (20) , T (6) and T (3) under the condition of
a CuS04 concentration of 0.4 mM and an S.A.
concentration of 4 mM. The S.A. was added directly
before the measurements of the fluorescent spectrum and
20 the absorption spectra for the first time. After 8, 14,
24 and 35 minutes, the fluorescent spectra and the
absorption spectra were measured. The results are shown
in Figs. 5 and 6. In Fig. 5, the upper graphs each show.
the fluorescent spectrum with an ordinate axis of an
25 RFU value (absolute value), the middle graphs each show
the fluorescent spectrum with an ordinate axis of an
RFU value (relative value) and the lower graphs each
show the absorption spectrum. Fig. 6 shows a change
with elapsed time of the peak RFU value (A), and shows
a change with elapsed time at a wavelength of 346 nm
5 (B) .
[0094] As shown in the Figures, fluorescence is
almost disappeared in all oligo-DNAs of (T20), T(6) and
(T3) after 30 minutes. In particular, the fluorescence
is quickly disappeared in the oligo-DNAs having short
10 base lengths. After 35 minutes, the fluorescent spectra
were measured. Immediately thereafter, 1.8 p1 of 44 mM
S.A. solution was again added to the sample for
measurement. The fluorescence could be again detected.
From this, the disappearance of the fluorescence could
15 be considered due to oxidation of Cu(1) ions to Cu(I1)
ions. In each of the fluorescent spectra of the oligo-
DNAs T(6) and T(3), as the peak intensity was decreased,
a new peak was observed at a short wavelength side.
[0095] On the other hand, in each of the absorption
20 spectra of the respective oligo-DNAs, a decrease in the
peak intensity was observed with elapsed time. The
absorption spectra were more gradually decreased as
compared with the fluorescent spectra.
[0096] Fig. 7 (A) to (C) show two-dimensional
25 fluorescent spectra acquired in oligo-DNAs T(20), T(6)
and T(3) by the type F-4500 spectrof1uorophotomete.r.
Fig. 8 shows excitation spectra (broken lines) and
fluorescent spectra (solid lines) obtained in the
respective oligo-DNAs. The spectrum was measured at a
space of 1 nm for a fluorescent wavelength, and at a
5 space of 2 nm for an excitation wavelength.
[0097] As shown in the Figures, it was confirmed
that the patterns of the fluorescent spectra were
changed depending on the base lengths of the oligo-DNAs.
It was also confirmed that the patterns of the
10 excitation spectra were changed depending on the base
lengths.
[0098] In order to further examine a relationship
between the base sequences and the spectra, the oligo-
DNAs each having a three base length sequence by a
15 combination of adenine (A) and thymine (T) described in
SEQ ID NOS: 11 and 18 were measured for the
fluorescence. The results are shown in Figs. 9 and 10.
In Fig. 9, an ordinate axis (A) represents an RFU value
in each wavelength measured by the Nanodrop and an
20 ordinate axis (B) represents a value provided by
dividing the RFU value in each wavelength by a maximum
RFU value. Fig. 10 shows an average value and a
standard error by measuring the maximum value of the
RFU and the peak wavelength for three times.
25 [0099] As shown ,in the Figures, it was confirmed
that the fluorescence intensity and the peak wavelength
were changed depending on the base sequences of the
oligo-DNAs.
[OlOO] Fig. 11 shows the results of the measurement
obtained in the oligo-DNAs in~luding~sequencoef SEQ ID
5 NOS: 19 and 20. It was confirmed that the oligo-DNAs
having the sequence described in SEQ ID NO: 20
containing uracil (U) emitted the fluorescence having
the spectrum shape and the peak position similar to
that of the oligo-DNAs having the sequence described in
10 SEQ ID NO: 19 containing thymine (T), although the
fluorescence intensity in the oligo-DNAs having the
sequence described in SEQ ID NO: 20 was faint.
[@lo11

15 This Example showed that the orange-colored
fluorescence having a wavelength of about 500 nm to 700
nm was observed by ultraviolet irradiation when DNAs
were mixed with a HEPPSO buffered solution containing
sodium chloride into which CuS04 and the S.A. were mixed.
20 It was confirmed that the fluorescence intensity
depended on the concentration of CuS04, and the
fluorescence intensity and the spectrum were also
influenced by the base sequences of the nucleic acids.
[0102] The fluorescence was observed in the oligo-
25 DNAs containing at least thymine (T), adenine (A) or
uracil (U). In the experiment where the oligo-DNAs each
having a three base length containing thymine (T) and
adenine (A) were used, the fluorescence was observed in
any sequence. In addition, it is shown that the
fluorescence intensity and the spectra were influenced
5 not only by the amount of thymine (T) or adenine (A),
but also by the position (sequence order) on the oligo-
DNAs .
[0103] With time elapsed after the addition of the
S.A., the fluorescence intensity was decreased with
10 time, but was recovered by re-addition of the S.A. In
the meantime, Cu(1) ions are very unstable in the
presence of oxygen, and are changed into Cu(I1) or
solid copper as soon as the reduction effect of the S.A.
is lost. From this, it is considered that the ,
15 fluorescence is derived from the composite of Cu(1)
ions and the nucleic acids. In order to detect the
fluorescence by the interaction between copper and the
nucleic acids, it may be desirable that a contact of
the reaction solution with oxygen in the air be
20 minimized.
[Example 21
[0104] Example 2 illustrates that orange-colored
fluorescence similar to that observed in Example 1 was
emitted by ultraviolet irradiation under certain
25 conditions when a solution containing nucleic acids was
contacted with solid copper.
[01051

As the copper that was contacted with the nucleic
acids, copper powder (Copper, Powder, -75um, 99.9% /
5 Cat.No.030-18352 / manufactured by Wako Pure Chemical
Industries, Ltd., Osaka, Japan) was used.
As the RNAs, Rat Brain Total RNAs (Cat.No.636622,
Takara Bio Inc., Otsu, Japan) were used by dissolving
it to DEPC treated water (Cat.No.312-90201 / Wako Pure
10 Chemical Industries, Ltd., Japan.
PIPES, ACES, BES, TAPSO, HEPPSO, EPPS, TAPS, CAPS,
TES, Tricine and OPSO were purchased from DOJINDO
Laboratories (Kumamoto, Japan). Each of these was used
by adjusting the pH pursuant to the protocols provided
15 by the manufacturer. Other reagents were the same as in
Example 1.
[0106] The nucleic acids were contacted with copper
by mixing a variety of nucleic acids, salts and copper
powder into a total amount of 40 microliters solution,
20 and agitating it for 15 minutes. The amount of the
copper powder added was 375 mg per milliliter of the
solution, unless otherwise noted. The amount of the
salt, or sodium chloride (NaCl), was 500 mM, unless
otherwise noted.
25 [0107] After the sample was centrifuged to settle
the copper powder, a supernatant was measured for the
spectrum of the fluorescence and the intensity. The
measurement of the spectra of the fluorescence and the
intensity was performed in the similar steps as in
Example 1.
5 [01081

The reaction solution to which 1.5 mg/ml of ssDNAs
were added was measured for the fluorescence three
times. The results are shown in Fig. 12 (abscissa axis:
10 wavelength, ordinate axis: RFU). As shown in the Figure,
when the sample containing the nucleic acids was
contacted with solid copper and then UV-excited, the
fluorescence having a peak around 600 nm could be
detected.
15 [0109] Next, a reaction solution was prepared by
adding the copper powder in the amount of 375 mg, 250
mg, 125 mg, 62.5mg, 37.5 mg, 12.5 mg qnd 0 rng based on
1 mL of the reaction solution. To the reaction solution,
1.5 mg/ml of ssDNAs were added. The fluorescence was
20 measured for three times. The result was shown in Fig.
13. As shown in the Figure, the fluorescence intensity
depended on the amount of the copper powder. In the Cu
powder used in this Example, apparent fluorescence was
observed when the amount was 37.5 mg/ml or more. On the
25 other hand, no apparent fluorescence was observed when
the amount was 12.5 mg/ml or less.
[OllO] Then, the type and the concentration of the
salt in the reaction solution were changed. To the
reaction solution, 1.5 mg/ml of ssDNAs were added. The
intensities of the fluorescence detected were compared.
5 The results are shown in Fig. 14. (A) shows the
fluorescence intensity detected in the reaction
solution to which 0.5, 0.25, 0.1, 0.05, 0.025 and 0 M
sodium chloride (NaC1) were added. (B) shows the
fluorescence intensity detected in the reaction
10 solution to which 0.45 M sodium chloride (NaCl), 0.45 M
potassium chloride (KCl), 0.45 M magnesium chloride
(MgC12) and 45% ethanol (EtOH) were added. The
fluorescence intensity was represented by the RFU at
604 nm, and measured for three times. The result was
15 shown as the average and the standard error. As shown
in the Figure, the fluorescence intensity depended on
the amount of sodium chloride. Also, the fluorescence
was detected under the coexistence of potassium
chloride and magnesium chloride as well as sodium
20 chloride.
[Olll] Fig. 15 shows comparison results of the
fluorescence intensity detected when the concentration
of the nucleic acids added to the reaction solution was
changed. (A) shows the fluorescence intensity detected
25 in the reaction solution to which 5, 2.5, 1, 0.5, 0.25,
0.1, 0.05, and 0 mg/ml of ssDNAs were added. (B) shows
the fluorescence intensity detected in the reaction
solution to which 2.5, 0.25, and 0 mg/ml of RNAs were
added. The abscissa axis represents the concentration
of the nucleic acids, and the ordinate axis represents
5 the RFU at a fluorescent wavelength of 604 nm. The
measurement was performed for three times. The
concentration of sodium chloride (NaC1) was 0.25M, and
the amount of the copper power was 200 mg per 1 ml. The
condition was used in the following experiments, unless
10 otherwise noted. As shown in the Figure, the
fluorescence intensity depended on the concentration of
DNAs and the concentration of RNAs.
[0112] Next, the reaction solution to which 0.1 mM
oligo-DNAs having different sequences described in SEQ
15 ID NOS: 1, 2, 5, 6 and 9 was measured for the
fluorescence. The results are shown in Fig. 16. An
ordinate axis (A) represents an RFU value measured by
the Nanodrop, and an ordinate axis (B) represents a
relative RFU value when the peak height was set to 1.
20 As shown in the Figure, the fluorescence intensity and
the peak wavelength were influenced by the base
sequences. In particular, it could be confirmed that
when the percentage of thymine (T) was high, the
fluorescence intensity was high and the peak wavelength
25 became longer.
[0113] The reaction solution to which oligo-DNAs
having sequences described in SEQ ID NOS: 1, 2, 5 and 6
was also measured using the type F-4500
spectrofluorophotometer. Fig. 17 shows the results of
the fluorescent spectra (slit width of 2.5 nm) within
5 400 nm to 700 nm when the excitation light of 360 nm
(slit width of 10 nm) was irradiated. Again, it could
be confirmed that when the percentage of thymine (T)
was high, the fluorescence intensity was high and the
peak wavelength became longer in the sequence
10 containing thymine (T) and adenine (A). Fig. 18 shows
the results by scanning the excitation light at 330 nm
to 390 nm (slit width of 3 nm) and 400 nmto 700 nm
(slit width of 2.5 nm) to measure excitation -
fluorescent spectra. (A) represents three dimensionally,
15 and (B) represents a contour. An axis EX represents an
excitation wavelength (nm), an axis EM represents a
fluorescence wavelength (nm) and a height direction
represents the fluorescence intensity. Based on the
results, it could be read that the excitation and the
20 fluorescent spectra and the intensity were changed by
the different base sequences of the DNAs.
[0114] In order to further examine a relationship
between the base sequences and the spectrum, the oligo-
DNAs each having a combination sequence of cytosine (C)
25 having eight bases and thymine (T) having 12 bases
described in SEQ ID NOS: 21 and 26 were measured for
the fluorescence. The results are shown in Fig. 19. As
shown in the Figure, the fluorescence intensity
differed when the sequence was different even if the
base composition of the DNAs was the same.
5 [0115] Next, the double-stranded DNAs including a
mismatch were measured for the pattern of the
fluorescent spectrum. As the double-stranded DNAs,
three types: a mixture ((e) + (f)) of oligo-DNAs each
having a sequence shown in SEQ ID NO: 1 and oligo-DNAs
10 each having a sequence shown in SEQ ID NO: 2, a mixture
((d) + (f)) of oligo-DNAs each having a sequence shown
in SEQ ID NO: 5 and oligo-DNAs each having a sequence
shown in SEQ ID NO: 2, and a mixture ((e) + (c)) of
oligo-DNAs each having a sequence shown in SEQ ID NO: 1
15 and oligo-DNAs each having a sequence shown in SEQ ID
NO: 6 were used. Any of the oligo DNAs were mixed at a
final concentration of 0.5 mg/ml. The results are shown
in Fig. 20. An ordinate axis (A) represents an RFU
value measured by the Nanodrop, and an ordinate axis
20 (B) represents a relative RFU value when the peak
height was set to 1. An abscissa axis represents a
wavelength (nm) . As shown in the Figure, the
fluorescence intensity in the double-stranded DNAs was
lower than that in the single-stranded DNAs. However,
25 in the double-stranded DNAs having a mismatch of
thymine (T), the strong fluorescence was confirmed.
[0116] The intensities of the fluorescence detected
were compared, when the types of the buffer and the pH
in the reaction solution were changed. The results are
shown in Fig. 21. (A) shows relative values of peak RFU
5 values of a sample (+) containing ssDNAs and a sample
( - ) containing no nucleic acids under each buffered
condition. (B) shows a relative value of a peak RFU
value of a sample containing oligo-DNAs having the
sequence shown in SEQ ID NO: 1 under the same condition.
10 (C) shows a relative value of a peak RFU value of a
sample containing oligo-DNAs having the sequence shown
in SEQ ID NO: 2 under the same condition. The
concentration of each buffer was 50 mM, the final
concentration of the ssDNAs was 0.5 mg/ml, and the
15 final concentration of the oligo-DNAs was 25 mM. The
relative value of the peak RFU value means that the
peak RFU value measured under no buffered condition is
set to 1. The fluorescence intensity depended on the
types of the buffer. The fluorescence was almost not
20 detected when no nucleic acids exist in the buffer.
[0117]

Based on the results in this Example, it revealed
that the fluorescence could be detected under adequate
25 conditions including the salt concentration, when the
nucleic acids were contacted with solid copper powder,
as is the case that the nucleic acids were contacted
with Cu(1) ions. It seemed that the fluorescence
observed in each case of copper ions and solid copper
was provided by the same mechanism, because their
5 properties such as wavelength properties and sequence
dependency are almost the same. Also, the fluorescence
was observed when the RNAs were used as the nucleic
acids. In addition, in the double-stranded DNAs, strong
fluorescence was observed when the mismatch exists
10 especially in thymine (T). This suggested that binding
with the complementary sequence might inhibit the
formation of the fluorescent substance by binding the
nucleic acids with copper. Also, it is considered that
the increase in the fluorescence intensity at the
mismatch site could be applied to a method of detecting
for mutation in the base sequences of the nucleic acids.
[0118] In the experiments for comparing the
fluorescence under each buffered condition, the
fluorescence was observed in the buffer of PIPES, BES,
HEPPSO, EPPS, TAPS, CAPS, TES and POPSO. In particular,
strong fluorescence was detected in the buffer of PIPES,
HEPPSO, EPPS and POPSO. The fluorescence could be
observed within a pH range of 7.0 to 10.5. It was found
that a change in the fluorescence intensity depending
25 on the type of the butfer and the pH showed a different
pattern depending on the base sequences of the nucleic
SP313000W000
acids. On the other hand, the buffer having a property
to chelate and stabilize Cu(I1) ions has a tendency
that the fluorescence is not observed. Although no data
is provided in this Example, the fluorescence was
5 almost not observed when the reaction solution
containing, for example, a Tris buffer, EDTA or the
like was used.
[Example 31
[0119] In Example 3, it was confirmed that the
10 fluorescence could be detected after the nucleic acids
were brought into contact with copper sputtered on the
surface of the glass, and the properties of the
fluorescence were analyzed.
[0120]
15
As the DNAs, the ssDNAs described in Example 1
were used. As the RNAs, the RNAs described in Example 2
were used.
Copper was sputtered on the surface of the glass
20 using an apparatus, SH-350 manufactured by ULVAC, Inc.
(Kanagawa, Tokyo) on which a Cu target, 99.99% (Kojundo
Chemical Laboratory Co., Ltd, Saitama, Japan) was
mounted. In the sputtering, a thickness was set to 40
nm, and an adequate sputtering time was set based on a
25 deposition speed measured in advance. The glass for
sputtering silver was manufactured by Kyodo
International, Inc., Kanagawa, Japan.
[0121] On a slide glass on which copper or silver
was sputtered or an untreated slide glass, a sample
solution was placed, and a gap cover glass, 24x25 No.4
5 / #CG00024 / Matsunami Glass Ind., Ltd., Osaka, Japan
was covered thereon. After it was allowed to be stood
for about 5 minutes, the fluorescence was observed. For
the observation, an inverted microscope Ti-U (Nikon Co.,
Tokyo, Japan) was used. For capturing the fluorescence,
10 a filter set UV-1A (Ex: 365/10, DM: 400, BA: 400/~ikon)
was used. For capturing and recording an image, a
digital CCD camera Retiga 2000R (QImaging, BC, Canada)
and a 20x objective lens was used.
[0122]
15
Fig. 22 shows images captured after the sample
containing 5 mg/ml of DNAs and 0.5 M of NaCl was
allowed to stand for 5 minutes on the copper sputtered
glass. Fig. 23 shows images captured after the sample
20 containing 5 mg/ml of RNAs and 0.5 M of NaCl was
allowed to stand for 5 minutes on the copper sputtered
glass.
[0123] As shown in Fig. 22(A), when the sample
containing DNAs was used, smooth fluorescence was
25 observed on the entire captured image. On the other
hand, as shown in Figs. 23 (A) and (B), when the sample
73
containing RNAs was used, the fluorescence having a
specific wave-like pattern within the captured image
was observed. A prospective cause of the pattern
specific to the RNAs was that the single-stranded RNAs
5 were hybridized each other to form the higher order
structure.
[0124] Next, the fluorescence intensity within the
captured image was converted into numerals. Each
captured image was divided into nine sections as shown
10 in Fig. 22 (B) . One of the nine sections (C in the
Figure) was set to be a measuring range. An average
value of the fluorescence intensity within the
measuring range was calculated. For each sample, five
parts on the slide were captured to calculate the
15 average value from each image. The resultant five
average values were further averaged and calculated for
standard deviation.
[0125] Fig. 24 shows the fluorescence intensity
acquired when the sample containing the DNAs or the
20 RNAs was contacted with copper or silver sputtered on
the glass. In Fig. 24, "DNA/CuUf "RNA/CuU, and " ( - ) /Cu"
denote the sample containing 5 mg/ml DNAs, the sample
containing 5 mg/ml of RNAs, and the sample containing
no nucleic acids; the fluorescence intensity being
25 measured on the Cu sputtered glass. In
addition, "DNA/A~", "RNA/AgU, and " ( - ) /Ag" denote the
sample containing 5 mg/ml of DNAs, the sample ,
containing 5 mg/ml of RNAs, and the sample containing
no nucleic acids; the fluorescence intensity being
measured on the Ag sputtered glass. Each sample
5 contained 0.5 M NaC1. Since the fluorescence intensity
in the "DNA/CuU was significantly greater than those of
the other samples, its exposure time was 1 minute. In
all samples excluding the "DNA/Cu", the exposure time
was 5 seconds.
10 [0126] As shown in the Figure, in the Cu sputtered
glass, the "DNA/Cu0 and the 'RNA/CuU had the
fluorescence intensity higher than the "(-)/CuU.
Especially in the DNA sample, the strong fluorescence
was detected. On the other hand, in the Ag sputtered
15 glass, the "DNA/AgU and the "RNA/AgU showed no increase
in the fluorescence intensity as compared with the ' ( -
) /Ag". As compared with the " ( - ) /CuU, the ( - ) /Ag"
showed the higher measured value. This may be caused by
a background derived from a reflected light, a
20 scattered light or autofluorescence on the Ag sputtered
surface.
[0127] Next, a change in the fluorescence intensity
with elapsed contact time of the nucleic acids with
copper was examined. A point of time when the sample
25 containing 5 mg/d of ssDNAs and 0.5 M of NaCl was
placed between the Cu sputtered glass and the gap cover
glass was designated as a starting point to measure the
fluorescence intensity per predetermined time. The
image was captured every 15 seconds, and a shutter for
excitation light was opened and closed per capturing
5 session. The x10 objective lens was used, and the
exposure time was 1 second. In every time, one image
captured was used to measure the fluorescence intensity.
The results are shown in Fig. 25.
[0128] As shown in the Figure, the fluorescence
10 intensity was gradually increased for several minutes
after the sample was introduced, and reached the
maximum value within about three minutes.
[0129] After a predetermined time was elapsed from
the contact of the nucleic acids with copper, a change
15 in the fluorescence intensity by temperature change was
examined. Immediately after the image has been captured,
it held at room temperature. After 50 seconds, a heat
block heated to 65OC was gently placed over the Cu
sputtered glass. After 100 seconds, the heat block was
20 removed. The image was captured every 5 seconds. After
150 seconds, the measurement was stopped for now and
the shutter for excitation light was closed. After 900
seconds, the measurement was again made. The results
are shown in Fig. 26.
25 [0130] As shown in the Figure, the fluorescence
intensity was gradually decreased for the first 50
seconds. This might be caused by fluorescence
photobleaching. During the next 50 seconds, the
fluorescence was disappeared at a speed apparently
different from the fluorescence photobleaching. After
5 the heat block was removed and it returned to room
temperature, the fluorescence was gradually recovered.
After 900 seconds, the fluorescence intensity was
returned to a level that color degraded fluorescence
intensity was subtracted from initial fluorescence
10 intensity. These results show that the fluorescence
emitted from the nucleic acids contacted with copper
was heat sensitive, and was reversibly disappeared as
the temperature increased.
[Example 41
15 [0131] Example 4 illustrates that the cell nuclei
could be fluorescently observed by introducing the
sample containing cells onto the copper-sputtered glass.
[0132]

As PBS, Dulbeccors Phosphate Buffered Saline,
Ca/Mg free (Invitrogen Corporation, CAI USA) was used.
In an onion thin skin experiment, a commercially
available onion thin skin was carefully peeled by a
pair of tweezers, soaked into distilled water and
25 rinsed for using 'it. The onion thin skin was placed on
the Cu sputtered glass, was soaked into the PBS, was
covered by a cover glass, and was then observed.
In an experiment of a human leukocyte sample,
IMMUNO-TROL Cells (Cat.No.6607077, Beckman Coulter,
Inc., Fullerton, CA, USA) were treated as follows:
5 Firstly, 500 microliters of the IMMUNO-TROL Cells were
separated, cleaned with PBS, and settled using a
centrifugal machine (1200rpm, 5min) . Thereafter, a
supernatant was discarded to flake pellets, water
hemolysis treatments are repeated two times to provide
10 a sample. The sample was diluted with PBS, thereby
preparing a leukocyte sample. The water hemolysis
treatment was performed as follows: After the pellets
obtained as the result of the centrifugation were
sufficiently flaked, 9 ml of deionized water was added,
15 was mixed upside down for 30 seconds, 1 mL of lox PBS
Buffer (Nippon Gene Co., Ltd., Tokyo, Japan) was added
and fully agitated. The cells were centrifuged (1200
rpm, 5 min) and were settled to remove a supernatant.
The leukocyte sample was placed on the Cu sputtered
20 glass, was covered by a cover glass, and was then
observed.
[0133] The copper sputtered glass, the cover glass,
the microscope etc. were the same as in Example 3. In
the sputtering, a thickness was set to 20, 40, or 100
25 nm. The thickness was set to 40 nm in the following
experiments, unless otherwise noted. When Cu was
sputtered only on a part of a slide glass surface, a
polyimide tape was adhered on the slide glass surface
excluding a 5mm square in a center part, thereby
performing the sputtering. Then, the polyimide tape was
5 removed. Thus, the Cu sputtered glass having a Cu layer
only formed on the 5 mrn square in the center part was
produced.
LO1341 The onion thin skin was fluorescently
observed using an excitation filter: 365/10 nm, a
10 dichroic mirror: 400 nm, and a fluorescent filter:
590LP. The leukocyte sample and Jurkat cells were
fluorescently observed using a filter set UV-1A (Ex:
365/10, DM: 400, BA: 400/Nikon).
[0135]
15
Fig. 27 shows images of the onion thin skin on the
copper sputtered glass fluorescently observed and
captured. (a) and (b) show observed images on the Cu
sputtered glass. (c) and (d) show observed images on a
slide glass without sputtering Cu thereon. (a) and (c)
are bright field observed images. (b) and (d) are
fluorescent images. (a) to (d) are images captured
using a x10 objective lens. (e) is an image captured
using a x40 objective lens.
[0136] As shown in the Figures, strong fluorescence
specific to the cell nuclei was observed on the cells
over the Cu sputtered glass. Although slight
fluorescence was observed on a part of cell walls and
the like, it is considered as autofluorescence of the
cell walls and the like, because it was observed on the
5 cells over the slide glass without sputtering Cu
thereon.
[0137] Next, animal cells were observed. Fig. 28
shows images acquired by fluorescently observing and
capturing the human leukocyte sample on the copper
10 sputtered glass. (a) is a bright field observed image.
(b) is a fluorescent image. The x40 objective lens was
used.
[0138] In the fluorescent image, segmented
neutrophils specific to the leukocyte were apparently
15 observed.
[0139] Fig. 29 shows images observed by using the Cu
sputtered glass where Cu was sputtered only on a part
of a slide glass surface. On the Cu sputtered glass,
human leukocyte cell strains, i.e., Jurkat cells, were
20 spread, were covered by the cover glass, and were then
observed using the x20 objective lens. The images were
captured at a boundary between a Cu deposited area and
a no Cu deposited area on the Cu sputtered glass. (a)
and (c) are bright field observed images; black areas
25 occupying more than half are areas where light is not
transmit, because the Cu layers are formed. (b) and (d)
are fluorescent images.
[0140] Strong fluorescence was observed only on the
cell nuclei of the cells in the Cu deposited area. Fig.
30 shows observation results of the Jurkat cells using
5 the Cu sputtered glass on which the Cu layer was formed
in a thickness of 20 nm (a) or 100 nm (b) . The
fluorescence from the cell nuclei was observed at
either thickness.
[0141]
The results in this Example show that the
fluorescence can also be detected by bringing the cell
nuclei into contact with copper. It is clear that the
phenomenon occurs only on the glass substrate on which
15 copper is sputtered, and is the result of the action
between the cell nuclei and copper.
[0142] As a result of the fluorescence observation
of the onion thin skin cells and the leukocyte cells, a
difference between the shapes of cell nuclei in the
20 cells was apparently shown. From this, according to the
method of detecting nucleic acids of the present
technology, different shapes of cell nuclei depending
on the types of the cells can be identified.
101431 Although not shown in this Example, in the
25 experiment using the slide glass having copper
sputtered on a part thereof, after the fluorescence was
8 1
observed from only the cells on the Cu deposited area,
the slide glass was inclined to move the cells from the
Cu deposited area to the no Cu deposited area. After
moving, the fluorescence was continuously observed.
5 From this, even if the site where copper is contacted
with the cells is spaced from the site where the cells
are fluorescently observed, it is found that the
fluorescence can be detected by disposing a means for
moving the sample between the both sites.
10 [0144] After the fluorescence from the cell nuclei
of the cells between the Cu sputtered glass and the
cover glass was confirmed, the cover glass was removed
and the solution containing the cells were exposed to
air. Then, the fluorescence was quickly disappeared.
15 Also in the experiment using Cu(I1) ions and S.A. in
Example 1, it was found that the fluorescence was
disappeared after the reaction solution was exposed to
air for a long time. The disappearance of the
fluorescence could be considered due to oxidation of
20 Cu(1) ions by the contact with air. Accordingly, the
fluorescence generation may be inhibited by bringing
I
the sample solution into contact with air (in
particular, exposing to oxygen contained in the air).
It is considered that the method of detecting nucleic
25 acids according to the present technology is preferably
performed by limiting the contact with air, e.g., in
the microchip.
[Example 51
[0145] In Example 5, it was confirmed that the
fluorescence could be emitted from oligo-DNAs having a
5 two base length under the similar experimental
conditions as in Example 1.
[0146]

Seven types of oligo-DNAs purchased from
10 Invitrogen Corporation were measured for the
fluorescence using the similar materials and method as
in Example 1. The base sequences of the oligo-DNAs used
are T(20) (SEQ ID NO: I), T (10) (SEQ ID NO: 19), T (6)
(SEQ ID NO: lo), T (5) (SEQ ID NO: 27), T (4) (SEQ ID NO:
15 28), T(3) (SEQ ID NO: 12), T(2) (SEQ ID NO: 29) . Here,
a CuS04 concentration was set to 0.4 rnM, an S.A.
concentration was set to 4 mM, and the NanoDrop 3300
was used for measurement.
[0147]
20
Fig. 31 shows measurement results of T(20).
Respective concentrations of the oligo-DNAs are (a) 100
pMf (b) 50 1.IMI (c) 50 pM, (dl 25 pMI (e) 12.5 pM and
(f) 6.25 pM. In each graph, an abscissa axis represents
25 a wavelength (nm), and an ordinate axis represents
fluorescence intensity (RFU value). Figs. 32 to 37 show
measurement results of respective oligo-DNAs ~ ( 1 0 ) ~
T (6), T (5), T (4), T (3) and T(2) . Numerical values shown
in graphs in the Figures represent the concentrations
of the oligo-DNAs.
5 [0148] Fig. 38 shows fluorescent spectra obtained in
respective oligo-DNAs under the concentration condition
where the fluorescence intensity was highest. An
abscissa axis represents a wavelength (nm), and an
ordinate axis represents a relative value (peak RFU
10 value was set to 1). (a) represents a fluorescent
spectrum of T (20) , (b) represents a fluorescent
spectrum of T(10), (c) represents a fluorescent
spectrum of T(6), (d) represents a fluorescent spectrum
of T (5) , (e) represents a fluorescent spectrum of T (4) ,
15 (f) represents a fluorescent spectrum of T(3) and (g)
represents a fluorescent spectrum of T(2).
[0149] Fig. 39 shows graphs for plotting the peak
RFU values of respective oligo-DNAs in the respective
concentrations. An abscissa axis represents a
20 concentration of each oligo DNAs (vM), and an ordinate
axis represents a peak RFU value (logarithmic value).
(a) represents a result of T (20), (b) represents a
result of T(10), (c) represents a result of T(6), (d)
represents a result of T(5), (el represents a result of
25 T(4), (f) represents a result of T(3) and ( g )
represents a result of T (2) .
84
[01501

From the results of this Example, it was revealed
that the fluorescence was observed from oligo-DNAs
5 having a thymine two base length. It was found that the
shape of the fluorescent spectrum was little changed
even when the concentration of the oligo-DNAs was
changed, but the fluorescent peak tended to be shifted
to a short wavelength side as the base length became
10 shorter (see Fig. 38). It was discerned that the
intensity of the fluorescent spectrum tended to be
dependent on the concentration of the oligo-DNAs, but
reached plateau and was inversely reduced when the
concentration exceeded the certain value (see Fig. 39).
15 Also, it was observed that the fluorescence intensity
was decreased when the concentration of DNAs was too
high in the experiment where the copper powder was used
in Example 2 (see Fig. 15) .
[Example 61
20 [0151] In Example 6, an experiment was made using
oligo-DNAs having a three base length configured of T
and C or of T and G under the similar experimental
conditions as in Example 1.
[0152]
25
Oligo-DNAs purchased from Invitrogen Corporation
were measured for the fluorescence using the similar
materials and method as in Example 1. The sequences of
the oligo-DNAs used are TTT (SEQ ID NO: 12), TTC, TCT,
CTT, TCC, CTC, CCT, CCC, TTG, TGT, GTT, TGG, GTG, GGT, GGG.
5 A CuS04 concentration was set to 0.4 mM, an S.A.
concentration was set to 4 mM, an oligo-DNA
concentration was set to 0.5 mM, and the NanoDrop 3300
was used for measurement.
[0153]
10
The results are shown in Figs. 40 and 41. In Fig.
40, (a) represents a result of TTT (SEQ ID NO: 12), (b)
represents a result of TTC, ( c ) represents a result of
TCT, (d) represents a result of CTT, (e) represents a
15 result of TCC, (f) represents a result of CTC, (g)
represents a result of CCT, and (h) represents a result
of CCC. In Fig. 41, (a) represents a result of TTT (SEQ
ID NO: 12), (b) represents a result of TTG, (c)
represents a result of TGT, (d) represents a result of
20 GTT, (e) represents a result of TGG, (f) represents a
result of GTG, (g) represents a result of GGT, and (h)
represents a result of GGG. An abscissa axis represents
a wavelength (nm), and an ordinate axis represents a
logarithmic value of the fluorescence intensity (RFU
25 value) .
[0154] In the oligo-DNAs having a mixed sequence of
T and C, the fluorescence intensity was strongest at
TTT, then, CTT, CCT and TCT. Weak fluorescence was
identified at TTC and CTC (see Fig. 40). On the other
hand, no fluorescence having a peak at around 600 nm
5 was identified at TCC and CCC. In the oligo-DNAs having
a mixed sequence of T and G, the fluorescence having a
moderate intensity was identified at TTG, weak
fluorescence was identified at GTT, but no fluorescence
having a peak at around 600 nm was identified at other
10 sequences (see Fig. 41) .
[0155]

In the oligo-DNAs having a mixed sequence of T and
C, CTT including T at second and third positions showed
15 the fluorescence intensity higher than those of TCT and
TTC. Also, TCT and CCT including T at a third position
showed the fluorescence intensity higher than those of
TTC and CTC including T at a second position. From
these, it is considered that, in the oligo-DNAs having
20 a mixed sequence of T and C, T at a third base position
highly contributes to fluorescence, and T at a second
base position secondary contributes thereto.
[0156] In the oligo-DNAs having a mixed sequence of
T and GI no fluorescence having a peak at around 600 nm
25 was identified excluding TTG and GTT. The fluorescence
intensity was generally lower than that of the oligoDNAs
having a mixed sequence of C and T. From this, it
is considered that G has actions to absorb fluorescence
energy and quench fluorescence.
[Example 71
5 [0157] In Example 7, it was confirmed that
fluorescence was quenched by a quench pigment.
[0158]

Oligo-DNAs T(10) (SEQ ID NO: 19) purchased from
10 Invitrogen Corporation and oligo-DNAs (T(lO)BHQ2)
(Sigma-Aldrich Corporation) provided by modifying 3'
ends of the oligo-DNAs T(10) with Black Hole Quencher-2
(BHQ2) were used to measure the fluorescence using the
similar materials and method as in Example 1. A CuS04
15 concentration was set to 0.4 mM, an S.A. concentration
was set to 4 mM, an oligo-DNA concentration was set to
0.05 mM and the NanoDrop 3300 was used for measurement.
[0159]

The results are shown in Fig. 42. An abscissa axis
represents a wavelength (nm), and an ordinate axis
represents a fluorescence intensity (RFU value).
Obvious fluorescence was observed at T(10), but no
fluorescence was detected from T(lO)BHQ2 modified by
25 the quencher.
[0160]

BHQ2 is a quencher that is known to effectively
absorb light especially in the range of about 560 nm to
650 nm. It is considered that the fluorescence observed
5 at T(10) was no more observed at T(lO)BHQ2 by an effect
of the BHQ2. The result suggests that it is possible to
combine the action of copper with the FRET.
[Example 81
[0161] In Example 8, fluorescence intensities and
10 spectrum shapes of thymine (T) and uracil (U) were
again compared, and it was confirmed that the
intensities were different, but the spectrum shapes
were identical between the both. Furthermore,
methylated cytosine (MeC) and inosine (I) were examined
15 for fluorescence generation, and it was revealed that
both did not generate fluorescence.
[0162]

The results of measuring T (10) and U(9)G each for
three times are shown in Fig. 43. (a) is a graph having
an abscissa axis representing a wavelength (nm), and an
10 ordinate axis representing fluorescence intensity (RFU
value). (b) is a graph having an ordinate axis
representing an average value of the fluorescence
intensity (RFU value) as a relative value (peak RFU
value of each oligo-DNA was set to 1). It was confirmed
15 that U(9G) emitted the fluorescence having the lower
strength but the similar spectrum shape than/to that of
T (10) .
[0164] The results of measuring T(10), C(10) and
C(4)MeC(6) are shown in Fig. 44. An abscissa axis
20 represents a wavelength (nm), and an ordinate axis
represents fluorescence intensity (RFU). Noticeable
fluorescence was observed from T(10), but no
fluorescence was observed from C(10) and C (4)MeC (6) .
[0165] The results of measuring T (lo), A(10) and
25 I(9)G are shown in Fig. 45. An abscissa axis represents
a wavelength (nm), and an ordinate axis represents
fluorescence intensity (RFU) . Noticeable fluorescence
was observed from T(10), weak fluorescence was observed
from A(10), but no fluorescence was observed from I(9) G.
[0166]
5
The nucleic acids having a uracil sequence emitted
fluorescence having a lower intensity but a similar
spectrum shape, as compared with that emitted from the
nucleic acids having a thymine sequence. Example 1
10 supports this result. In addition, it was confirmed
that the nucleic acids having a cytosine sequence or a
cytosine and methylated cytosine sequence emitted no
fluorescence.
[0167] From these results, it shows that uracil can
15 be identified from cytosine and methylated cytosine by
detecting fluorescence using copper. This suggests that
the method of detecting nucleic acids according to the
present invention can detect a replacement of cytosine
( C ) with uracil (U) by a bisulfate reaction, and
20 analyze methylation of DNA molecu~es.
[Industrial Applicability]
[0168] According to the present technology, only by
bringing a sample into contact with copper, it is
possible to easily detect or measure presence or
25 absence of nucleic acids and the number, the base
sequences in the sample; and the shape, the
distribution, the number and size of cell nuclei in the
4-
sampie .
[0169] This technology contributes to enhance
analysis and analytical research of nucleic acids orea
cells in a variety of fields including a medical field
(pathology, tumor immunology, transplantation, genetics,
regenerative medicine, chemical treatment, etc.), a
drug discovery field, a clinical examination field, a
food field, an agricultural field, an engineering field,
a forensic medicine field and a criminal identification
field.