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Accepted Manuscript
Title: A Strategy for Development of Electrochemical DNABiosensor based on Site-specific DNA Cleavage of Restriction
Endonuclease
Authors: Jinghua Chen, Jing zhang, Huanghao Yang, Fengfu
Fu, Guonan Chen
PII: S0956-5663(10)00301-5
DOI: doi:10.1016/j.bios.2010.05.033
Reference: BIOS 3807
To appear in: Biosensors and Bioelectronics
Received date: 12-3-2010
Revised date: 7-5-2010
Accepted date: 24-5-2010
Please cite this article as: Chen, J., zhang, J., Yang, H., Fu, F., Chen, G., A
Strategy for Development of Electrochemical DNA Biosensor based on Site-specific
DNA Cleavage of Restriction Endonuclease, Biosensors and Bioelectronics (2008),
doi:10.1016/j.bios.2010.05.033
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A Strategy for Development of Electrochemical DNA1
Biosensor based on Site-specific DNA Cleavage of 2
Restriction Endonuclease3
4
Jinghua Chena,b
, Jing zhangc, Huanghao Yang
a*, Fengfu Fu
a, Guonan Chen
a*5
a. Ministry of Education Key Laboratory of Analysis and Detection Technology for 6
Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection7
Technology for Food Safety, Department of Chemistry, Fuzhou University,8
Fuzhou, 350002, China9
b. Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical10
University, Fuzhou 350004, China.11
c. Pharmaceutical department of Fujian College of Medical Occupation and12
Technology, Fuzhou 350101, China13
14
Abstract15
A new strategy for development of electrochemical DNA biosensor based on16
site-specific DNA cleavage of restriction endonuclease and using quantum dots as17
reporter was reported in this paper. The biosenser was fabricated by immobilizing a18
capture hairpin probe, thiolated single strand DNA labeled with biotin group, on a gold19
electrode. BfuCI nuclease, which is able to specifically cleave only double strand DNA20
but not single strand DNA, was used to reduce background current and improve the21
sensitivity. We demonstrated that the capture hairpin probe can be cleaved by BfuCI22
* Cooresponding author, e-mail: gnchen@fzu.edu.cn (G. Chen); hhyang@fzu.edu.cn (H. Yang); Tel.: +86 591
87893315; fax: +86 591 83713866
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nuclease in the absence of target DNA, but can not be cleaved in the presence of target23
DNA. The difference before and after enzymatic cleavage was then monitored by24
electrochemical method after the quantum dots were dissolved from the hybrids. Our 25
resuts suggested that the usage of BfuCI nuclease obviously improved the sensitivity26
and selectivity of the biosensor. We successfully applied this method to the27
sequence-selective discrimination between perfectly matched and mismatched target28
DNA including a single-base mismatched target DNA, and detected as low as 3.3×10-14
29
M of complementary target DNA. Furthermore, our above strategy was also verified30
with fluorescent method by designing a fluorescent molecular beacon (MB), which31
combined the capture hairpin probe and a pair of fluorophore (TAMRA) and quencher 32
(DABCYL). The fluorescent results is consistent with that of electroanalysis, further 33
indicated that the proposed new strategy indeed works as our expected.34
Keywords: Electrochemical DNA Biosensor, Site-specific DNA Cleavage, Restriction35
Endonuclease, Cymbidium mosaic virus36
37
1. Introduction38
Methods for the sequence-specific DNA detection have attracted significant39
attention due to possible applications in fields ranging from virus detection to the40
diagnosis of genetic diseases [Heller et al., 2002; Balakin et al., 1998]. Consequently,41
various techniques have been employed for the detection of DNA hybridization, such42
as chromosome analysis [Jorge et al., 1996], fluorescence in situ hybridization (Jilani43
et al., 2008) and real-time quantitative reverse transcription PCR (Rong et al., 2002).44
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But there were some limitations in these techniques, such as time-consuming, poor 45
precision and expensiveness. So it is very significant to develop new effective46
methods.47
Within recent years, several inventive designs for DNA sensors based on an48
electrochemical readout have appeared due to the fact that electrochemical detectors49
are simple, reliable, cheap, sensitive and selective for genetic detection [Miao et al.,50
2003, 2004]. These sensors can be prepared by immobilizing single-stranded DNA51
probes on different electrodes and using electroactive indicators or other methods to52
measure the hybridization events between the DNA probes and their complementary53
DNA fragments. Consequently, a variety of sensing strategies have been developed,54
aiming at the improvement of sensitivity and selectivity. For example, DNA55
hybridization detection deals with the use of electroactive indicators interacting56
directly and specifically with the DNA duplex, such as intercalators, DNA groove57
binders, metal complexes and threading agents, have been recently studied58
(Demeunynck et al., 2003; Li et al., 2007; Liu et al., 2004; Niu et al., 2006; Chen et al.,59
2003, 2004). While intercalator-based DNA sensing protocols take the advantages of 60
design simplicity and operation convenience, they often suffer from high background61
signals that are associated with non-specific binding of intercalators to unhybridized62
ssDNA.63
In an attempt to circumvent this problem, DNA hairpins were proposed and64
popularly employed. DNA hairpins have been found to exhibit extraordinary stability,65
better selectivity and higher specificity than similar assays performed using66
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single-stranded DNA probe. Furthermore, DNA probes with hairpin structure exhibit a67
particularly high sensitivity to detect mismatch. Consequently, their hybridization68
processes can significantly enhance the specificity and improve the signal-to-noise69
ratio, thus enabling the detection of as few as picomolar DNA targets. The detection70
limit can be further pushed down to the femtomolar level by coupling the hairpin-type71
sensors with signal amplification offered by using either enzymes (Liu et al., 2008) or 72
inorganic nanoparticles labels (Gao et al., 2006).These strategies greatly improved the73
sensitivity, however, practical application of these strategies might be hampered due to74
poor stability of enzymes or relatively poor specificity for the detection of single base75
mismatch and single-nucleotide polymorphisms (SNPs). Moreover, it is important to76
develop a simple and rapid approach to realize simultaneous detection of multiple77
targets. Existing sensors are often not amenable to detection in complicated samples78
such as DNA species related to virus. So, new sensitive and selective DNA79
hairpin-type strategies still need to be developed.80
Cymbidium mosaic virus (CymMV) is one of the most prevalent and economically81
important orchid viruses (Wong et al., 1994). It infects numerous commercially82
important orchid genera and has attained a worldwide distribution (Zettler et al., 1990).83
CymMV cause flower colour breaking, size reduction and stunted growth, thus84
reducing the quality of the orchids and greatly affecting the economy of the orchid85
industry. Therefore, it is very significant to develop a new effective method for 86
detection of CymMV. Herein, we reported a new strategy for development of 87
electrochemical DNA biosensor for detection of DNA species related to virus88
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(cymbidium mosaic virus, CymMV, in this case) using site-specific DNA cleavage of 89
restriction endonuclease (REN)–BfuCI. A restriction enzyme (or restriction90
endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at91
specific recognition nucleotide sequences known as restriction sites (Kessler and92
Manta,1990). In this case, BfuCI is able to specifically cleave only double strand DNA,93
but not single strand DNA. It have been widely used in life processes, such as the94
replication, transcription, recombination and repair of nucleic acids, however using95
BfuCI in electrochemical biosensors have not been reported. In our strategy, the96
capture probe, thiolated hairpin DNA sequence labeled with biotin group at the other 97
end, was immobilized on a gold electrode through S-Au bonding (Figure 1). The stem98
of hairpin DNA is a special double-stranded DNA that contains a BfuCI REN99
recognition site (-mer sequence: 5′-GATC-3′) (Shizuka et al., 2008).The electrode was100
then blocked with 2-mercaptoethanol (MCH) to form a mixed monolayer. MCH has101
been used as a spacer to minimize nonspecific binding and maximize the efficiency of 102
hybridization of capture and target probes (Cheng et al., 2007). Next, the immobilized103
biotin hairpin probes were hybridized with various target DNA sequences, then the104
hybridized electrode surface was incubated with REN to discriminate between105
perfectly matched target DNA (PM, complementarity with the loop part of hairpin106
probe) and mismatched target DNA (MM), including a single-base mismatched (SM)107
target DNA.108
109
(Figure 1 should be inserted in here)110
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111
2. Experimental112
2.1. Chemicals and Apparatus113
All oligonucleotides were synthesized by TaKaRa biotechnology Co., Ltd. (Dalian,114
China), and their base sequences were illustrated in Table 1. BfuCI buffer were115
obtained from BoCai Biotechnology Co. Ltd. (Shanghai, China). Avidin-QDs (with a116
CdSe/ZnS core-shell structure about 10 nm) were obtained from Wuhan Jiayuan117
Quantum Dots Co., Ltd. (Wuhan, China). Tris-(hydroxymethyl) aminomethane was118
purchased from Cxbio Biotechnology Co. Ltd. (Denmark). Ethylenediaminetetraacetic119
acid (EDTA), mercaptohexanol (MCH) and tris (2-carboxyethyl) phosphine120
hydrochloride (TCEP) were purchased from Sigma-Aldrich (USA). The buffer 121
solutions are as follows: Hybridization buffer was the mixture of 100 mM NaCl and 10122
mM TE (pH 8.0), buffers for DNA immobilization buffer is the mixture of 10 mM TE,123
10 mM TCEP, 100 mM NaCl and 10 mM MgCl2 (pH 7.4), MgCl2 was added into the124
electrolyte to induce the formation of the hairpin structure of cDNA assembled onto125
the electrode surface, as reported previously (Dai et al., 1998; Dao et al., 1992; Gao et126
al., 1999; Ramsing et al., 1989). Washing buffer was the mixture of 0.1 M NaCl and 10127
mM PB (pH 7.4). Solution contained 0.1 mol/L KCl and 2 mmol/L128
Fe(CN)63-
/Fe(CN)64-
was used for electrochemical impedance spectroscopy (EIS)129
characterization. All solutions were prepared with MilliQ water (18.2 MΩ/cm130
resistivity) from a Millipore system.131
The electrochemical measurements for electrochemical impedance spectroscopy132
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(EIS), stripping voltammetry and differential pulse voltammetry (DPV) were carried133
out on a CHI 660C electrochemical working station (CH Instrument Company, USA)134
using a three-electrode system consisted of a platinum wire as an auxiliary electrode,135
an Ag/AgCl electrode as reference electrode and a 2-mm-diameter Au disk electrode as136
working electrode (for EIS) or a glassy carbon electrode as working electrode (for 137
stripping voltammetry and DPV). The spectra and intensity of fluorescence were138
measured with a Eclipse spectrofluorometer (Varian).139
2.2. Electrode Preparation140
The whole fabrication process of this biosensor is outlined in Figure 1. A gold disk 141
electrode (GE) was firstly polished to obtain mirror surface with 0.05 μm alumina142
powder, followed by sonication in ethanol and water for 5 min respectively. Then, the143
GE was electrochemically cleaned to remove any remaining impurities (Fan et al.,144
2003). After drying with nitrogen, the electrode was immediately used for DNA145
immobilization. Firstly, 5 µL S1 solution was first spread on the pre-cleaned gold146
electrode surface for 12 hours in the 100 % humidity. Next, this electrode was147
immersed in 1 mmol/L MCH for 2 hour to remove the nonspecific DNA adsorption148
and optimize the orientation of the capture probes to make hybridization easier (Zhang149
et al., 2006). Then, the S1-immobilized electrode was immersed in hybridization buffer 150
containing complementary T1, single-base mismatch T2 or uncomplementary T3,151
respectively. The hybridization was allowed for 30 min with stirring at 45
o
C.152
Afterwards, the sensor was then immersed in a solution of BfuCI reaction buffer 153
(0.067 units/uL) in NEBuffer 41×(50 mM potassium acetate, 20 mM Tris-aceate, 10154
mM magnesium acetate, 1 mM dithiothreitol, pH 7.9) and rinsed with wash buffer.155
Prior to electrochemical measurement, the modified electrode was incubated with 3 µL156
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of Avidin-QDs for 25 min at room temperature and washed with wash buffer to remove157
the physical adsorption of QDs and dried under a stream of nitrogen.158
2.3. Electrochemical Detection159
The Au electrode modified with hybrids labeled with biotin-avidin-system was160
immersed into a colorimetric tube containing 200 μL of 0.2 M nitric acid solution for 5161
min. The dissolved Cd2+
solution was transferred into 2.0 mL of the 0.1 M pH 5.3162
HOAC-NaAC buffer supporting electrolyte solution containing 10 μg/mL HgCl2.163
Stripping voltammetric measurements of the dissolved Cd2+
were performed using an164
in situ plated mercury film on a glassy carbon electrode following a pretreatment at 0.6165
V for 1 min, and a accumulation at -1.4 V for 5 min. The positive (DPV) scan was166
performed after a 15 s rest period from -0.8 to -0.5V (vs. Ag/AgCl), with pulse167
amplitude of 50 mV and pulse width of 50 ms. The anodic stripping peak current i p, a168
located at about -0.68 V was taken as the analytical response.169
Electrochemical impedance experiments were performed in the presence of 2 mM170
[Fe(CN)6]4-/3-
. The biased potential was 0.22 V (versus Ag/AgCl) and the amplitude171
was 5.0 mV. The electrochemical impedance spectra (EIS) were recorded in the172
frequency range of 0.1-105
Hz with a sampling rate of 12 points per decade. A Nyquist173
plot (Zre vs Zim) was drawn to analyze the impedance results.174
2.4. Investigation of specificity by fluorescence175
Under the optimum condition, the molecular beacons were added in NEBuffer 41×176
(50 mM potassium acetate, 20 mM Tris-aceate, 10 mM magnesium acetate, 1 mM177
dithiothreitol, pH 7.9) containing BfuCI (0.067 units/uL), complementary DNA or 178
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complementary DNA+BfuCI (0.067 units/uL), respectively. The hybridization was179
allowed for 30 min with stirring at 45oC. Afterwards, in the determined excitation and180
emission wavelengths (λ ex=521 nm, λ em=586 nm), the fluorescence intensity of 181
molecular beacons and the fluorescence intensity hybridization buffer were detected182
individually. Compare the change of the fluorescence intensity and investigate the183
specificity.184
3. Results and discussion185
3.1. Strategy for design of this Electrochemical DNA Biosensor186
In the absence of PM target, the capture probe exits predominantly in the hairpin187
form (loop-stem structure, at certain concentrations and buffer conditions). So it can be188
easily cleaved by REN. Thus, avidin labeled QDs (Avidin-QDs) cannot bind to the189
capture probe through the biotin-avidin-system. However, in the presence of the PM190
target, the loop part of the hairpin probe was hybridized with the target strand to form191
the matched duplex DNA. Meanwhile, the stem is opened to form single strand and192
resistant to the cleavage of REN. Thus the Avidin-QDs can then bind to the capture193
probe. So, the hybridization events can be monitored by stripping voltammetry after 194
the QDs were dissolved from the hybrids. In contrast to prior electrochemical DNA195
detection schemes based on DNA hairpins (Immoos et al., 2004; Fan et al., 2003), this196
strategy generates an electrochemical signal upon recognition of the target DNA (i.e.,197
signal “on” device).198
3.2 EIS of different modified electrodes199
Electrochemical impedance technique was employed to characterize the fabrication200
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in whole process. Figure 2 shows the EIS changes for surface-modified process.201
Compared with the bare Au electrode (Figure 2a), the probe S1 modified and MCH202
treated Au electrode (S1/MCH/GE) shows a larger eT resistance (Figure 2b), mainly203
due to the electrostatic repulsion between negative charges of the DNA backbone and204
the Fe(CN)63−/4−
probe. After the probe modified electrode was immersed into the205
BfuCI reaction buffer solution, the eT resistance (Figure 2c) decreased. The resistance206
decrease could be attributed to the fact that the stem of the hairpin probe structure207
contains a cleavage site for BfuCI and was subsequently cleaved by this enzyme. The208
cleavage resulted in the decrease of the negative charges of DNA on the GE surface.209
Under optimal condition, the S1 can hybridize with target DNA T1 and become210
double-stranded DNA (S1-T1/MCH/GE). At this time, the monolayer of DNA on the211
electrode surface became denser, and the negative charges on the electrode surface212
increased. Thus the electron-transfer resistance is enhanced further, and the value of 213
Ret increases (Figure 2d). When the S1-T1/ MCH/GE was directly combined with214
BfuCI, no significant difference of eT resistance was observed (Figure 2e). It may be215
that the formation of S1-T1 structure essentially does not contain the REN cleave site.216
Therefore, it can not be cleaved by BfuCI, which indicated that the hybridization217
events could be monitored by employing this novel electrochemical strategy.218
219
(Figure 2 should be inserted in here)220
221
3.3. Sensitivity of the DNA Biosensor222
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The sensitivity of the DNA biosensor was detected as shown in Figure 3. The results223
showed that the peak current value of DPV increased with the concentration of the224
target DNA. The linear range for logarithm of target DNA was 1.0×10-13
~1.8×10-12
M225
with the equation of I(μA)=0.1366logC(0.1 pM) + 0.1953 (I was the current intensity,226
and C was the concentration of target DNA, r=0.9956, n=6). The relative standard227
deviations (RSD) of the sensor for 10 replicate determination of 1.0×10−12
M target228
DNA was 2.8 %. The detection limit of 3.3×10-14
M target DNA could be estimated229
using 3σ. This detection limit is lower than some existing electrochemical biosensors230
based on quantum dots as reporter (Sun, et al., 2008; Wang et al., 2003; Zhu et al.,231
2004).232
233
234
(Figure 3 should be inserted in here)235
236
3.4. Selectivity of the DNA Biosensor237
The selectivity of the present biosensor was investigated by using the DNA probe238
(S1) to hybridize with the same concentration of complete complementary target DNA239
sequence (T1), the single-base mismatched DNA sequence (T2) and the240
noncomplementary DNA sequence (T3), respectively. As shown in Figure 4, a241
well-defined peak current signal of Cd2+
was obtained for the complementary sequence242
(Figure 4, curve a). It can be also known that the present biosensor has high243
hybridization specificity, it can easily discriminate the complementary from244
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single-base mismatch target DNA. In the presence of oligonucleotide containing a245
single-base mismatch, significantly decreased voltammetric signal can be observed246
(Figure 4, curve b), which indicates that the complete hybridization is not247
accomplished due to the base mismatch. In addition, as expected, nearly no response of 248
peak current can be observed for the capture probe hybridization with249
noncomplementary sequence oligonucleotide (Figure 4, curve c), since no successful250
hybridization occurs due to the sequence mismatch between the capture probe and the251
noncomplementary sequence.252
253
(Figure 4 should be inserted in here)254
255
The selectivity and specificity of the hairpin DNA probe was also studied by using256
fluorescence method, which was valuable in optimization of the hairpin DNA probe257
design. The fluorescent molecular beacons (MB) was designed by a combination of 258
DNA hairpin structure and a pair of fluorophore (TAMRA) and quencher (DABCYL).259
The ends of the stem of the hairpin were modified by fluorophore and quencher,260
individually. Before hybridization, the structure of DNA probe kept hairpin, and the261
quencher was close to the fluorophore. At this time, the efficiency of fluorescence262
quenching played a key role. So there were little fluorescent signals on the DNA probe263
(Figure 5, curve 1). After hybridization with the complementary DNA, with the264
hybridization of DNA and the change of the hairpin structure, the distance between the265
fluorophore and the quencher was increased. So the efficiency of fluorescence266
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quenching between the fluorophore and the quencher was decreased. Thus the267
fluorescence intensities were increased. (Figure 5, curve 3). It is worth mentioning that268
the fluorescence intensities were significantly increased after MB was directly269
incubated with BfuCI nuclease (Figure 5, curve 2). It could be attributed to the fact that270
when the MB is incubated with solutions containing BfuCI, the BfuCI carries out271
catalytic reactions to cleavage of the MB at the cleave site. Then the strand is broken272
into two pieces and dissociated from the MB. As a result, the amount of the quencher 273
decreases, resulting in higher fluorescence intensities. However, after hybridization274
with the PM target DNA, the stem of the MB was opened, meanwhile, the new double275
helix structure of the DNA system does not contain the REN cleave site. Therefore, it276
can not be cleaved by REN, the obtained fluorescence intensity was no significantly277
changed.(see Figure 5, curve 4). The results were in accordance with those obtained by278
electrochemical method. Of course, the performance of the hairpin DNA probe can279
also be monitored with fluorescent method. However, fluorescent method needs280
complex process of fluorescent labeling and purification, which are time consuming,281
labor intensive and cost high. Compared to fluorescent method, the proposed282
electroanalysis has many advantages such as inexpensive instrument, lower detection283
limit and simplicity due to ease of obtaining electrical signal.284
(Figure 5 should be inserted in here)285
4. Conclusions286
In summary, we have introduced here a novel strategy for development of 287
electrochemical DNA biosensor based on hairpin probe and site-specific DNA288
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cleavage of restriction endonuclease. This biosensor was used to detect DNA species289
related to cymbidium mosaic virus. The results illustrated that the proposed sensor has290
the advantages of higher sensitivity and lower background current. Given the291
simplicity in design of the proposed electrochemical sensor, it is fairly easy to292
generalize this strategy to detect a spectrum of targets. Furthermore, this design could293
be also used to construct novel optical DNA biosensors. So, it might have a promising294
future for investigation of DNA hybridization and would play the potential295
predominance in diagnosis of virus or diseases.296
Acknowledgements297
This work was financially supported by National Basic Research Program of China298
(No.2010CB732403), NSFC (20735002, 40940026, 20775019) of China, the Hi-Tech299
Research and Development Program of China (No. 2006AA09Z168), the National300
Science Foundation of Fujian Province (2009J01023) and the Foundation of Fujian301
Education Department (JA09116).302
303
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Figure captions:354
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Figure 1 Diagram of the procedure for the fabrication of DNA biosensor.355
Figue 2 Impedance spectra (Nyquist plot) of bare GE (a), the S1/MCH modified GE356
(b), the S1/MCH modified GE after being immersed into the BfuCI in reaction buffer 357
solution (c), the S1-T1/MCH modified GE(d) and S1-T1/MCH modified GE after 358
being immersed into the BfuCI in reaction buffer solution (e) in the presence of 2 mM359
[Fe(CN)6]4-/3-
. The biased potential was 0.22V (versus Ag/AgCl) in the frequency360
range of 0.1–105
Hz and the amplitude was 5.0 mV.361
Figure 3 DPV for different target concentrations of T1 sequence (×10-13
mol/L): (f-a)362
background, 1.0, 3.0, 6.0, 10.0, 14.0 and 18.0. Inset: Plot of I versus logarithm of 363
concentration. Error bars =±relative standard deviation.364
Figure 4 DPV of hybridization with different kinds of target DNA. (a) complementary365
sequence; (b) one-base mismatched sequence; (c) non-complementary sequence; All366
the concentrations of target DNA used were 1.8×10−12
M.367
Figure 5 Fluorescent graph of MB hybridizing with different gene fragments. samples368
are MB(curve 1); MB after incubed in REN(curve 2); MB+complementary369
DNA(curve 3); MB+complementary DNA after incubed in REN(curve 4). Buffer 370
solution: NEBuffer 41X (50 mM potassium acetate, 20 mM Tris-aceate, 10 mM371
magnesium acetate, 1 mM dithiothreitol, pH 7.9). λ ex = 521 nm, λ em= 586 nm.372
373
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Figure 1377
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Figure 2390
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0 4000 8000 12000 16000 20000 24000
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Z l m / O h m
e
Rs
Ret
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ac
b d
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Figure 3404
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-0.80 -0.75 -0.70 -0.65 -0.60 -0.55 -0.50 -0.450.10
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Figure 4419
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Figure 5434
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20
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F
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Table 1 Details of the DNA sequences448
Capture probe (S1)a
5′-HS-AAA A GA TCT T CA AAC TGC GGA CAT T AA GAT C-3′
molecular beacon b
5'-TAMRA-GATCTT CAA ACTGCGGACATT AAGATC-DABCYL-3'
Target (T1) 5′- AAT GTC CGC AGT TTG-3′
single-base mismatch (T2) 5′-AAT GTC CAC AGT TTG-3′
non-complementary (T3) 5′- CCG TCA ATT AAG CCA-3′
a The program was used to predict the solution-phase conformation of this sequence.449
This program predicts that under the hybridization conditions adopted for the analysis,450
only one secondary structure (the hairpin one) is thermodynamically stable, confirming451
the suitability of the chosen sequence (Zuker et al., 2003).452
b Tetramethoxyl Rhodamine (TAMRA) as fluorophore, 4-(2-methyl453
on-amino-azobenzene) benzoate (DABCYL) as quencher.454
455
456
457
458