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Fluorescence detection of single nucleotide

polymorphisms using a universal molecular beacon

Yang-Wei Lin

1

, Hsin-Tsung Ho

2,3

, Chih-Ching Huang

4

and Huan-Tsung Chang

1,

*

1

Department of Chemistry, National Taiwan University 1, Section 4, Roosevelt Road, 2Department of Laboratory Medicine, Mackay Memorial Hospital,3Mackay Medicine, Nursing and Management College, Taipei and4Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan

Received March 27, 2008; Revised August 5, 2008; Accepted August 6, 2008

ABSTRACT

We present a simple and novel assay—employing a universal molecular beacon (MB) in the presence of Hg2+—for the detection of single nucleotide poly-morphisms (SNPs) based on Hg2+–DNA complexes inducing a conformational change in the MB. The MB (T7-MB) contains a 19-mer loop and a stem of a pair of seven thymidine (T) bases, a carboxyfluores-cein (FAM) unit at the 5’-end, and a 4-([4-(dimethyla-mino)phenyl]azo)benzoic acid (DABCYL) unit at the 3’-end. Upon formation of Hg2+–T7-MB complexes through T–Hg2+–T bonding, the conformation of T7-MB changes from a random coil to a folded struc-ture, leading to a decreased distance between the FAM and DABCYL units and, hence, increased effi-ciency of fluorescence resonance energy transfer (FRET) between the FAM and DABCYL units, result-ing in decreased fluorescence intensity of the MB. In the presence of complementary DNA, double-stranded DNA complexes form (instead of the Hg2+–T7-MB complexes), with FRET between the FAM and DABCYL units occurring to a lesser extent than in the folded structure. Under the optimal conditions (20 nM T7-MB, 20 mM NaCl, 1.0 kM Hg2+, 5.0 mM phosphate buffer solution, pH 7.4), the linear plot of the fluorescence intensity against the con-centration of perfectly matched DNA was linear over the range 2–30 nM (R2= 0.991), with a limit of detection of 0.5 nM at a signal-to-noise ratio of 3. This new probe provides higher selectivity toward DNA than that exhibited by conventional MBs.

INTRODUCTION

The past decade has witnessed the development of many advanced biomolecular recognition probes for highly sensitive and selective detection of DNA molecules (genes) of interest (1–6). One such set of promising

probes are single-stranded DNA molecular beacons (DNA-MB) that form hairpin-shaped structures to recog-nize targeted DNA molecules. To allow the monitoring conformation changes in DNA-MB upon reactions with targeted DNA, a fluorophore and a quencher are cova-lently conjugated at the termini of each DNA-MB strand. DNA-MBs act as fluorescence resonance energy transfer (FRET)-based switches that are normally in the closed or ‘fluorescence off’ state, but switch to the open or ‘fluores-cence on’ state in the presence of target (complimentary) DNA strands (7).

When DNA-MBs are used for the detection of single nucleotide polymorphisms (SNPs), problems associated with their nonspecific binding to DNA-binding proteins and endogenous nuclease degradation occur, leading to false-positive signals and their limited applicability in complex biological samples (8–10). MBs containing nuclease-resistant backbone residues, such as negatively charged phosphorothioates and neutral peptide nucleic acids, have been developed, but they sometimes exhibit toxicity, self-aggregation and nonspecific binding to single-stranded DNA (ss-DNA)-binding protein (SSB) (11–13). To provide high sensitivity and fast hybridization kinetics, hybrid molecular probes consisting of two ss-DNA sequences tethered to two ends of a poly(ethylene glycol) chain have been developed (14). The two ss-DNA sequences are complementary to adjacent areas of a target sequence in such a way that hybridization of the probe with the target brings the 50- and 30-ends of the probe in

close proximity. Nevertheless, hybrid molecular probes are more difficult to prepare and are more expensive than conventional DNA-MBs.

Probes based on the Hg2+-induced conformational change of a DNA molecule through thymidine (T)– Hg2+–T coordination have been realized for the detection of Hg2+ions (15–18). A DNA sensor has been employed for the detection of Hg2+through the enhanced efficiency of FRET as a result of formation of T–Hg2+–T complexes (15). Recently, we presented a simple and rapid colouri-metric assay—employing poly-Tnand 13 nm-diameter Au NPs in the presence of salt—for the detection of Hg2+

*To whom correspondence should be addressed. Tel: +886 2 33661171; Fax: +886 2 33661171; Email: changht@ntu.edu.tw

ß 2008 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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ions based on Hg2+–DNA complexes inducing the aggre-gation of Au NPs (17).

In this article, we present a simple and novel assay— employing T7-MB in the presence of salt and Hg2+—for the detection of SNPs based on Hg2+–DNA complexes inducing a conformational change in T7-MB. The T7-MB contains a stem of a pair of 7-mer T bases that interact with Hg2+and a loop of 19-mer DNA bases that recog-nize targeted DNA. According to our previous study (18), for obtaining stable DNA–Hg complexes that allow selec-tive detection of target DNA, the minimum number of T is 14. Therefore, 7-mer bp of Ts in the stem region are neces-sary in the stem region for providing a proper function. The T7-MB probe contains a donor of carboxyfluorescein (FAM) at the 50-end, and a quencher of

4-([4-(dimethyla-mino)phenyl]azo)benzoic acid (DABCYL) at the 30-end

(the sequence of the MB listed in Table 1). The T7-MB is a random-coil structure that changes into a folded struc-ture in the presence of Hg2+ ions through T–Hg2+–T bonding (19–21). As a result of the decreased distance between the donor and quencher, the fluorescence of FAM in the Hg2+–T7-MB complexes becomes weaker because of FRET occurring between the FAM and DABCYL units. When the DNA loop of T7-MB interacts with a targeted DNA more strongly than do the T7units in the stem with Hg2+, a double-stranded DNA forms, rather than the folded structure. In this case, the FAM and DABCYL units are separated far apart, resulting in FAM fluorescing strongly, as depicted in Scheme 1. We investigated the effect of the Hg2+ concentration on the sensitivity and selectivity of the T7-MB probe, and compared its sensing performance toward SNPs with that of conventional DNA-MBs.

MATERIALS AND METHODS Chemicals

Mercury(II) chloride (HgCl2) and magnesium(II) chloride (MgCl2) used in this study were purchased from Aldrich (Milwaukee, WI, USA). Sodium phosphate dibasic anhy-drous and sodium phosphate monobasic monohydrate, obtained from J. T. Baker (Phillipsburg, NJ, USA), were used to prepare the phosphate buffer (5.0 mM, pH 7.4). The T7-MB, DNA-MBx (x = 13), perfectly matched DNA (DNApm) and mismatched DNA (DNAmmx) (see Table 1 for sequences) were purchased from Integrated DNA Technology, Inc. (Coralville, IA, USA). The sequences in T7-MB and DNA-MBx that do not have any biological targets were randomly designed to provide optimum selectivity toward the target DNAs and hybrid-ization kinetics (4). Milli-Q ultrapure water was used in all experiments.

Analysis of samples

Aliquots (400 ml) of 5.0 mM phosphate buffer (pH 7.4) containing NaCl (0–250 mM) and MB (20 nM) were main-tained at ambient temperature for 10 min. Aliquots (50 ml) of tested DNA (1.0 mM) were added to the solutions, which were then incubated for 30 min. The final ratio of the concentrations of the MB and the tested DNA was 1:5.

An aliquot (50 ml) of Hg2+(0–1.5 mM) was added to each solution, which was then incubated for 2 h prior to fluor-escence measurements (Cary Eclipse; Varian, CA, USA) at various temperatures (10–908C). To evaluate the resis-tance to endogenous nuclease degradation, aliquots (450 ml) of 5.0 mM phosphate buffer (pH 7.4) containing NaCl (20 mM), MgCl2 (5.0 mM), T7-MB or DNA-MB (20 nM) and Hg2+(1.0 mM) were maintained at ambient temperature for 2 h. An aliquot (50 ml) of DNase I (final concentration: 5.0 mg/ml) was added to each solution and then the mixtures were subjected to fluorescence measure-ments after certain periods of time, as indicated in the Results and discussion section. To evaluate the nonspecific binding to SSB, 5.0 mM phosphate buffer (pH 7.4, 450 ml) solutions containing NaCl (20 mM), SSB (100 nM) and T7-MB or DNA-MB (20 nM) were maintained at ambient temperature for 30 min. An aliquot (50 ml) of Hg2+ (1.0 mM) was added to each solution, which was then incubated for 2 h prior to fluorescence measurement.

RESULTS AND DISCUSSION Sensing behavior

Two aliquots of the T7-MB (20 nM) were separately added to 5.0 mM phosphate buffers containing 20 mM NaCl solution (pH 7.4) in the absence and presence of targeted DNA (DNApm; 100 nM) and then the mixtures were equi-librated for 30 min at ambient temperature. Two aliquots of Hg2+ (final concentration: 1.0 mM) were then added separately to the two mixtures. In the absence of the target DNA, the fluorescence of FAM (excitation wave-length: 475 nm) was low, as indicated in Figure 1 (spec-trum a). In the presence of the targeted DNA, the fluorescence (spectrum b) of FAM was higher than that in the absence of the target DNA. These results support the sensing mechanism illustrated in Scheme 1. When using a single base mismatched DNA (DNAmm1) having the sequence listed in Table 1 as a control, the fluorescence of FAM (spectrum c) was only slightly higher than that in the absence of the targeted DNA, suggesting that the T7-MB probe has high specificity toward DNApm. In addi-tion, the selectivity of T7-MB toward DNApmto DNAmm1

Table 1. DNA sequences of MBs and trget DNA

Name Sequence (50–30) T7-MB FAM-TTTTTTTTCTAAATCACTATGGTCGCTTTTT TT-DABCYL DNA-MB1 FAM-ACTTAGTTCTAAATCACTATGGTCGCACTA AGT-DABCYL DNA-MB2 FAM-ACCTAGCTCTAAATCACTATGGTCGCGCTA GGT-DABCYL DNA-MB3 FAM-GCCGAGCTCTAAATCACTATGGTCGCGCTC GGC-DABCYL DNApm GCGACCATAGTGATTTAGA DNAmm1 GCGACCATAATGATTTAGA DNAmm2 GCGACCATACTGATTTAGA DNAmm3 GCGACCATATTGATTTAGA DNAmm4 GCGACCATAGAGATTTAGA DNAmm5 GCGACCATAGCGATTTAGA

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increased upon increasing in the ratio of the targeted DNA to T7-MB, and achieved a maximum when the tar-geted DNA was used in 5-fold excess (inset of Figure 1). The selectivity values of T7-MB (20 nM) toward DNApm over DNAmm1 were 3.0, 3.0, 4.2, 9.5 and 46 when the molar ratios of the target DNA to T7-MB were 0.1, 0.5, 1, 2 and 5, respectively. The selectivity increased upon increasing the concentration of the targeted DNA, because the hybrid structure of T7-MB with DNApm is more stable than that with DNAmm1. The use of four other single base mismatched DNA strands (DNAmm2–5) provided similar results to those obtained using DNAmm1. Furthermore, when using a random DNA sequence (50-ACCTGGAAGAGTATTGCAA-30) as a control to

test the specificity of our T7-MB, we did not observe any change in the fluorescence. The highly specific nature of our T7-MB probe suggested that it would have great potential for use in SNPs studies.

Effect of Hg2+concentration

The sensing capability of our T7-MB probe for DNA depends on the interplay of the complexes formed between T7and Hg2+and between the DNA sequence in the loop

and the tested DNA. Thus, we expected that the specificity and sensitivity of our T7-MB probe would depend on the concentration of Hg2+, because it affects the amount of Hg2+–T7-MB complex formed. We investigated the effect of Hg2+ at various concentrations (0–1.5 mM) on the fluorescence of the FAM unit in the T7-MB in the absence of tested DNA. Upon increasing concentration of Hg2+in the presence of 20 nM T7-MB (Figure 2A, closed square), the fluorescence of FAM initially decreased rapidly (from 0 to 0.5 mM) and then decreased more gradually (from 0.5 to 1.5 mM). This result suggests that the folded DNA structure was more stable in the presence of higher con-centrations of Hg2+. To support this hypothesis, we con-ducted melting temperature measurements; here, we define Tmas the temperature at which the fluorescence of FAM reaches 50% of its original value. Upon increasing the temperature, the fluorescence intensity increased as a result of breaking the T–Hg2+–T bonds (Figure 2B). Upon increasing the Hg2+ concentration, the value of Tm increased, reaching a plateau at the concentration of Hg2+of 1.0 mM (inset to Figure 2B). T7-MB DNApm DNAmm : FAM : DABCYL : DNAmm : DNApm NaCl NaCl Hg2+ Hg2+

Scheme 1. Schematic representation of the working principles of the T7-MB. 500 550 600 650 0 100 200 300 400 IF (a. u.) Wavelength (nm) a b c 0 2 4 6 8 10 0 10 20 30 40 50 (IF 1 − IF0 )/( IF2 − IF0 ) [DNAtarget]/[T7−MB]

Figure 1. Fluorescence spectra of the T7-MB (20 nM) in (a) the absence

of target DNA and (b, c) the presence of (b) DNApm(100 nM) and (c)

DNAmm1(100 nM). Inset: the values of (IF1–IF0)/(IF2–IF0) of T7-MB in

the presence of DNApm (IF1) and DNAmm1(IF2), as functions of

con-centration ratio of DNAtargetto T7-MB. The solution contained 5 mM

phosphate buffer (pH 7.4), 1.0 mM Hg2+and 20 mM NaCl.

0 300 600 900 1200 1500 0 200 400 600 [Hg2+] (nM) IF (a. u.) IF (a. u.) 0 10 20 30 40 50 (I F 1 I F 0 )/( I F 2 I F 0 ) 500 550 600 650 0 200 400 600 90 85 80 75 70 60 55 50 40 30 20 15 Temperature (°C) Wavelength (nm) 100 300 500 700 900 1100 1300 1500 15 25 35 45 55 65 Tm ( °C) [Hg2+] (nM) A B

Figure 2. (A) Plots of (closed square) the fluorescence intensity at 518 nm of T7-MB (20 nM) and (open square) the values of (IF1– IF0)/(IF2–IF0)

of T7-MB in the presence of DNApm(IF1) and DNAmm1(IF2), both as

functions of the concentration of Hg2+ (0–1.5 mM). (B) Fluorescence spectra of the T7-MB (20 nM) recorded a various temperatures. Inset:

plot of the value of Tmof T7-MB as a function of the concentration of

Hg2+(0–1.5 mM). Other conditions were the same as those described in

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The results in Figure 2 suggest that the concentration of Hg2+is an important factor determining the specificity of the T7-MB. Thus, to determine the optimal Hg2+ concen-tration under the tested conditions, we plotted (IF1– IF0)/ (IF2– IF0) against the Hg

2+

concentration, where IF0, IF1, and IF2are the fluorescence intensities of the FAM unit in T7-MB in the absence of the targeted DNA and in the presence of DNApm and DNAmm1, respectively. A higher value of this ratio indicates better specificity of the T7-MB probe toward DNApm over DNAmm. Figure 2A (open square) indicates that the ratio was maximized at an Hg2+ concentration of 1.0 mM; at higher concentrations (e.g. 10 mM), the T7-MB prefers to complex with Hg2+, reducing its ability to recognize its target DNA. In addition, the temperature also affected the specificity of the T7-MB. The specificity of the T7-MB probe toward DNApm over DNAmm achieved a plateau at ambient temperature (25–308C). At higher temperature, the T–Hg2+–T bonds were broken as a result of decreas-ing the specificity (Figure S1). Thus, the optimal condi-tions—providing the highest specificity of the T7-MB toward its target DNA—involved the use of 20 nM T7-MB in 5.0 mM phosphate buffer (pH 7.4) containing 1.0 mM Hg2+and 20 mM NaCl at ambient temperature.

Next, we separately investigated the kinetics of forming folded structures of the T7-MB with and without targeted DNA in the presence of Hg2+. The fluorescence intensity of the T7-MB decreased immediately once Hg2+ was added. However, the fluorescence intensities took 1.5 and 2.0 h to achieve constant values in the presence of DNApm and DNAmm1, respectively (Figure S2). Figure S2 reveals that the folded rate of the T7-MB with DNAmm1was slower than that with DNApm. The kinetics of this probe is slow, because some undesired Hg-oligonu-cleotide complexes may be kinetically preferred formed, especially in the case of DNAmm1 (20). Based on these kinetics, we employed an equilibrium time of 2.0 h in the following experiments.

Sensitivity and specificity

We investigated the sensitivity of the T7-MB at different concentrations toward DNApm. Figure 3 indicates that the fluorescence intensity increased upon increasing the con-centration of DNApm when using 20 nM T7-MB. We obtained a linear response (R2= 0.991) of the fluorescence intensity against the concentration of DNApm over the range 2–30 nM, (inset to Figure 3), with a limit of detec-tion of 0.5 nM at a signal-to-noise ratio of 3. The LODs of DNApmby using T7-MB at the concentrations of 10.0 and 50.0 nM were 0.48 and 1.20 nM, respectively. High con-centration of T7-MB probe produced high background fluorescence intensity, leading to decreases in the sensitiv-ity. When using low concentrations (<20 nM) of T7-MB, poor selectivity toward DNApmis problematic. Relative to other existing methods for the detection of DNA using DNA-MBs (the optimum conditions as shown in Figure S3), the T7-MB probe provides at least a 3-fold improvement in sensitivity. The relative standard devia-tion for quantitadevia-tion of DNA using the T7-MB probe was <0.8%.

To compare the present system to a conventional DNA-MB probe for the study of SNPs, we employed the two systems separately for the detection of DNApm and five mismatched strands DNAmm1–5. Because the stability of DNA-MBxprobe depends on the GC content in the stem, three different DNA-MBx probe (x = 1–3; no Hg

2+ ) as listed in Table 1 were chosen. The performances of the four MB probes were evaluated according to the values of (IF–IF0)/IF0, where IF0is the florescence intensity of the FAM in T7-MB or DNA-MB in the absence of target DNA and IFvalues are those in the presence of DNApm or DNAmm1–5, respectively. Figure 4A reveals that our T7 -MB probe exhibits enhanced specificity over the conven-tional DNA-MBx under the optimal conditions (20 nM T7-MB in the presence of 1.0 mM Hg2+ or DNA-MBx (x = 1–3), 20 mM NaCl and 5.0 mM phosphate buffer solution, pH 7.4 at 358C). We further conducted similar experiments under physiological conditions (150 mM NaCl, 5.0 mM KCl, 1.0 mM MgCl2, 1.0 mM CaCl2 and 25 mM Tris–HCl buffer solution, pH 7.4). The specificity values of T7-MB and DNA-MBx (x = 1–3) toward DNApm over DNAmm1 were 69-fold for the T7-MB probe (20 nM in the presence of 100 mM Hg2+), and 1.0-, 1.1- and 1.2-fold for DNA-MBx (20 nM; x = 1–3), respectively. We also compared the stabilities of the T7-MB and DNA-MB2probes in the presence of the endo-nuclease DNase I (Figure 4B). The DNA-MB2degraded rapidly once DNase I was added, whereas the T7-MB remained unaffected for at least 20 min under otherwise identical conditions. After 2 h, at least 50% of the T7-MB in the presence of Hg

2+

remained in its folded structure, based on changes in the fluorescence intensity. This behavior arose mainly because the folded structure of the T7-MB is more stable than the random-coil structure of the DNA-MBx. We finally compared the resistance of the T7-MB and DNA-MB2 probes toward nonspecific binding proteins. DNA-MBxare subjected to nonspecific binding to SSB. Binding of the DNA-MB2to SSB caused

500 550 600 650 0 100 200 300 IF (a. u.) Wavelength (nm) 0 100 200 300 400 500 100 200 300 400 IF (a. u.) [DNApm] (nM) [DNApm] (nM) 30 20 15 10 5 2 0

Figure 3. Fluorescence spectra of T7-MB (20 nM) recorded at various

concentrations of DNApm. Inset: plot of the fluorescence intensity at

518 nm of T7-MB (20 nM) as a function of the concentration of

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it to remain in a randomly coiled structure, leading to a false-positive signal (Figure 4C). For simplicity, we nor-malized the fluorescence intensities of the two MBs in the presence of SSB to their respective values in the absence of SSB. Interestingly, our results reveal that the T7-MB was barely affected after the addition of excess SSB, indicating that this probe is superior to conventional MBs for detect-ing target DNA strands within biological samples contain-ing high amounts of SSB. Table 2 compares our present approach with four popular approaches [conventional DNA-MB, locked nucleic acid (LNA)-MB, superquench-ers-MB and hybrid-MB] to SNPs study with respect to detection limit, specificity and resistance to SSB and nucle-ase digestion. The specificity of our method is superior to the other four methods. The sensitivity of our approach is comparable to those of superquenchers-MB and hybrid-MB approaches, and is better than those of conventional DNA-MB and LNA-MB approaches. Like our approach, LNA-MB and hybrid-MB resist to the binding of SSB and nuclease digestion. However, the LNA-MB and hybrid-MB are more difficult and expensive to prepare. Nevertheless, the use of toxic Hg2+ions, albeit in small amounts, in our probe system is a disadvantageous fea-ture. This disadvantage can be overcome by using different DNA sequences that respond to the presence of lower-toxicity metal ions such as Ag+and K+ions (22–27).

CONCLUSIONS

We have developed a new sensing strategy for SNPs study using T7-MB probe in the presence of Hg2+. This new approach is simple, sensitive, selective and cost-effective for studying SNPs. The T7-MB probe in the presence of Hg2+has greater resistance toward nuclease digestion and undergoes less nonspecific binding with SSB. When com-pared with the conventional MB approaches, the T7-MB probe provides a greater specificity toward perfect-matched DNA over misperfect-matched DNA and is more stable in the presence of high concentrations of salt.

0 5 10 15 20 DNA-MB3 DNA-MB2 DNA-MB1 T7-MB (IF x − IF0 )/ IF0 DNAmm1 DNAmm2 DNAmm3 DNAmm4 DNAmm5 DNApm 0 5 10 15 20 25 30 0 50 100 150 200 250 300 350 b a add DNApm add DNase I IF (a. u.) Time (min) B 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 DNA-MB2 + SSB DNA-MB2 T7-MB + SSB T7-MB IF (normalized) C A

Figure 4. (A) Fluorescence enhancements of T7-MB and DNA-MBx

(20 nM) in the presence of DNAmm1, DNAmm2, DNAmm3, DNAmm4,

DNAmm5 and DNApm. The final concentration ratios of the T7-MB

and DNA-MBxto the tested DNA were 1:5. The fluorescence

measure-ments of T7-MB and DNA-MBx were at ambient temperature and

358C, respectively. (B) Digestion of (a) T7-MB and (b) DNA-MB2

(20 nM) by DNase I (5.0 mg/ml) in the presence of 5.0 mM MgCl2.

(C) Responses of the two MBs toward the presence of SSB. The final ratio of the concentrations of MB and SSB was 1:5. Other conditions were the same as those described in Figure 1.

Table 2. Comparison of SNPs studies using T7-MB and other four

different approaches Type of MB Detection limit of the DNApm (nM) Specificitya Resistance SSB Nuclease Reference Conventional DNA-MB 1.5 7.0 No No In this study

T7-MB 0.5 69 Yes Yes In this study

LNA-MB 10 10 Yes Yes Wang, L.,

et al. (6)

Superquenchers-MB

0.1 30 No No Yang, C.J.,

et al.(7) Hybrid-MB 0.8 25 Yes Yes Yang, C.J.,

et al. (14)

aSpecificity: (I

F1–IF0)/(IF2–IF0) where IF0, IF1 and IF2 are the

fluorescence intensities of the fluorophore units in the MBs without the targeted DNA and with DNApmand DNAmm, respectively.

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When SNPs study under physiological conditions is needed, the stability and specificity of the T7-MB probe can be further improved by carefully controlling Hg2+ concentrations and/or the stem length. The superior char-acteristics of the T7-MB probe show its great potential for use in SNPs studies.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

National Science Council of Taiwan (NSC 96-2627-M-002-013 and NSC 96-2627-M-002-014); National Taiwan University for PDF support (96R0066-37 to Y.-W.L.). Funding for open access publication charge: National Science Council of Taiwan (NSC 96-2627-M-002-013 and NSC 96-2627-M-002-014).

Conflict of interest statement. None declared.

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數據

Figure 1. Fluorescence spectra of the T 7 -MB (20 nM) in (a) the absence of target DNA and (b, c) the presence of (b) DNA pm (100 nM) and (c) DNA mm1 (100 nM)
Figure S2 reveals that the folded rate of the T 7 -MB with DNA mm1 was slower than that with DNA pm
Figure 4. (A) Fluorescence enhancements of T 7 -MB and DNA-MB x

參考文獻

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