Anal. Chem. 1999, 71, 796-800
Detection of PCR Products in Solution Using Surface Plasmon Resonance Eriko Kai,† Shinya Sawata,‡ Kazunori Ikebukuro,† Tetsuya Iida,§ Takeshi Honda,§ and Isao Karube*,†
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, National Space Development Agency of Japan, 2-1-1 Sengen, Tsukuba-city, Ibaraki 305-0047, Japan, and Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadagaoka, Suita-city, Osaka 565-0871, Japan
Polymerase chain reaction (PCR) products were detected using a flow injection-type sensor based on surface plasmon resonance. Asymmetric PCR was used to amplify the target DNA sequence, and two products with different length were produced. The novelty of our DNA detection system was that our target DNA was double stranded but the probe binding site, located in the 3′-terminus, was single stranded. This avoids the formation of intra- and intermolecular complexes. This novel design permitted us not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of Escherichia coli O157:H7. Polymerase chain reaction (PCR) amplification is a useful technique for the diagnosis of diseases caused by genetic factors. Generally, detection of particular DNA sequences is carried out using electrophoresis of PCR products and amplified using primers that are sequence-specific for the DNA of the pathogen. The general method for determining DNA amplification by PCR is based on the relative mobility of PCR products in gel electrophoresis. Although it is simple and effective for detection of PCR products, it is not possible to determine whether the sequence of the amplified target DNA is the same as that intended. In addition to this, a carcinogenic chemical such as an ethidium bromide is used for this method. Therefore, gel electrophoresis is not suitable. For general and routine analysis, Southern blotting, a method for the sequencespecific detection of DNA, is capable of solving this problem.1 However, Southern blotting is not suitable for the rapid analysis of many samples because the technique involves multiple steps, and reproducibility is sometimes lost in several of these steps. We present herein a flow injection-type detection system, based on surface plasmon resonance (SPR),2,3 for the sequence-specific detection of DNA. †
The University of Tokyo. National Space Development Agency of Japan. § Osaka University. (1) Perry-O’Keefe, H.; Yao, X. W.; Coull, J. M.; Fuchs, M.; Egholm, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14670-14675. (2) Liedberg, B.; Nylander, C.; Lundstrom, I. Sens. Actuators, 1983, 4, 299304. (3) Jonsson, U.; Malmqvist, M. Advances in Biosensors; JAI Press: Greenwich, CT, 1992; Vol. 2, pp 291-336. ‡
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Several groups have reported the sequence-specific detection of target single-stranded DNA, using various transducers, for example, SPR,4,5 resonant mirror,6 grating coupler,7 fiber optic evanescent wave,8,9 fiber optic fluorescence,10 piezoacoustics,11 and voltammetry.12-14 All of these systems were based on the detection of the physical changes caused by hybridization of the target DNA with probe DNA immobilized on the transducers. Although the principles of detection of these DNA sensors were different, the minimum size of the target single-stranded DNA and the detection limits were similar.15 Most importantly, they all relied upon the formation of a double-stranded complex upon hybridization of the immobilized probe with the target DNA. Since it is necessary that the two strands form an intermolecular complex without the formation of intramolecular complexes or homodimerization, generally speaking, it is difficult to detect the long homogeneous sequences, such as poly(A) or poly(T), or heterogeneous DNA oligomers of less than 25 base pairs.15 These limitations apply not only to target DNA but also to the probe DNA. Thus, the variety of DNA sensors based on the hybridization of PCR products with a probe DNA is very limited and there were few papers reported on DNA detection systems by amplification of PCR products. One novel system employed peptide nucleic acid (PNA) as the probe, instead of a DNA oligomer. It was possible to hybridize the probe and target DNA under conditions that did not permit the formation of inter- or intramolecular complexes of the amplified DNA itself.16 The unique properties of the PNA/DNA hybrid allowed the successful detection of PCR products. This system (4) Nilsson, P.; Persson, B.; Uhlen, M.; Nygren, P. A. Anal. Biochem. 1995, 224, 400-408. (5) Gotoh, M.; Hasegawa, Y.; Shinohara, Y.; Shimizu, M.; Tosu, M. DNA Res. 1995, 2, 285-293. (6) Watts, H. J.; Yeung, D.; Parkes, K. Anal. Chem., 1995, 67, 4283-4289. (7) Bier, F. F.; Scheller, F. W. Biosens. Bioelectron. 1996, 11, 669-679. (8) Graham, C. R.; Leslie, D.; Squirrell, D. J. Biosens. Bioelectron. 1997, 7, 487493. (9) Abel, A. P.; Weller, M. G.; Duveneck, G. L.; Ehrat, M.; Widmer, H. Anal. Chem. 1996, 68, 2905-2912. (10) Piunno, P. A. E.; Krull, U. J.; Hudson, R. H. E.; Damha, M. J.; Cohen, H. Anal. Chem. 1995, 67, 2635-2643. (11) Su, H.; Williams, P.; Thompson, M. Anal. Chem. 1995, 67, 1010-1013. (12) Hashimoto, K.; Mikawa, K.; Goto, M.; Ishimori, Y. Supra-molec. Chem. 1993, 2, 265-270. (13) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chim. Acta 1994, 286, 219-224. (14) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-3833. (15) Bier, F. F.; Fu ¨ rste, J. P. EXS. 1997, 80, 97-120. 10.1021/ac9807161 CCC: $18.00
© 1999 American Chemical Society Published on Web 01/09/1999
Figure 1. Strategy for DNA detection using asymmetric PCR and SPR.
Table 1. Combinations of Primers for Asymmetric PCR major primers
minor primers
sample
P-d aP-a P-c aP-a P-b aP-a P-a aP-a
aP-b P-d aP-b P-c aP-b P-b aP-b P-a
asym-A1(s) asym-B1(a) asym-A2(s) asym-B1(a) asym-A3(s) asym-B3(a) asym-A4(s) asym-B4(a)
base pairs
} } } }
143 101 256 214 284 242 391 349
UPD1
sequence determination of DNA17-23 or the preparation of probe DNA.24-26 This is the first report wherein asymmetric PCR is adopted for the detection of amplified of target DNA using our novel system and its application to the detection of the gene encoding verotoxin 2 in Escherichia coli O157:H7.
UPD2 UPD3 UPD4
was useful for the determination of DNA, although it did entail the use of expensive PNA. This paper presents another solution to the problem. While Perry-O’Keefe et al.16 focused on the probe, we focused on the target DNA. Our strategy is shown in Figure 1. The novel target DNA, which we called “unilateral protruding DNA” (UPD), was double stranded, except for the probe binding site located in 3′end of the longer strand. The UPD was quite thermostable in biologically neutral conditions, thus preventing the formation of intramolecular complexes and homodimers. The single-stranded region comprised 42 nucleic acid bases, complementary to the probe. We named the asymmetric PCR products, which included the longer “sense” single-stranded DNA as the predominantly amplified species, asym-A(s), and named the other, which contained “antisense” single-stranded DNA as the major product, asym-B(a) (Table 1). Samples for our sensor were prepared in combination of sense strand DNA with antisense strand produced by asymmetric PCR simultaneously. UPD could bind to probe DNA and could be existed stably since it has long double-stranded parts. This products cannot be prepared by normal PCR procedure, and this is profitable to DNA detection systems because we can design and synthesize any UPD independently on template DNA sequence. Asymmetric PCR was used to amplify predominantly one strand of DNA. Usually, asymmetric PCR is utilized for the direct (16) Perry-O’Keefe, H.; Yao, X. W.; Coull, J. M.; Fuchs, M.; Egholm, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 147670-14675.
MATERIALS AND METHODS Preparation of Target DNA. E. coli O157:H7 was cultured overnight in a 1.5-mL scale. After being harvested by centrifugation, the cell walls were ruptured using sodium dodecyl sulfate (SDS) and proteinase K. The E. coli O157:H7 genomic DNA was purified by phenol extraction and ethanol precipitation. Purified genomic DNA was redissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and stored at -20 °C until required. Oligonucleotide Primers for PCR. Our system demanded two pairs of oligonucleotide primers to produce each target DNA. Four different targets were designed differing in the number of bases. Thus we synthesized six oligonucleotide primers, each comprising 21 nucleic acid bases. 1. P-a (5′-CGG TAT CCT ATT CCC GGG AGT-3′) 2. P-b (5′-TCT CAG GGG ACC ACA TCG GTG-3′) 3. P-c (5′-TTA ACC ACA CCC CAC CGG GCA-3′) 4. P-d (5′-GCC GGG TTC GTT AAT ACG GCA-3′) (17) Innis, M. A.; Myambo, K. B.; Gelfand, D. H.; Brow, M. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9436-9440. (18) Rivetti, C.; Walker, C.; Bustmante, C. J. Mol. Biol. 1998, 280, 41-59. (19) Bianchi, N.; Rutigliano, C.; Tomassetti, M.; Feriotto, G.; Zorzato, F.; Gambari, R. Clin. Diagn. Virolog. 1997, 8, 199-208. (20) Eggerding, F. A.; Peters, J.; Lee, R. K.; Inderlied, C. B. J. Clin. Microbiol. 1991, 29, 945-952. (21) Biabchi, N.; Mischiati, C.; Feriotto, G.; Fiorentino, P. D.; Biase, S.; Appicella, N.; Gambari, R. J. Virol. Methods. 1994, 47, 321-329. (22) Holzenberger, M.; Levi-Minzi, S. A.; Herzog, C. P. Deak, S. B.; Robert, L.; Boyd, C. D. PCR Methods UPAl. 1993, 3, 107-114. (23) Hamelin, R.; Jego, N.; Laurent-Puig, P.; Vidaud, M.; Thomas, G. Oncogene 1993, 8, 2213-2220. (24) Bianchi, N.; Mischiati, C.; Feriotto, G.; Gambari, R. Int. J. Oncol. 1994, 4, 903-907. (25) Scully, S. P.; Joyce, M. E.; Abidi, N.; Bolander, M. E. Mol. Cell. Probes 1990, 4, 485-495. (26) Petronis, A.; Heng, H. H.; Tatuch, Y.; Shi, X. M.; Klempan, T. A.; Tsui, L. C.; Ashizawa, T.; Surh, L. C.; Holden, J. J.; Kennedy, J. L. Am. J. Med. Genet. 1996, 67, 85-91
Analytical Chemistry, Vol. 71, No. 4, February 15, 1999
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Figure 2. Part of DNA coding verotoxin 2 subunit A from E. coli O157:H7 strain. White boxes indicate DNA oligomers which act as PCR primers. The underline indicates probe DNA (PB). Combinations of these primers for asymmetric PCR are shown in Table 1.
5. aP-a (5′-CTG TCC GTT GTC ATG GAA ACC-3′) 6. aP-b (5′-GAA CGT TCC AGC GCT GCG ACA-3′) Their binding sites on the template DNA are indicated in Figure 2. The primers were designed so that the PCR of each sample (eight combinations; see Table 1 and Figure 2) could be performed at the same annealing temperature. The melting temperature (Tm) of each primer was calculated using the equation Tm (°C) ) [2(A + T) + 4(G + C)].27 Oligonucleotide Probe. We designed a 21-nucleic acid bases probe (PB) using the same methodology as for the primers. The sequence was 5′-CGT TGC AGA GTG GTA TAA CTG-3′, and the 5′-terminus was labeled with biotin. SPR Detection System. We used a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden) SPR system employing Biacore SA5 (Research grade) sensor chips, on which streptavidin molecules were preimmobilized at a density of ∼1 ng mm-2.4 Asymmetric PCR. Asymmetric PCR is usually performed under conditions in which the concentration of the primer used in the amplification of the desired strand is 10 times higher than that of the other primer.18,19 In this study, the concentration of the major primer was 5 µM whereas that of the minor primer was 0.5 µM. We carried out all PCR using a Perkin-Elmer GeneAmp PCR system 9600 (Perkin-Elmer, Norwalk, CT) system, employing TaKaRa Ex Taq polymerase (TaKaRa Shuzo, Kyoto, Japan). PCR conditions were 40 times rounds of 94 °C for 1 min, 59 °C for 30 s, and 72 °C for 1 min using the highest ramp rate between each temperature. Amplification products of DNA were checked using electrophoresis in an 1% agarose (genetic technology grade FMC SeaKem) gels and detected by ethidium bromide staining. Immobilization of Probe DNA. A 10 µmol L-1 aliquot of PB in 10 mM Tris-HCl buffer (pH 7.4) was injected into the BIAcore 2000, and the sensor chip was then treated with HBS (10 mM HEPES buffer pH 7.4, including 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20) running buffer at 1 µL min-1 for 50 min. It (27) Marmur, J.; Doty, P. J. J. Mol. Biol. 1962, 5, 109-118.
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was subsequently washed with 10 µL of 50 mM NaOH solution at 5 µL min-1. We calculated the changes in resonance angle (expressed in resonance units, RU equals to 0.0001°) using only one measurement per sample because all four flow cells were incorporated independently on one sensor chip. The changes in RU upon immobilization of the probe PB were 529.1 in flow cell 1 (Fc1), 608.8 in Fc2, 473.2 in Fc3, and 514.7 in Fc4. Preparation of Analyte DNA Samples. The unilateral protruding DNA 1 (UPD1) was prepared from the sense strand DNA, asym-A1(s) (143 nucleic acid bases) and the anti-strand DNA, asym-B1(s) (101 nucleic acid bases). The sense strand asymA1(s) was amplified using the primer P-d (major) and aP-b (minor). The antisense strand asym-B1(s) (101 nucleic acid bases) was amplified using primer as aP-a (major) and P-d (minor). After asymmetric PCR, analyte DNA samples were added to a solution containing the two asymmetric PCR products in equal volumes. The total volume was then adjusted to 0.1 mL in TES buffer (10 mM Tris-HCl buffer pH 7.5 contain 0.3 M NaCl and 10 mM EDTA); these samples were denatured at 95 °C for 10 min and then cooled to 25 °C over 30 min. Detection of PCR Products. Analyses of SPR data were carried out semiautomatically using the BIAcore 2000 system. The flow rate was fixed at 5 µL min-1, and a total of 50 µL of each sample was injected. TES buffer was employed as a sample buffer, and HBS was used as a running buffer. All RU values used in this paper are the average of three data from the four flowcells. RESULTS AND DISCUSSION We employed the gene encoding the verotoxin 2 subunit A in E. coli O157:H7 as a template for PCR. The sequence is shown in Figure 2.28 The major reason we adopted this DNA as an analyte was the urgent requirement for a system capable of the rapid detection of the verotoxin gene. Generally speaking, to determine the target DNA, it is very important that the DNA did not seem to be involved in any gene regulation processes, because such sequences tend to form characteristic secondary structures in order to be recognized easily by gene regulatory proteins.29-31 Amplification of DNA Samples Using Asymmetric PCR Products. We first determined whether the four kinds of symmetric and asymmetric PCR products solution were being amplified. Figure 3 shows the electrophoresis patterns of symmetric PCR products (lanes 1 and 2), asymmetric PCR products (lanes 3 and 4), and UPD1 prepared by our novel method (lane 5). The products in lane 1 (sym-B1) were amplified using P-d and aP-a primers, while the products in lane 2 (sym-A1) were amplified using P-d and aP-b. There were two bands in each lane except lane 5, although the upper bands in lanes 1 and 2 were very small. We consider that these bands were pseudoasymmetric PCR (28) Lin, Z.; Yamazaki, S.; Kurazono, H.; Ohmura, M.; Karasawa, T.; Inoue, T.; Sakamoto, S.; Suganami, T.; Takeoka, T.; Taniguchi, Y.; Takeda, T. Microbiol. Immunol. 1993, 37, 451-459. (29) Smith, S. B.; Cui, Y.; Bustamante, C. Science, 1996, 271, 795-799. (30) Chasman, D. I.; Flaherty, K. M.; Sharp, P. A.; Kornberg, R. D. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8174-8178. (31) Nikolov, D. B.; Hu, S.-H.; Lin, J.; Gasch, A.; Hoffmann, A.; Horikoshi, M.; Chua, N.-H.; Roeder, R. G.; Burley, S. K. Nature 1992, 360, 40-46.
Table 2. Comparison among UPD1, Pseudo-UPD1, and Asym-A1(s) Prepared Symmetric and Asymmetric PCR Products target DNAa
Figure 3. Electrophoresis patterns of symmetric and asymmetric PCR products in 1% agarose gel. Lane M, λ DNA digested by EcoT14 I; lane 1, sym-B1; 101-bp amplified using 100 nM P-d and 100 nM aP-a as primers; lane 2, sym-A1; 143-bp symmetrically amplified using 100 nM P-d and 100 nM aP-b; lane 3 asym-B1(a) asymmetrically amplified using 50 nM P-d and 500 nM aP-a; lane 4 asym-A1(s) asymmetrically amplified using 500 nM P-d and 50 nM aP-b; lane 5, target PCR product (UPD1) prepared from asym-B1(a) (lane 3) and asym-A1(s) (lane 4).
products caused by the difference between the annealing and melting temperatures (Tm) of the primers. As the lower bands were the main products in lanes 1 and 2, we considered that they were the intended product. These bands also appeared as minor features in lanes 3 (asym-B1(a)) and 4 (asym-A1(s)) because PCR symmetrically proceeds for ∼25 cycles and then the production of single-stranded DNA predominates next cycle in 40 cycles.17 The mobility of the DNA indicated that the lower bands in lanes 1-4 were the same product. On the other hand, upper bands in lanes 1 and 3 were slightly longer than those in lanes 2 and 4, while lane 5 (i.e., UPD1) showed no upper band. These results could be explained by assuming that these upper bands were single-stranded DNAs with complementary sequences. Therefore, we assigned the upper band in lane 3 as the 101 single-stranded bases “antisense” sequence and that in lane 4 to the 143 singlestranded bases “sense” sequence. These results obviously indicate that the PCR products prepared using our protocol corresponded to our intended design (see Figure 1), even though they were slightly contaminated. Sequence-Specific Detection of Target DNA. The used primers set are shown in Table 1. The PCR product sym-A1(s) was symmetrically amplified using same concentrations of primers P-d and aP-b. We found that the RU change upon binding of pseudo-UPD1 was lower than that of UPD1. The average value of the response to pseudo-UPD1-a prepared using sym-A and sym-B1 was 46.8, ∼10 times lower than that of 467 UPD1 (Table 2). Although pseudo-UPD1-b (prepared from sym-A1 and asym-B1(a)) possessed the PB site, the 5′-terminus of the antisense strand prevented the probe from hybridizing to its binding site, since the concentrations of sense and antisense strands amplified symmetrically were much higher than that of asym-B1(a). Pseudo-UPD1-c consisted of symmetrically amplified sym-B1 (using P-d and aP-a), and asym-A1(s) was also prepared. This sample does not have a probe DNA binding site on the sense strand, because it is located “downstream” of the aP-a binding site (see Figures 1 and 2). Consequently, no RU change was detected when it was allowed to hybridize with a sensor chip
name
used strand
avb
SD
UPD1 pseudo-UPD1-a pseudo-UPD1-b pseudo-UPD1-c
asym-A1(s) asym-B1(a) sym-A1 sym-B1 sym-A1 asym-B1(a) asym-A1(s) sym-B1 asym-A1(s)
467.0 46.8 13.3 357.0 126.3
4.52 3.66 0.97 43.1 15.0
45.2 13.5
1.76 0.02
control experiments not react PCR products, 40 µL not contained PCR products
a A 0.04-mL aliquot each of asym-A1(s), asym-B1(a), sym-A, and symB1 used for preparation of PCR product. b Average of 20 values.
Table 3. Influence of the Length of Target DNA on SPR Response SPR response target DNAa
avb
SD
UPD1 UPD2 UPD3 UPD4
467 475 593 524
4.52 1.60 25.8 51.3
a A 0.04-mL aliquot each of asym-A1(s), asym-B1(a), asym-A2(s), asym-B2(a), asym-A3(s), asym-B3(a), asym-A4(s), asym-B4(a) used for preparation of asymmetric PCR product. b Average of 20 values.
possessing PB on its surface. These results confirmed that the RU change observed in our system was caused by the sequencespecific hybridization of the probe DNA and the UPD. On the other hand, three kinds of pseudo-UPD1, using four kinds of symmetric and asymmetric PCR products were prepared and observed under the same conditions. We also investigated sense single-stranded DNA alone, i.e., asym-A1(s). These results were shown in Table 2. Continuous experiments with same conditions repeated 20 times, those results showed good reproducibility of the sensor response to the UPD and pseudo-UPD1 detected (Tables 2 and 3). Relative standard deviations of the RU were less than 12% (n ) 20). So the change of RU value was reproducible. Comparing UPD1 with asym-A1(s), UPD1 is more thermostable than the asym-A1(s) because it has a 101-nucleic acid base double-stranded region which asym-A1(s) does not have. The low response to asym-A1(s) suggests that the single-stranded DNA may have formed intramolecular complexes or homodimers. It is preferable that the analyte samples form double-stranded DNA except for the PB site for SPR detection system. Moreover, in the case of designing primers, it should not include a complementary sequence to the probe DNA. In addition, in SPR detection, the sensor response depends on the mass change at the surface of a thin gold layer where the DNA probe are immobilized and therefore, depends on the molecular weight of the DNA. In Table 3, UPD1 (MW ) 8.26 × 104) showed a 3 times bigger response than asym-A1(s) (MW ) 4.83 × 104), while it had just a twice bigger molecular weight than the asym-A(s). It strongly suggests that UPD binding to PB becomes thermostable since it had double-stranded region except Analytical Chemistry, Vol. 71, No. 4, February 15, 1999
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Figure 4. Typical sensorgram showing the hybridization of UPD1 at 40 µL of PCR products with immobilized PB. Experimental conditions were described in Materials and Methods.
for the 42-nucleic acid base probe binding site. Since it has good stability, UPD can be most effective in DNA detection systems in the case where oligomers are used as sensor probes. Dependence of the Sensor Response on the Length of the UPD. We prepared four kinds of UPD, differing in the number of base pairs (Table 1), and investigated the influence of their lengths on the response of our system (Table 3). Figure 4 illustrated a typical sensorgram showing the interaction between UPD1 and immobilized PB. It needs a very short time to detect the target DNA sequences, only 15 min. Overall, the response increased with the length of target DNA but tended toward saturation (Table 3). However, the length of the target DNA did not appear to affect the RU value to a great extent, although increases in the SPR response were evident. Also, we demonstrated control experiments: 0 µL of PCR products (value of response equal to 13.5 RU, SD equal to 0.02, n ) 20) and 40 µL of nonreacted PCR products containing all the material for PCR except for Taq polymerase (45.2 RU, SD ) 1.76, n ) 20). Considering these results, we employed UPD1, the shortest and simplest product, as our standard DNA sample in the following experiments to eliminate the possibility of unspecified hybridization. Dependence of the Sensor Response on Both the Amount and Temperature of the Target DNA Solution. The results of amount and temperature for investigations (using UPD1) are shown in Figure 5. The horizontal axis indicates the concentration of UPD1. The increase in RU depended on the amount of target present. According to the calibration curve using UPD1 (data not shown), when we diluted 40 µL of UPD 1 to 100 µL, this corresponded to 7.5 × 10-7 M. We deliberately adopted the solution volume of asym-A1(s) used for the preparation of UPD1 instead of its concentration, because we considered that a demonstration of the quantitative correlation between the response and the amount of DNA in the (32) Pertl, B.; Kopp, S.; Kroisel, P. M.; Hausler, M.; Sherlock, J.; Adinolfi, M. Am. J. Obstet. Gynecol. 1992, 177, 899-896. (33) Morrys, T.; Robetetson, B.; Gallagher, M. J. Clin. Microbiol. 1996, 34, 29332936.
800 Analytical Chemistry, Vol. 71, No. 4, February 15, 1999
Figure 5. Dependence of RU at each temperature on amount of UPD1. Symbols indicate each RU at 25 (closed square), 30 (closed circle), 35 (closed triangle), and 40 °C (closed diamond).
PCR product solution was more relevant than the precise measurement of target DNA. Therefore, the concentrations of UPD in the figures were calculated from the calibration curve. Subsequently, since the detection limit of sensor response was S/N ) 3, the lowest concentration of UPD1 that could be detected was 1.5 × 10-7 M. Our method was very simple to estimate the concentration of the target DNA compared with other reports32,33 Last, we estimated association and dissociation rates for hybridization kinetics (immobilized PB and complementary sequence 21 nucleic acid bases) using BIA evaluation software (Biacore AB) within a temperature range from 25 to 40 °C. Under the 40 °C condition, the association rate (ka) was the smallest value (ka ) 7.19 × 104 M-1 s-1) and the dissociation rate (kd) was the largest value (kd ) 1.34 × 10-5 s-1) of all. It showed a good correlation compared with BIAcore Application Note 306 (Biacore AB), showing the case of DNA/DNA hybridization of 23 nucleic acid bases. This is sufficient evidence that our system is useful as a sensor for the detection of hybridized DNA amplified by asymmetric PCR within a temperature range from 25 to 40 °C. CONCLUSIONS In this paper, we demonstrate the detection of the sequencespecific DNA samples using a novel design of PCR products. This is the first flow injection system capable of detecting PCR products quantitatively. The fundamental concept of our DNA detection system is that the ability to efficiently detect DNA is entirely dependent on the structure and binding properties of singlestranded DNA. We have attempted to minimize these influences by amplifying the target DNA as a double strand, which possesses a short single-stranded region as the probe binding site. The results reported in this paper indicate that this strategy is successful and that our novel system may become a powerful tool for the detection of DNA sequences. Received for review July 2, 1998. Accepted November 18, 1998. AC9807161
