TY - JOUR
AU - Kambara, Hideki
AB - Abstract A DNA analysis platform called ‘Bead‐array’ is presented and its features when used in hybridization detection are shown. In ‘Bead‐array’, beads of 100‐µm diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1–300 µl) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent‐labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. ‘Bead‐array’ enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks. Received February 11, 2002; Revised June 18, 2002; Accepted June 27, 2002 INTRODUCTION The initial sequence of the human genome has already been identified and in the post‐sequence era, analysis of the functions and variations of genes is becoming more important especially in relation to our health and diseases ( 1 , 2 ). The gene expression profile and single nucleotide polymorphism are two major targets of the analysis and they both require large‐scale experiments. Various research efforts are ongoing to clarify the relation between genes, variations and physiological phenomena, and this information, once acquired, can be used for genetic testing. Thus, the development of a cost‐effective genetic‐testing system is required ( 3 , 4 ). One of the most promising methods for genetic testing is the DNA micro‐array technology. DNA micro‐array is an array of DNA probes that are respectively synthesized using either photolithographic techniques ( 5 – 8 ) or liquid‐spotting methods ( 9 , 10 ). It has been used for various applications including gene expression profiling ( 6 , 9 , 10 ) and mutation detection ( 7 , 8 ). Although it is a very powerful and attractive device, it is very expensive and a practical fabrication method for producing a cost‐effective device is still required. In addition, it is impossible to rearrange any of the probes in the array in accordance with changes in the analysis target. This requirement seems to be overcome by micro‐spheres having DNA probes. The combination of color‐coded microbeads and a flow cytometer ( 11 , 12 ), massive parallel signature sequence, which uses microbeads for cDNA cloning and parallel sequencing reactions ( 13 , 14 ), and fixed microbeads mounted on the terminal wells of optical fibers ( 15 , 16 ) have been reported. In this study, we demonstrate the excellent characteristics of a new DNA probe array format using beads with DNA probes. This format, ‘Bead‐array’, is an array of DNA probes on beads in a capillary with an order determined by the probe species and hybridization is performed by the reciprocal flow of the sample. Outline of the probe array in a capillary (‘Bead‐array’) A schematic view of ‘Bead‐array’ is shown in Figure 1 . It is an array of beads conjugated with DNA probes in a narrow capillary in a determined order according to the probe species (Fig. 1 A). Each bead has a different DNA probe to capture different DNA targets. The bead‐array is designed so as to decrease the volume of reaction space to <0.1 µl to enable fast hybridization. The bead size was determined to be 100 µm, slightly smaller than the inner diameter of the capillary. In the present experiment, 1 to 100 beads are placed in a capillary, which occupy <1‐cm length in the capillary (Fig. 1 B). The capillary is connected to a sample reservoir, buffer reservoir, waste reservoir and a syringe pump with capillary tubing (Fig. 1 C). The device is placed in a thermal chamber. A sample solution including target DNAs tagged with fluorophores is introduced into the bead‐array from the sample reservoir and continuously flowed reciprocally by operating the syringe pump. After the hybridization is completed, the beads are washed with buffer solutions introduced from the buffer reservoirs. Finally, the bead‐array is illuminated by a light source and the fluorescence intensity of each bead is measured with a CCD camera. MATERIALS AND METHODS Immobilization of DNA probes to glass‐bead surfaces DNA probes were immobilized on glass‐bead surfaces by the methods described by Okamoto et al. ( 17 ) and Chrisey et al. ( 18 ) with some modifications. Glass beads with an average diameter of 103 µm (Matsunami Glass Ind. Ltd, Japan) were immersed in a 1% aqueous solution of detergent, Extran®MA02 (Merck Japan, Japan), overnight at room temperature. They were washed ultrasonically for 20 min, followed by a rinse in deionized water for 20 min with ultrasonication. Then, they were heated at 80°C in 1 N NaOH for 20 min and then rinsed with deionized water. To dry, the glass beads were placed in a vacuum at room temperature. To add an amino group to the surface, the washed glass beads were placed in a 1% aqueous solution of 3‐aminopropyltrimethoxysilane (Wako Chemical, Japan) for 60 min at room temperature. Then, the beads were rinsed with deionized water, dried in a vacuum at room temperature and then baked at 120°C for 60 min. A maleimide group was then added to the bead surfaces with heterobifuctional crosslinker. Eighty milligrams of the amino group‐induced beads were placed in 2 ml of 50% dimethylsulfoxide/50% ethanol solution with 0.8 mg of N ‐(11‐maleimidoundecanoxyloxy) succinimide (Dojindo, Japan) for 120 min at room temperature, rinsed with 50% dimethylsulfoxide/50% ethanol solution and ethanol, and dried in a vacuum. Synthesized DNA probes with the thiol group at 5′ terminus (Genset Japan, Japan) were dissolved in 1× phosphate‐buffered saline (PBS) (0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M Na 2 HPO 4 and 0.002 M KH 2 PO 4 , pH 7.0) at a final concentration of 40 µM. One milligram of the maleimide group‐induced beads was placed in 5 µl of the DNA probe solution for 60 min. Then, the beads were rinsed with 1× PBS and deionized water and dried in a vacuum. These DNA probe‐fixed beads were kept in an airtight container at 4°C until use. Bead‐array production and its setting for hybridization experiments An array holder consisting of a capillary tube with an inner diameter of 150 µm (TPS150365; Polymicro Technologies, USA) was used to hold the bead‐arrays and its capillary coating was removed to make a window for detecting fluorescence. The length of the window region of an array holder was up to 1 cm and the beads were located at the window region in the array holder by placing two stainless steel wires (50 µm) inserted in the array holder at both sides of the beads. To prevent the array holder from breaking, a stainless steel guide was attached to it during this operation. The array holder was connected to two capillaries with inner seal connecters (GL Science, Japan). One of the capillaries was introduced into a sample reservoir that was generally used in Light Cycler™ (Roche, Switzerland) or a 0.5‐ml Eppendorf tube. Another capillary was attached to a gastight syringe (Hamilton, USA) with a capillary union (MSV/008; GL Science). They were set in a chamber (L‐75; GL Science) with a thermo‐controller (ST280; GL Science) during hybridization and washing. A syringe pump (sp‐230iw; WPI, USA) controlled the movement of the syringe to make a liquid flow in the capillary. The sample flow rate was such that all the sample volume flowed through the bead‐array in 1 min with a repeated reciprocal flow. DNA hybridization and washing conditions Hybridization was performed using 4× SSC (1× SSC: 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.0)–0.1% sodium dodecyl sulfate (SDS) buffer at 45°C. Hybridization times, target concentrations and sample volumes are described in the figure legends. After hybridization, the beads were washed sequentially with buffers containing 1× SSC–0.03% SDS, 0.2× SSC, 0.05× SSC, and with deionized water. They were then dried in a vacuum before fluorescence intensity measurement. For reference experiments with beads in Eppendorf tubes, the hybridization and washing conditions were the same except that 0.5 mg of beads was placed in a 200‐µl sample solution in 0.5‐ml Eppendorf tubes during hybridization. Fluorescence detection and intensity measurement A fluorescence microscope (IX‐70; Olympus, Japan) with a CCD camera (DV434‐BV; Andor Technology, UK) was used to measure fluorescence intensity. A bead‐array or a bead was placed on a non‐fluorescence glass slide (S‐0313Neo; Matsunami Glass Ind. Ltd), centered in the view and its image was taken through an objective lens of 10× magnification with the CCD camera by setting the exposure time to 200 ms. A Hg lamp (Ushio Inc., Japan) and a set of WIG filters (Olympus) were used, which are suitable for exciting color tags such as Texas Red with a green light and detecting in a red light region. The fluorescence intensity on a bead was represented by the average signal from the bead surface subtracted by the signal from the bead before the hybridization experiment. The average and standard deviation were calculated over the fluorescence intensity of three beads under the same experimental conditions. The data were normalized by the fluorescence intensity of the bead in case that complementary target hybridization with its concentration as 1 × 10 –6 M, the sample volume as 10 µl, and the reaction time as 10 min. Estimation of the DNA probe density on glass beads To estimate the DNA probe density on glass‐bead surfaces, color‐tagged DNA probes, 5′‐thiol/3′‐Texas Red DNA, were immobilized on beads by the same method used in 5′‐thiol DNA probe production. The total number of DNA probes immobilized on beads was estimated by observing the fluorescence intensity change in the DNA probe solution through the immobilization process. The difference was due to the immobilization of the probes on the bead surfaces. A fluorescence spectrometer (F‐4500; Hitachi, Japan) with an excitation wavelength of 560 nm and an emission wavelength of 610 nm was used. The probe density on the bead surfaces was estimated from the decreased amount of DNA probes in the solution and the total surface area of beads. The experimental details were as follows: 0.8 nmol of color‐tagged DNA probe was dissolved into 22 µl of 1× PBS (pH 7.0). One milligram of maleimide group‐induced beads was placed in 5 µl of the color‐tagged DNA probe solution for 60 min at room temperature for probe fixing. The beads were washed as described in ‘Immobilization of DNA probes to glass‐bead surfaces’ and all the washing solution was collected and diluted to 1 ml with 1× PBS. From the fluorescence intensity and volume of this solution, compared with those of 5 µl of the color‐tagged DNA probe solution diluted to 1 ml with 1× PBS, the number of the color‐tagged probes fixed to the bead surface through the immobilization process was estimated. From the weight density of a glass bead, 2.5 g ml –1 , and the average diameter of a bead, 103 µm, the surface area of 1‐mg beads was estimated as 2.33 × 10 7 µm 2 . Finally, the DNA probe density on glass‐bead surfaces was evaluated by dividing the number of the color‐tagged probes fixed to the bead surface by the estimated surface area of beads. Synthesized DNA sequences and their terminal groups To demonstrate hybridization efficiency, two sets of complementary DNA (Genset, France) were synthesized. The probes had the functional group including thiol, HS‐(CH 2 ) 6 ‐OP(O 2 )O‐, at the 5′ termini to be immobilized on the beads and the targets had fluorophore tags, Texas Red‐(CH 2 ) 6 ‐OP(O 2 )O‐, at their 5′ termini for fluorescence detection. The sequences of probes were originally designed for capturing p53 mRNA at two different regions and therefore the sequences of the synthesized targets were perfectly complementary to the sequences of the probes. Their Tm values were estimated by the nearest neighbors method at a DNA concentration of 100 pM and a Na + concentration of 1000 mM. To estimate the DNA probe density on glass‐bead surfaces, color‐tagged DNA probes, 5′‐thiol/3′‐Texas Red DNA, were synthesized. The sequences of synthesized DNA were as follows: probe A, GCCTCACAACCTCCGTCA; target A, TGACGGAGGTTGTGAGGC ( Tm = 71.2°C); probe B, AAGAAGCCCAGACGGAAA; target B, TTTCCGTCTGGGCTTCTT ( Tm = 69.7°C); color‐tagged probe, CACTTCACTTTCTTTCCA. RESULTS AND DISCUSSION Illustrations of hybridization on bead‐arrays Hybridization experiments on bead‐arrays were performed and images after hybridization were taken with a CCD camera on a fluorescence microscope. The transparent and fluorescence images are shown in Figure 2 . In each bead‐array, one bead in the middle had a probe different to that on the other beads. A target complementary to the probe on the bead at the center was introduced into the capillary. As seen in Figure 2 , only the bead with a probe complementary to the target was observed on the florescence image and other beads with non‐complementary probes showed no fluorescence. The fluorescence from the beads was clearly detected from outside of the capillaries. Hybridization features on a bead‐array The hybridization rate on bead‐arrays was investigated and compared with that on beads in Eppendorf tubes. The target concentration was 1 × 10 –8 M. The results are shown in Figure 3 . With a complementary target, the fluorescence intensity reached a plateau in 1 min on bead‐arrays. On the contrary, the fluorescence intensity had not reached a plateau even after 300 min in Eppendorf tubes. In this hybridization condition, at least 300 times faster hybridizations were achieved by bead‐arrays compared with the same beads in Eppendorf tubes. The hybridization rate should be slow on a flat DNA micro‐array without any sample flow. With a non‐complementary target, the fluorescence intensity was very small. The hybridization rate on bead‐arrays at lower target concentrations was also investigated. The target concentration was set to 1 × 10 –11 M. The results are shown in Figure 4 , in parallel with the results of 1 × 10 –8 M. Even at the lower target concentration, the fluorescence intensity reached a plateau in 1 min. Next, the sensitivity of bead‐arrays was investigated by varying target concentrations with a fixed hybridization time of 10 min. The results are shown in Figure 5 . In the case of a target complementary to the probe, the fluorescence intensity linearly increased for the target DNA concentration from 1 × 10 –13 to 1 × 10 –9 M and reached a plateau. As will be shown below, the DNA probe on one bead was ∼1 × 10 –14 mol, which is equal to the target number in the 1 × 10 –9 M condition, so all target molecules in the sample solution could not hybridize to the probes on a bead any more in the plateau region. Even when the target concentration was lowest, 1 × 10 –13 M, which corresponds to the target number of 1 amol, the fluorescence intensity from the complementary target was distinct from that of the non‐complementary target. In the case of the target without complementary sequence, the fluorescence intensity also increased as the target concentration increased, but not as high as in the case of the matched target. The discrimination ratio between complementary and non‐complementary hybridization was estimated as the ratio of target concentrations that resulted in the same fluorescence intensity. The ratio was from ∼4 × 10 3 to ∼4 × 10 4 , and so discrimination was clear. The effects of the sample volume and the number of beads were also investigated. First, a case where the sample concentration was constant and the sample volume was varied was investigated. The results are shown in Figure 6 . The fluorescence intensity increased linearly as the sample volume, namely the total number of target DNAs increased. The fluorescence intensities obtained here were almost equal to those in Figure 5 , if the horizontal axes of both graphs are expressed as the total number of targets in a solution. Next, a case where the same amounts of target were diluted in different volumes was investigated. The number of targets was set to 100 fmol or 100 amol. The results are shown in Figure 7 . If the number of targets in the sample solution was the same, the fluorescence intensity was the same, irrespective of the dilution volume. This result suggests that the hybridization in a bead‐array progresses very fast and probes capture targets in a sample solution almost completely even from a volume as large as 300 µl. Finally, the number of the same probe attached to beads in the capillary was varied. The number of beads was varied from 1 to 100. The results are shown in Figure 8 . As the number of beads was increased, the fluorescence intensity decreased. This was because the target molecules were distributed among the surfaces of the beads. The fluorescence intensities obtained here were again almost equal to those in Figure 3 , if the horizontal axes of both graphs are expressed as the total number of target molecules per bead. The kink in this graph corresponds to the point of saturation in the data where the target concentration was varied. DNA probe density on a bead and hybridization efficiency of a bead‐array To study the efficiency of hybridization on a bead‐array, the DNA probe density on beads was estimated. The fluorescence intensities of the diluted probe solution before and after probe immobilization were 99.85 and 76.61, and the volume of the solutions were 1000 and 1244.5 µl, respectively. As the number of the DNA probes in the solution before the immobilization process was 1.818 × 10 –10 mol, the number of probes immobilized on a bead surface was estimated to be 7.1 × 10 –12 mol. By dividing this number by the bead surface area, 2.33 × 10 7 µm 2 , the average DNA probe density on a glass‐bead was evaluated as 1.8 × 10 5 probes/µm 2 . Also, the total number of probes on a bead was calculated as 6.0 × 10 9 , which is equal to 10 fmol probes. The fluorescence intensity on this bead measured by the fluorescence microscope was 1.9074. In the following discussion, the estimated fluorescence intensity based on an assumption that all target molecules in the solution are captured on a bead and the fluorescence intensity observed by the experiment are compared. Here, we take an example where a target concentration is 1 × 10 –11 M and the sample volume is 10 µl. The number of targets is 100 amol. The number of targets was less than the number of probes on one bead. If all the target was captured on a bead, its density on the bead surface is calculated as 1.81 × 10 3 targets/µm 2 by dividing the number of targets by the bead surface area. If the fluorescence intensity on the bead surface is proportional to the density of the fluorophore, the fluorescence intensity on a bead is calculated as 0.00194 by using the DNA probe density and observed fluorescence intensity of a bead with a fluorophore‐tagged probe. The fluorescence intensity observed in this condition was 0.00187 ± 0.00010, which coincides well with the estimated value. This suggests that almost all the target molecules in solution hybridize to probes on a bead and are captured by using a bead‐array. All experimental results show that hybridization in a bead‐array was very fast and reached the equilibrium in a very short time. Because of this rapid reaction, the fluorescence intensity on the bead surface after hybridization corresponded only with the number of target molecules per bead and not with the concentration of targets. In the case where the number of targets was less than the number of probes on a bead, almost all target molecules were captured on bead surfaces. These features were due to the configuration of the bead‐array. By forcing target molecules to flow very near to the probes on the bead surface, targets reach the probes without a diffusion limitation, which is a problem in many hybridization systems with liquid and solid interactions. CONCLUSION We have presented a DNA analysis platform named ‘Bead‐array’. Due to its structure and usage, the bead‐array has two good features. The first feature is the separation of the probe immobilization process and the probe‐arraying process. By distributing beads after immobilizing DNA probes to many beads in a homogeneous reaction, this procedure enables the cost‐effective production of DNA analysis devices, even for different probe combinations for corresponding experimental purposes. The other feature is its small reaction volume with continuous sample flow. This feature solves the slow diffusion process of a target that limits the reaction rate in most devices, even for a relatively large sample volume, such as hundreds of microliters. As shown here, the fast reaction rate leads the reaction to equilibrium and almost all targets are captured on the probes on the beads. A reproducible and quantitative analysis based on the number of targets in solution has been achieved. These features are very important when making highly efficient and sensitive devices. We propose to use this platform to develop a highly sensitive and fast, disposable device. By changing the probes, many applications are also possible. It especially suits multiplex analysis of genes for research use and diagnostic tests. ACKNOWLEDGEMENTS This work was performed as a part of a research and development project of the Industrial Science and Technology Program supported by the New Energy and Industrial Technology Development Organization, Japan. The authors thank Hironori Shindo at Matsunami Glass Ind. Ltd for supplying the glass beads. The authors also thank Professor Masafumi Yohda at Tokyo University of Agriculture and Technology for his helpful advice. View largeDownload slide Figure 1. ( A ) Schematic view of a bead‐array. One hundred beads with different probe DNA are arrayed in a capillary in the intended order. The size of the bead and the inner diameter of the capillary is ∼100 µm. The length of the bead‐filled capillary is ∼1 cm and the reaction volume inside the capillary is <0.1 µl. ( B ) Microscopic image of a bead‐array. The inner diameter of the capillary was 150 µm and the average bead size was 103 µm. A part of the 100 probe‐attached beads arrayed in a capillary is shown. ( C ) Schematic view of a bead‐array system. A bead‐array is connected to a sample reservoir, buffer reservoir and waste reservoir (on the left) and a syringe pump (on the right). Sample solution from the sample reservoir moves back and forth inside the bead‐array during hybridization and buffer solution from the buffer reservoir is introduced during washing. The fluorescence intensity on each bead is measured with a detection system after hybridization. View largeDownload slide Figure 1. ( A ) Schematic view of a bead‐array. One hundred beads with different probe DNA are arrayed in a capillary in the intended order. The size of the bead and the inner diameter of the capillary is ∼100 µm. The length of the bead‐filled capillary is ∼1 cm and the reaction volume inside the capillary is <0.1 µl. ( B ) Microscopic image of a bead‐array. The inner diameter of the capillary was 150 µm and the average bead size was 103 µm. A part of the 100 probe‐attached beads arrayed in a capillary is shown. ( C ) Schematic view of a bead‐array system. A bead‐array is connected to a sample reservoir, buffer reservoir and waste reservoir (on the left) and a syringe pump (on the right). Sample solution from the sample reservoir moves back and forth inside the bead‐array during hybridization and buffer solution from the buffer reservoir is introduced during washing. The fluorescence intensity on each bead is measured with a detection system after hybridization. View largeDownload slide Figure 2. Microscopic images of the bead‐array after hybridization. ( A ) Bead‐array A: the third bead from the left was conjugated with probe A and the other beads were conjugated with probe B. Sample solution with target A, which is complementary to probe A, was used for hybridization. ( B ) Bead‐array B: the fourth bead from the left was conjugated with probe B and the other beads were conjugated with probe A. Sample solution with target B, which is complementary to probe B, was used for hybridization. Hybridization was performed at 45°C for 60 min and the concentration of target tagged with Texas Red was 1 × 10 –8 M. Images were taken with a CCD camera and a fluorescence microscope. Only beads with a complementary probe to each target can be observed in the fluorescence image and the position of the rest of beads are shown with dotted circles. View largeDownload slide Figure 2. Microscopic images of the bead‐array after hybridization. ( A ) Bead‐array A: the third bead from the left was conjugated with probe A and the other beads were conjugated with probe B. Sample solution with target A, which is complementary to probe A, was used for hybridization. ( B ) Bead‐array B: the fourth bead from the left was conjugated with probe B and the other beads were conjugated with probe A. Sample solution with target B, which is complementary to probe B, was used for hybridization. Hybridization was performed at 45°C for 60 min and the concentration of target tagged with Texas Red was 1 × 10 –8 M. Images were taken with a CCD camera and a fluorescence microscope. Only beads with a complementary probe to each target can be observed in the fluorescence image and the position of the rest of beads are shown with dotted circles. View largeDownload slide Figure 3. Effect of the reaction time. Hybridization was performed at 45°C and the concentration of targets tagged with Texas Red was 1 × 10 –8 M. The sample volume was 10 (bead‐array) or 200 µl (beads in Eppendorf tubes). The reaction time was varied from 0 to 120 (bead‐array) or 300 min (beads in Eppendorf tubes). Probe‐A‐attached beads were used. Open circles, target A (complementary) hybridization in bead‐arrays; open diamonds, target A hybridization with beads in Eppendorf tubes; closed circles, target B (non‐complementary) hybridization with bead‐arrays; closed diamonds, target B hybridization with beads in Eppendorf tubes ( n = 3). View largeDownload slide Figure 3. Effect of the reaction time. Hybridization was performed at 45°C and the concentration of targets tagged with Texas Red was 1 × 10 –8 M. The sample volume was 10 (bead‐array) or 200 µl (beads in Eppendorf tubes). The reaction time was varied from 0 to 120 (bead‐array) or 300 min (beads in Eppendorf tubes). Probe‐A‐attached beads were used. Open circles, target A (complementary) hybridization in bead‐arrays; open diamonds, target A hybridization with beads in Eppendorf tubes; closed circles, target B (non‐complementary) hybridization with bead‐arrays; closed diamonds, target B hybridization with beads in Eppendorf tubes ( n = 3). View largeDownload slide Figure 4. Effect of the reaction time. Hybridization was performed at 45°C and the concentration of targets tagged with Texas Red was 1 × 10 –8 or 1 × 10 –11 M. The sample volume was 10 µl. The reaction time was varied from 0 to 120 min. Probe A‐attached beads and target A were used. Open circles, 1 × 10 –8 M (target number 100 fmol); open squares, 1 × 10 –11 M (target number 100 amol) ( n = 3). View largeDownload slide Figure 4. Effect of the reaction time. Hybridization was performed at 45°C and the concentration of targets tagged with Texas Red was 1 × 10 –8 or 1 × 10 –11 M. The sample volume was 10 µl. The reaction time was varied from 0 to 120 min. Probe A‐attached beads and target A were used. Open circles, 1 × 10 –8 M (target number 100 fmol); open squares, 1 × 10 –11 M (target number 100 amol) ( n = 3). View largeDownload slide Figure 5. Effect of the target concentration. Hybridization was performed at 45°C for 10 min. The concentration of target tagged with Texas Red was varied from 1 × 10 –13 to 1 × 10 –6 M. The sample volume was 10 µl. Probe A‐attached beads were used. Open circles, target A (complementary); closed circles, target B (non‐complementary) ( n = 3). View largeDownload slide Figure 5. Effect of the target concentration. Hybridization was performed at 45°C for 10 min. The concentration of target tagged with Texas Red was varied from 1 × 10 –13 to 1 × 10 –6 M. The sample volume was 10 µl. Probe A‐attached beads were used. Open circles, target A (complementary); closed circles, target B (non‐complementary) ( n = 3). View largeDownload slide Figure 6. Effect of the sample volume. Hybridization was performed at 45°C for 10 min. Probe A‐attached beads and target A were used. The concentration of target was 1 × 10 –11 M and the sample volume was varied from 1 to 100 µl ( n = 3). View largeDownload slide Figure 6. Effect of the sample volume. Hybridization was performed at 45°C for 10 min. Probe A‐attached beads and target A were used. The concentration of target was 1 × 10 –11 M and the sample volume was varied from 1 to 100 µl ( n = 3). View largeDownload slide Figure 7. Effect of the sample dilution volume. Hybridization was performed at 45°C for 10 min. Probe A‐attached beads and target A were used. The number of targets in a sample was 100 fmol or 100 amol. The sample dilution volume was varied from 1 to 100 µl. Open circles, 100 fmol; open rectangles, 100 amol ( n = 3). View largeDownload slide Figure 7. Effect of the sample dilution volume. Hybridization was performed at 45°C for 10 min. Probe A‐attached beads and target A were used. The number of targets in a sample was 100 fmol or 100 amol. The sample dilution volume was varied from 1 to 100 µl. Open circles, 100 fmol; open rectangles, 100 amol ( n = 3). View largeDownload slide Figure 8. Effect of the number of beads. Hybridization was performed at 45°C for 10 min. 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TI - DNA probes on beads arrayed in a capillary, ‘Bead‐array’, exhibited high hybridization performance
JF - Nucleic Acids Research
DO - 10.1093/nar/gnf086
DA - 2002-08-15
UR - https://www.deepdyve.com/lp/oxford-university-press/dna-probes-on-beads-arrayed-in-a-capillary-bead-array-exhibited-high-W6BQolGdf8
SP - e87
EP - e87
VL - 30
IS - 16
DP - DeepDyve
ER -