Light-Induced Molecular Cutting: Localized Reaction on a Single DNA

Jul 16, 2003 - A short focused pulse of light was used to selectively cut lambda-phage DNA ... enzyme and the chelating agent compete for free Mg2+ io...
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Anal. Chem. 2003, 75, 4188-4194

Light-Induced Molecular Cutting: Localized Reaction on a Single DNA Molecule Vijay Namasivayam,†,§ Ronald G. Larson,† David T. Burke,‡ and Mark A. Burns*,†

Departments of Chemical Engineering, Human Genetics, and Electrical Engineering and Computer Science, The University of Michigan, Ann Arbor, Michigan 48109-2136

A short focused pulse of light was used to selectively cut lambda-phage DNA molecules at specific restriction sites. Lambda DNA (48.5 kbp) was stretched and placed in a solution containing a restriction enzyme (Sma 1), caged magnesium ions (using a DM-Nitrophen complex), and a chelating agent (EDTA). When a pulse of UV light was directed at a particular location on the stretched DNA molecule, magnesium ions were released into solution. A series of binding reactions then occur in which the enzyme and the chelating agent compete for free Mg2+ ions. Since Sma 1 functions only in the presence of Mg2+, as is true of most endonucleases, the site(s) in the vicinity of the pulse (typically ∼6 µm) were cut while other sites (three total for this DNA/enzyme pair) were not. The ratio of the concentration of the chelating agent to that of the magnesium ions was used to control the radius of this reaction zone with higher ratios leading to smaller, localized reaction areas. This optically based reaction mechanism could be useful to understand single molecule enzymatic kinetics, and when coupled with other DNA analysis techniques, this could be used to construct complex genotyping and sequencing devices that would analyze parts of single DNA molecules. Recent advances in single molecule manipulation techniques offer great promise for enhancing our understanding of the functioning and behavior of individual members of a molecular ensemble. Optical traps1 have been developed to trap biological macromolecules bound to polystyrene beads 1 µm in diameter. Scanning tunneling microscopy2 and atomic force microscopy3 techniques have been used to probe single molecules adsorbed onto a surface. These advances, coupled with improvements in fluorescence microscopy imaging of single molecules, have made molecular manipulation a reality.4,5 Of the “single” molecules * To whom correspondence should be addressed. Phone: (734) 764-4315. Fax: (734) 763-0459. E-mail: [email protected]. † Department of Chemical Engineering. ‡ Department of Human Genetics. § Department of Electrical Engineering and Computer Science. (1) Ashkin, A.; Dziedzic, J.; Bjorkholm, J.; Chu, S. Optical Lett. 1986, 11, 288290. (2) Lee, G.; Arscott, P. G.; Bloomfield, A.; Evans, D. F. Science 1989, 244, 475-477. (3) Washizu, M.; Yamamoto, T.; Kurosawa, O.; Shimamoto, N. Proc. Int. Conf. Sold-State Sens. Actuators 1997, 2C1.01, 473-476. (4) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (5) Weiss, S. Science 1999, 283, 1676-1683.

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studied, biological macromolecules such as DNA and RNA continue to attract widespread interest among researchers.6 Single molecule studies of DNA typically involve stretching or uncoiling them and performing enzymatic reactions on the extended molecules. DNA stretching studies provide a wealth of information on the elastic nature of the molecule.7-9 Enzymatic reaction studies at a single molecule level are aimed at understanding the functioning of molecular motors, such as DNA polymerases, RNA polymerases, and restriction enzymes, at low concentrations.10-13 In addition to providing fundamental information, single molecule studies have also led to practical insights for the development of new techniques for sequencing DNA. Current sequencing methods suffer from the limitations of long gel electrophoresis times and relatively short read lengths (1000 bases). Miniaturization of gel electrophoresis has considerably expedited sequencing but the read lengths remain the same. As a result, considerable time is wasted in complex computations to reassemble the sequence information. A very powerful approach would be to sequence a single molecule directly. One single molecule sequencing technique uses successive enzymatic degradation of a fluorescently labeled DNA molecule from one end and sequential detection of the released monomers.14 Alternatively, we could improve the sequencing speed by performing restriction digestion reactions on a stretched DNA molecule, collecting the fragments in order and sequencing each fragment by gel electrophoresis. By keeping track of the order of the fragments sequenced, postprocessing redundancies could be avoided. However, the success of this technique relies on the ability to perform localized reactions on single molecules. So far, studies on DNA-based single reactions have focused on performing restriction digestion along the length of the whole molecule for optical mapping.15 A technique to cut a stretched DNA molecule with an enzyme at one specific site will pave the way for sequencing at a single molecule level in particular and will expedite the overall process of sequencing in general. (6) Bustamante, C.; Macosko, J. C.; Wuite, G. J. L. Nat. Rev. Mol. Cell Biol. 2000, 1, 130-136. (7) Bustamante, C.; Marko, J. F.; Siggia, E. D.; Smith, S. Science 1994, 265, 1599-1600. (8) Perkins, T. T.; Smith, D. E.; Chu, S. Science 1997, 276. (9) Smith, S. B.; Finzi, L.; Bustamante, C. Science 1992, 258, 1122-1126. (10) Maier, B.; Bensimon, D.; Croquette, V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12002-12007. (11) Strick, T. R.; Croquette, V.; Bensimon, D. Nature 2000, 404, 901-904. (12) Wabuyele, M. B.; Soper, S. A. Single Mol. 2001, 2, 13-21. (13) Schafer, B.; Gemeinhardt, H.; Uhl, V.; Greulich, K. O. Single Mol. 2000, 1, 33-40. (14) Dorre, K. Bioimaging 1997, 5, 139-152. 10.1021/ac034180h CCC: $25.00

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In this paper, we present a novel technique to achieve sitespecific enzymatic cleavage of a single stretched lambda DNA molecule by controlling the delivery of magnesium ions to a desired reaction site. Most endonucleases work in the presence of magnesium ions, and by confining the free magnesium ions around one restriction site, we can preferentially cut the stretched DNA molecule at a given location. DM-Nitrophen is regularly used as a caging compound for sealing Ca2+/Mg2+ ions, and this caging complex can be broken down by UV light.16 DM-Nitrophen has, hence, enjoyed extensive use in neurophysiology to rapidly flood a cell with Ca2+ ions after exposure to UV light.17 In this work, we use the caged Mg2+ ion concept to cut a lambda DNA molecule at one specific location with an enzyme Sma 1, which has three recognition sites on the lambda DNA. We also address several competing factors that need to be optimized to improve robustness of this localized reaction system. MATERIALS AND METHODS DNA Stretching. Lambda phage DNA molecules (Gibco BRL) were first mixed with an intercalating fluorescent dye SYBR Green (Molecular Probes) in the ratio of 5 bp/1 dye molecule and diluted in Tris-HCl (pH 8) to a concentration of 10 pg/µL. At this concentration, we expect to see a few DNA molecules per microliter of the sample. A small sample volume (10 µL) was then placed on a 100-µm-thick glass cover slip (Fischer Scientific), and the cover slip was spun at a high speed (5000 rpm) for 30 s using a photoresist spinner. This spin-stretching procedure demonstrated by Yokota et al.18 results in stretching of the fluorescently stained lambda DNA molecules in the radial direction. Enzymes and Buffers. Restriction enzyme Sma I that works in the presence of magnesium ions at 25 °C was used as a candidate for this study. Lambda DNA has three restriction sites for Sma 1, and hence, a complete reaction would yield three cuts (four fragments). Commercial endonuclease enzymes are usually supplied with a buffer that contains Magnesium ions. In our study, we replaced the commercial buffer with a homemade buffer that contains caged magnesium ions. Magnesium ions were provided as caged compounds by mixing them with dimethoxynitrophenamine (purchased as DM-Nitrophen from Calbiochem) in the ratio of 1 (Mg)/10 (DM). The caged magnesium ions can be released from the photolabile DM-Nitrophen complex by irradiation with pulses of UV light (absorption maximum, 360 nm). The reaction system used in the macroscale control study had the following composition: 50 mM potassium acetate, 20 mM tris acetate, 10 mM magnesium, 100 mM DM-Nitrophen, 1 mM DTT (dithiothreitol), and 1 mM EDTA. In the final microscale single molecule reaction study, the concentration of EDTA was varied from 0 to 250 mM, while the remaining constituents of the buffer were maintained at the same concentrations as before. EDTA is added to chelate the photoreleased magnesium ions and to prevent them from diffusing out to the other restriction sites. (15) Jing, J.; Reed, J.; Huang, J.; Hu, X.; Clarke, V.; Edington, J.; Housman, D.; Anantharaman, T. S.; Huff, E. J.; Mishra, B.; Porter, B.; Shenker, A.; Wolfson, E.; Hiort, C.; Kantor, R.; Aston, C.; Schwartz, D. C. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8046-8051. (16) Zucker, R. Methods Cell Biol. 1994, 40, 31-63. (17) Ayer, R. K., Jr.; Zucker, R. S. Biophys. J. 1999, 77, 3384-3393. (18) Yokota, H.; Sunwoo, J.; Sarikaya, M.; van den Engh, G.; Aebersold, R. Anal. Chem. 1999, 71, 4418-4422.

Figure 1. Illustration of the concept of photoinduced localized reactions. Magnesium ions are added as caged compounds in the reaction mixture. Caged magnesium ions are released by a short pulse of UV. Free Mg ions work with the enzyme to cut the stretched DNA at a specific site. Diffusion of the released magnesium ions is controlled by adding a chelating agent (EDTA).

Fluorescence Microscopy and Imaging. The cover slip containing stretched DNA molecules was mounted on an inverted fluorescence microscope (Nikon TE 200) for visualizing single DNA molecules. Since we used SYBR green and YOYO-1 to stain the molecules, we used the appropriate blue/green filters (490 nm excitation/510 nm emission). A high magnification (100×, NA 1.3) oil immersion objective lens was used for this study. A black masking tape with a pinhole was placed on the 100× Objective lens to localize the light pulse over a 5-µm radius, thereby releasing the magnesium ions only around a desired restriction site. An electronic shutter (Uniblitz) was used to irradiate the sample with a 100-ms pulse of blue light and break the DMNitrophen complex. Images were acquired using a 12-bit highresolution cooled digital interline CCD (Micromax, 1300 YHS, Princeton Instruments, New Jersey) and captured on a computer Metaview imaging software. DNA Fragment Length Estimations. Images showing results of single molecule reactions were analyzed using NIH image 1.62. For each reaction experiment, sizes of the cut fragments were estimated by first marking each fragment as an area of interest and then measuring the net fluorescence intensity of that area. The net fluorescence intensity of a piece of DNA is given by the product of the number of pixels in the area of interest and the mean fluorescence intensity. Similarly, the net fluorescence intensity corresponding to the whole DNA molecule was also measured. By knowing the actual size of the molecule to be 48.5 kbp, the size of each fragment was estimated by calculating the ratio of the fluorescence intensities. RESULTS AND DISCUSSIONS Our localized reaction concept, as illustrated in Figure 1, relies on controlled delivery of magnesium ions to a specific reaction site on the stretched DNA. Magnesium ions are essential for most enzymatic reactions, and they are introduced, along with the enzymes, as caged ions within a compound called DM-Nitrophen. These caged magnesium ions can be then released upon photolysis of DM-Nitrophen complex, and when released, the free Mg2+ ions aid in enzymatic cleavage of the DNA molecule. By Analytical Chemistry, Vol. 75, No. 16, August 15, 2003

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Figure 2. Spin stretching results. (a) DNA molecules placed in off-center positions were completely stretched out (21 µm). (b) DNA molecules placed in the center were partly stretched. (c) In some cases, hairpin-shaped looped configurations were seen.

localizing the release of these Mg2+ ions and confining the Mg2+ ion concentration in a specific region, we can induce an enzymatic reaction to occur at a specific site and thereby cut a stretched DNA molecule at one specific site. In the following sections, we describe in detail the reaction system and the controlling factors that determine the resolution and robustness of this system. Macroscale Controls. Sma 1 was chosen as a target enzyme for this study since lambda DNA (48.5 kbp) has three restriction sites for Sma 1 (GenBank accession number J02459 or M17233). Complete digestion of lambda DNA with Sma 1 should hence result in four fragments of the following sizes: 8271, 8617, 12220, and 19397. As a standard for comparison, a macroscale reaction was first carried out with Sma 1 in the commercial buffer (that comes with the enzyme) under the recommended conditions (25 °C, 60 min). The resulting fragments were then separated on a linear polyacrylamide matrix on a capillary electrophoresis system, and all four fragments were seen. The reaction was then carried out in a homemade buffer that has caged magnesium ions and EDTA in addition to the other constituents of the commercial buffer. The caged magnesium ions were first released by placing the sample under a UV light source for 5 s, and the reaction was performed under the same operating conditions as before. Complete digestion of lambda DNA was observed. As a control, the reaction was carried out without shining UV light on the sample ions, and this resulted in no enzymatic cleavage. These experiments show that magnesium ions are necessary for digestion of lambda DNA with endonucleases such as Sma 1. 4190

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Spin-Stretching DNA. To study spatially localized reactions on single DNA molecules, we first need a simple and reliable method to stretch DNA molecules. A variety of techniques are currently available to stretch and fix long DNA molecules.8,15,19,20 For this study, we followed two published methods of stretching DNA. The first method is based on the drying droplet technique in which a small drop that contains lambda DNA molecules is placed on a cover slip and allowed to evaporate.15 The radial flow pattern generated from the center to the meniscus within the droplet during the drying process, has been known to stretch DNA molecules. Although a simple method, it requires careful cleaning of the glass surfaces prior to stretching, and second, it is highly influenced by the humidity and the temperature conditions in the lab. The second method is called the spin stretching technique in which a small drop containing DNA molecules is placed on a cover slip. The cover slip is fixed on a spinner and spun at a high speed (5000 rpm), and the flow pattern created during the spinning process stretches out the DNA molecules.18 Figure 2a shows a set of DNA molecules completely stretched out using the spin stretching technique. At the center of the drop, we observed partially stretched DNA molecules (Figure 2b), as reported by Yokota et al. and in some cases, we observed looped (hairpin shaped) DNA molecules as well (Figure 2c). Three modifications to the procedure published by Yokota et al. were (19) Washizu, M.; Kurosawa, O.; Arai, I.; Suzuki, S.; Shimamoto, N. IEEE Trans. Ind. Appl. 1995, 31, 447-456. (20) Bensimon, D.; Simon, A. J.; Croquette, V.; Bensimon, A. Phys. Rev. Lett. 1995, 74, 4754-4757.

Figure 3. Localized reaction hypothesis and results. (a) Illuminating zone 1 (that has one restriction site) should yield a single cut; illuminating zone 2 (two restriction sites) should yield two cuts. Illumination over the whole molecule (zone 3) should yield all three cuts, and no UV illumination should yield no cuts. (b) A single cut seen at ∼19 680 bp from one end of the molecule due to preferential illumination over zone 1. Error in size estimation is ∼(450 bp. (c) Two cuts seen due to illumination over zone 2. The sizes of the two fragments are estimated to be ∼8900 and 8170 bp. (d) Complete illumination over the whole molecule gives all three cuts. (e) Reaction without any UV illumination shows no cuts.

required in order to perform our enzymatic reactions. First, we used glass cover slips instead of mica sheets, since we needed an optically transparent surface to image the fluorescently stained DNA molecules using an inverted microscope. Second, we used calcium chloride solution instead of magnesium chloride as a prerinsing solution to treat the cover slips prior to DNA stretching. Since our reaction system relies on controlled release of caged Mg2+ ions, having MgCl2 on the cover slip would hinder the process. Third, we used an intercalating dye SYBR green (497/520) instead of YOYO-1 (491/509) to prestain the DNA molecules. Staining the DNA molecules with YOYO-1 inhibits enzymatic reactions. On the other hand, the presence of typical staining concentrations of the SYBR Green I dye does not significantly inhibit the ability of several restriction endonucleases, including HindIII and EcoRI to cleave DNA.21 However, since YOYO-1 is a more sensitive dye than SYBR and less prone to photobleaching, DNA molecules were poststained with YOYO-1 after the reactions to observe the cuts. In essence, we used SYBR green in the reaction mix to image the DNA molecules before the reaction (21) Struhl, S. Biotechniques 1985, 3, 452-453.

and also to position the stretched DNA molecule under the 100× objective. YOYO-1 was added after the reaction to avoid photobleaching and get better images of the results of the reaction. Spatially Localized Reactions. Our localized reaction hypothesis is depicted in Figure 3a. One of the three Sma 1 restriction sites is close to one end of the molecule (zone 1), and the remaining two sites are close to the other end of the molecule (zone 2). If we preferentially illuminate one end of the molecule with UV light and confine the released magnesium ions within zone 1, we should observe a single specific cut. On the other hand if we illuminate zone 2 and confine the free magnesium ions on this end of the molecule we should see two cuts. If we illuminate the entire molecule (zone 3) we should see all the three specific cuts, and finally, no illumination should result in no cuts at all. To test this hypothesis, a cover slip with stretched DNA molecules was placed on the stage of an inverted microscope. To generate a tightly focused illumination zone, a black masking tape with a pinhole was placed on the 100× objective lens. The masking tape with the pinhole placed on the 100× objective cuts down the illumination zone to a 5-µm radius spot. The light spot was then focused on a specific zone (zone 1 or zone 2) on a single stretched Analytical Chemistry, Vol. 75, No. 16, August 15, 2003

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DNA molecule. The shutter was closed, and a 10-µL drop of Sma 1 enzyme mixed in the homemade buffer containing caged magnesium ions, was placed (roughly) on the cover slip over the stretched DNA molecule. The shutter was then briefly opened for 100 ms (electronically) to irradiate the sample with a pulse of blue light and break the DM-Nitrophen complex. A 100-ms pulse should be sufficient because, in theory, the DM-Nitrophen complex can be broken down to release Mg ions with a 0.2 ms UV pulse. The enzyme drop was then allowed to incubate for 1 min at room temperature, during which time the released magnesium ions and the enzyme bound on the specific restriction sites on the DNA molecules and caused cleavage. After 1 min of incubation, the cover slip was washed with DI water twice. The DNA molecule was then stained with YOYO-1 to observe if there were any cuts. A fully stretched lambda DNA (48 502 base pairs) stained with an intercalating dye is about 21 µm in length.22 Figure 3b shows a typical single cut on a DNA molecule that was illuminated with UV light over zone 1. The dark spot on the DNA molecule indicates where the cleavage has occurred. In our experiments, we used an intercalating dye (SYBR green) to image the DNA after the reaction. SYBR green binds between the two strands of the DNA molecule and fluoresces only when bound between the strands. It is possible that once the restriction enzyme (Sma 1) chews the bases at the restriction site, SYBR green is no longer bound between the bases, and hence, the fluorescence is lost. Retraction of the DNA fragments after cleavage, although possible, seems unlikely in these experiments, since here, the DNA molecules are fully elongated and fixed on the surface of a glass cover slip. In optical mapping studies, researchers have observed similar dark nonfluorescent regions at the cleaved sites of stretched DNA molecules.23,15,24 Conventional restriction digestion reaction results are analyzed using size-estimation techniques, such as capillary electrophoresis. However, in single molecule studies, the results of the reaction are observed using fluorescence microscopy, and the sizes of the cut fragments are estimated using image analysis software.18 We used a similar image analysis technique to validate the results of our localized restriction digestion reactions. Images were acquired using a 12-bit high-resolution camera (Micromax 1300 YHS, Princeton Instruments). The expected spatial resolution in the images is on the order of ∼10 nm. A software package NIH image 1.62 was used to analyze the images showing the results of our single-molecule reactions. Figure 3b shows the result of a reaction after the DNA molecule was illuminated over zone 1. The restriction site in zone 1 is known to be 19 397 bp (8.4 µm) from one end. Calculations of the fragment lengths generated in these cases show that the estimated size of the fragment is 8.52 µm ((0.2 µm), which corresponds to 19 680 bp ((450 bp), confirming that this cut is specific and is caused by the endonuclease action. Similarly, if the light is focused over zone 2, we get a restriction pattern with two cuts, as shown in Figure 3c. The sizes of the two fragments (22) Perkins, T. T.; Smith, D. E.; Larson, R. G.; Chu, S. Science 1995, 268, 8387. (23) Aston, C.; Mishra, B.; Schwartz, D. C. Trends Biotechnol. 1999, 17, 297302. (24) Lin, J.; Qi, R.; Aston, C.; Jing, J.; Anantharaman, T. S.; Mishra, B.; White, O.; Venter, J. C.; Schwartz, D. C. Science 1999, 285, 1558-1562.

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are expected to be 3.73 µm (8614 bp) and 3.58 µm (8271 bp). From image analysis, we estimated the sizes to be 3.85 ((0.2) µm and 3.54 ((0.2) µm, which correspond to 8900 bp ((450) and 8170 bp ((450), respectively. When the UV light was illuminated over the entire molecule, we saw all the three expected cuts, as in Figure 3d, and when the sample was not subjected to any UV illumination, we saw no cuts, as in Figure 3e. Control Parameter: EDTA/Magnesium Ion Balance. An important parameter that determines the success of these localized reactions is the concentration of ethylenediaminetetraacetic acid (EDTA) in the reaction sample. Since the reaction sample is allowed to incubate over the stretched DNA for 1 min after UV irradiation, there is sufficient time for the released magnesium ions to diffuse out over the entire length of the molecule. To confine the free magnesium ions over a specific zone, EDTA was used as a chelating agent. However EDTA in excess concentration can chelate out all the free magnesium ions, inhibiting the reaction even in the desired location. Hence, a fine balance between the concentrations of magnesium ions and EDTA is crucial for the success of spatially localized reactions. In our experiments, the caged magnesium ion concentration was maintained at 10 mM (with DM-Nitrophen at 100 mM). From the manufacturer’s technical specifications, magnesium ions can be released from DM-Nitrophen with a UV pulse as short as 0.2 ms. In addition, the dissociation of the DM-Nitrophen complex and the release of Mg ions thereafter is almost an irreversible process (Kd of [Mg2+] with DM-Nitrophen is 2.5 µM, and with photolyzed DM-Nitrophen, is 3 mM25). Since we used a 100-ms UV pulse to break the DM-Nitrophen complex, we expected to see all the magnesium ions released into the solution. From our experiments, we have seen that without any EDTA in the solution, reactions are never localized, and all three cuts are seen irrespective of the zone illuminated. When the EDTA concentration was increased to 250 mM, no cuts were seen in almost all cases. When the EDTA concentration was fixed at an intermediate value of 100 mM, we could visualize the spatially localized reactions with one or two cuts on the stretched DNA molecule, as shown in Figure 3b,c. Spatial spread of the released magnesium ions can be modeled using finite element analysis. Localization of released magnesium ions is mainly influenced by two phenomena, the diffusion of released magnesium ions and the chelation of magnesium ions by EDTA. For this study, diffusion coefficient of magnesium ions in solution was assumed to be 10-5 cm2/s, and the initial concentration of magnesium at the point of release was taken to be 10 mM. Since EDTA was present in excess, the chelation reaction was assumed to be pseudo-first-order, and the rate constant was set at 104 s-1 (Kf was taken at pH 7.5). Timedependent spatial spread of UV-released magnesium ions was modeled for different EDTA concentrations in the bulk. Figure 4a shows the spatial spread of magnesium ions 10 s after release, with the bulk EDTA concentration set at 100 mM. From the figure, we can see that Mg ion concentration drops from 10 mM to 0.1 mM within a 6-µm radius (5 µm illumination + 1 µm diffusion) in this case. Simulation of the complicated diffusion/reaction of Mg ions can lead to insights on the minimum concentration needed for (25) Ellis-Davies, G. C. R.; Kaplan, J. H. J. Org. Chem. 1988, 53, 1966-1969.

Figure 4. Finite element analysis of localization of magnesium ions. (a) Simulation showing the two-dimensional spread of magnesium ions 10 s after release. (b) Estimated spatial spread of magnesium ions for different EDTA concentrations in the bulk.

enzymatic reactions at a single molecule level. As the magnesium ions diffuse away from the initial light-activated release location, the concentration decreases both in time and position. Thus, when EDTA concentration is high (∼250 mM), it is possible for no reaction to occur as a result of the swift chelation of Mg ions. However, at low to moderate EDTA concentrations, when reactions do occur at specific sites in the illumination zone, we can estimate the minimum concentration at which a single reaction will occur. Figure 4b shows the estimated spread of magnesium ions (from a 10 mM concentration at the point of release) for various EDTA concentrations in the bulk. For each curve, the distances plotted represent the maximum distance at which a concentration of 1, 0.1, or 0.01 mM Mg ions will be found. From the figure, we see that for EDTA concentrations above 100 mM, the spread of magnesium ions is tightly contained within 6 µm. These theoretical estimates indicate that the concentration outside the illumination

zone (near undesired cutting sites) is at or below one free Mg ion/0.5 µm3. Although the coupling of individual molecular events to bulk concentrations is difficult, this calculation, together with the observed position-selective cutting, indicates that at extremely low concentrations (0.01 mM) of Mg ions, stochastic molecular reactions can be suppressed. CONCLUSIONS In this work we have demonstrated for the first time a method to achieve spatially localized enzymatic reactions on a stretched DNA molecule. Our novel technique for performing spatially localized reactions is based on the controlled delivery of magnesium ions to a specific reaction site. The resolution of localization is determined by the UV-illumination area and the confinement of the released magnesium ions using the appropriate EDTA concentration. Although the technique has been demonstrated for a specific enzyme (Sma1) on lambda DNA, this method should Analytical Chemistry, Vol. 75, No. 16, August 15, 2003

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work for any long DNA molecule with most endonucleases that require the presence of free magnesium ions for their activity. Our UV-induced localized reaction scheme is optical, nonintrusive, and requires no additional equipment in addition to the microscope used for visualization. Such a system can be useful for understanding the kinetics of similar single molecule reactions. Finally, this system could also be integrated in a device that can take a single DNA molecule, stretch it, cut it at a specific location, and sequence the cut fragment. Such a device would serve as an excellent lowcost tool to expedite the pace of genomic sequencing.

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ACKNOWLEDGMENT We thank Yoshinori Yamaguchi (Department of Chemistry) and Madhavi Krishnan (Dept of Chemical Engineering) for their help. The work was supported by the NIH (R01-HG01406), NASA microgravity research (NAG3-2134) and the NSF (CTS-9987402).

Received for review February 23, 2003. Accepted May 27, 2003. AC034180H