A Dual-Beam Optical Microscope for Observation and Cleavage of

challenge in this research area is to use a laser beam to initiate or control chemical reactions at the single-molecule level. A second laser beam cou...
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Anal. Chem. 1998, 70, 1743-1748

A Dual-Beam Optical Microscope for Observation and Cleavage of Single DNA Molecules William A. Lyon, Michelle M. Fang, William E. Haskins, and Shuming Nie*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

An integrated epifocal and evanescent-wave optical microscope has been developed for real-time observation and bond cleavage studies of single DNA molecules. Large genomic DNA is stretched in a laminar flow stream and is immobilized on a polylysine-coated glass surface by strong electrostatic interactions. Unlike previous single-molecule dynamics and single-enzyme studies, this work takes advantage of the elastic nature of doublestranded DNA and measures DNA relaxation events that are triggered by two phosphodiester (P-O) bond breaks. The ability to follow chemical reactions on individual DNA molecules opens new possibilities for DNA mapping and for studying DNA-protein interactions. Single-molecule detection represents the ultimate limit in ultrasensitive chemical analysis1-10 and opens new possibilities for studying single-molecule dynamics and reactions.11-13 These studies overcome the problem of ensemble averaging and could provide structural and activity information that is not available from population-averaged measurements. Recent advances have observed single-molecule dynamics in free and restricted media14-16 and have studied the activity of single enzyme molecules.17,18 * Corresponding author; (e-mail) [email protected]. (1) Moerner, W. E. Science 1994, 265, 46-53, 1994. (2) (a) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553. (b) Goodwin, P. M.; Ambrose, W. P.; Keller, R. A. Acc. Chem. Res. 1996, 29, 607-613. (3) Betzig, E.; Chichester, R. J. Science 1993, 262, 1422-1425. (4) (a) Xie, X. S.; Dunn, R. C. Science 1994, 265, 361-364. (b) Xie, X. S. Acc. Chem. Res. 1996, 29, 598-606. (5) (a) Trautman, J. K.; Macklin, J. J.; Brus, L. E.; Betzig, E. Nature 1994, 369, 40-42. (b) Macklin, J. J.; Trautman, J. K.; Harris, T. D.; Brus, L. E. Science 1996, 272, 255-258. (6) Ha, T.; Enderle, Th.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6264-6268. (7) (a) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (b) Nie, S.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 2849-2857. (8) Barnes, M. D.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1995, 67, 418A423A. (9) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740-5747. (10) Cousino, M. A.; Jarbawi, T. B.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1997, 69, 544A-549A. (11) Keller, R. A.; Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Martin, J. C.; Wu, M. Appl. Spectrosc. 1996, 50, 12A-32A. (12) Nie, S.; Zare, R. N. Annu. Rev. Biophys. Biomol. Struc. 1997, 26, 565-594. (13) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555-559. (14) Xu, X.-H.; Yeung, E. S. Science 1997, 275, 1106-1109. (15) Dickson, R. M.; Norris, D. J.; Tzeng, Y.-L.; Moerner, W. E. Science 1996, 274, 966-969. (16) Schmidt, Th.; Schutz, G. J.; Baumgartner, W.; Gruber, G. J.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2926-2929. S0003-2700(98)00040-7 CCC: $15.00 Published on Web 03/20/1998

© 1998 American Chemical Society

Optical and mechanical measurements have also been demonstrated for single myosin molecular motors,13,19,20 single RNA polymerases,21 and single DNA molecules.22-25 A remaining challenge in this research area is to use a laser beam to initiate or control chemical reactions at the single-molecule level. A second laser beam could be used to detect or monitor singlemolecule events induced by the first laser. We report the development of an integrated epifocal and evanescent-wave optical microscope for direct observation and photocleavage of single DNA molecules. In this instrument, widefield evanescent illumination allows real-time observation of single DNA molecules, and a tightly focused laser beam initiates bond cleavage at desired positions along the DNA. While this experimental design has similarities with that used in laser microdissection of condensed human chromosomes,26 DNA cleavage events in our study can be triggered by only two phosphodiester (P-O) bond breaks, one on each DNA strand. This high sensitivity arises from the stretching and immobilization of single DNA molecules on a solid support. These DNA molecules relax to form a visible gap when two chemical bonds are cleaved. The use of linearly stretched DNA also improves the cut resolution to about 0.5-1.0 kbp, significantly better than that (∼1 million base pairs) reported by Berns and co-workers on metaphase chromosomes.26 In contrast, Gruelich and co-workers have used polycations such as polylysine and histone proteins to condense and aggregate λ-phage DNA into long and thick fibers.27 These fibrous bundles are visible in transmission optical microscopy and can be cut with a pulsed ultraviolet laser. (17) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681-682. (18) (a) Craig, D. B.; Arriaga, E.; Wong, J. C. Y.; Lu, H.; Dovichi, N. J. J. Am. Chem. Soc. 1996, 118, 5245-5253. (b) Craig, D. B.; Arriaga, E.; Wong, J. C. Y.; Lu, H.; Dovichi, N. J. Anal. Chem. 1998, 70, 39A-43A. (19) Spudich, J. A. Nature 1994, 372, 515-518. (20) Ishijima, A.; Doi, T.; Sakurada, K.; Yanagida, T. Nature 1991, 352, 301306. (21) Yin, H.; Wang, M. D.; Svoboda, K.; Landick, R.; Block, S. M.; Gelles, J. Science 1995, 270, 1653-1657. (22) (a) Perkins, T.; Smith, D. E.; Chu, S. Science 1994, 264, 819-822. (b) Perkins, T.; Quake, S. R.; Smith, D. E.; Chu, S. Science 1994, 264, 822826. (23) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795-799. (24) (a) Cluzel, P.; Lebrun, A.; Heller, C.; Lavery, R.; Viovy, J.-L.; Chatenay, D.; Caron, F. Science 1996, 271, 792-794. (b) Strick, T. R.; Allemand, J.-F.; Bensimon, D.; Bensimon, A.; Croquette, A. Science 1996, 271, 1835-1837. (25) Shi, X.; Hammond, R. W.; Morris, M. D. Anal. Chem. 1995, 67, 32193222. (26) (a) Berns, M. W.; Rounds, D. E. Sci. Am. 1970, 222, 98-110. (b) Berns, M. W.; Wright, W. H.; Steubing, R. W. Int. Rev. Cytol. 1991, 129, 1-44. (27) (a) Endlich, N.; Greulich, K. O. J. Biotechnol. 1995, 41, 149-153. (b) Ponelies, N.; Scheef, J.; Harim, A.; Leitz. G.; Greulich, K. O. J. Biotechnol. 1994, 35, 109-120.

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Figure 1. Schematic diagram of the dual-beam optical microscope for real-time observation and photocleavage of single DNA molecules.

Large DNA molecules are particularly well suited for exploring single-molecule chemical reactions because both observation and photocleavage involve multiple copies of a fluorescent tag. This should yield higher fluorescence intensities than a single fluorophore molecule. The ability to study chemical and biochemical reactions on large genomic DNA is important to gene mapping and fundamental studies of DNA-protein interactions.28,29 We also note that the present work is different from the dual-channel studies reported by Castro, Shera, Winefordner, and co-workers.30-32 Their results demonstrate that single molecules such as rhodamine dyes and DNA fragments can be sequentially detected by fluorescence and identified by electrophoretic mobility or twocolor optical detection. EXPERIMENTAL SECTION Instrumentation. Our dual-beam optical microscope was constructed by using an inverted Nikon microscope (Figure 1). Wide-field evanescent excitation was achieved with a prism by total internal reflection at the glass-liquid interface. A tightly focused laser beam was directed to the sample with an oil immersion objective (100×, NA ) 1.25) in the epi-illumination configuration (from below the microscope stage). Time-elapsed video images were acquired at 30 frames/s by using a video-rate intensified CCD camera (Photon Technology International, South Brunswick, NJ). These images were digitized by a high-speed frame grabber (Matrox, Dorval, Quebec, Canada) and analyzed by image-processing software (Matrox). A band-pass filter (560 DF 40, Chroma Tech, Brattleboro, VT) was placed in the optical path to reject the scattered laser light and to pass the (28) (a) Parra, I.; Windle, B. Nat. Genet. 1993, 5, 17-21. (b) Haaf, T.; Ward, D. C. Hum. Mol. Genet. 1994, 3, 629-633. (29) (a) Lilley, D. M. J., Ed. DNA-Protein Structural Interactions; IRL Press: New York, 1995. (b) Travers, A., DNA-Protein Interactions; Chapman and Hall Press: New York, 993. (30) Castro, A.; Shera, E. B. Anal. Chem. 1995, 67, 3181-3186. (31) Castro, A.; Williams, J. G. K. Anal. Chem. 1997, 69, 3915-3920. (32) Guenard, R. D.; King, L. A.; Smith, B. W.; Winefordner, J. D. Anal. Chem. 1997, 69, 2426-2433.

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Figure 2. Illustration of the DNA stretching and immobilization process in a laminar flow stream. See text for detailed discussion.

Stokes-shifted fluorescent light. The evanescent and epifocal laser beams were of the same wavelength at 488 or 514.5 nm, as provided by a continuous-wave (CW) argon ion laser (Omnichrome, Carlsbad, CA). The evanescent beam power density was on the order of 10 W/cm2, which was high enough for singlemolecule observation but not sufficient to cause DNA bond cleavage. The epifocal laser power density was ∼10 000 times higher, at 105 W/cm2. In addition to this intensity difference, the two laser beams could have different wavelengths (e.g., visible for observation and UV for bond cleavage) because they have entirely different optical paths. DNA Labeling and Stretching. Genomic T4 DNA molecules were stretched and immobilized on solid glass surfaces according to a hydrodynamic flow procedure. Glass cover slips were first cleaned in a sulfuric acid/hydrogen peroxide solution and coated with high-molecular-weight polylysine (MW ) 350 000). A 5.0µL aliquot of the polylysine solution (5.0 µg/mL) was spread evenly between two cleaned cover slips. Spontaneous adsorption of polylysine on glass was allowed to take place for 30-60 min. Fluorescent labeling of coliphage T4 DNA was achieved by using the intercalating dye POPO-3 (Molecular Probes, Eugene, OR).33 The DNA was labeled at a ratio of four base pairs per dye molecule (33) Glazer, A. N.; Rye, H. S. Nature 1992, 359, 859-861.

Figure 3. Real-time video images showing DNA stretching in a laminar flow stream and immobilization on a polylysine-coated glass surface. The time elapsed is 33 ms between images B and C and is 66 ms between images C and D. After the initial attachment, a DNA molecule is generally stretched and fixed in ∼100 ms. Variations in the stretched DNA length mainly occur at the attachment site where coiled DNA could exist.

by mixing an aliquot of DNA sample with a specific volume of freshly prepared 1 × 10-7 M dye solution. A small amount of the DNA mixture was diluted into a solution of 10 mM Tris buffer (pH 7.4) to obtain a final concentration of ∼10-10 M DNA. Approximately 5 µL of this diluted DNA sample was deposited on an uncoated cover slip, and then a coated cover slip was placed on top to initiate laminar liquid flow. This step resulted in both stretching and fixing of DNA molecules on the coated surface. The effect of the adsorbed polylysine density was studied by varying the polylysine concentration from 1 mg to 1 ng/mL of solution. This study showed that DNA molecules were best stretched into long and straight lines at a polylysine concentration of 5 µg/mL (further discussion in next section). To reduce photobleaching, approximately 10-100 mM mercaptoethanol was added prior to optical imaging. Reagents. All chemicals and biochemicals used in this work were obtained from commercial sources. Poly(L-lysine hydrobromide) (MW ) 350 000), Trizma base (tris(hydroxymethyl)aminomethane), Trizma hydrochloride (tris(hydroxymethyl)aminomethane hydrochloride), 2-mercaptoethanol, and coliphage T4 DNA were purchased from Sigma Chemical Co. (St. Louis, MO). Stock POPO-3 in dimethyl sulfoxide (DMSO) was obtained from Molecular Probes (Eugene, OR) and was diluted in Tris buffer (pH 7.4) immediately before use. Microscope cover slips (0.13 mm thick) were purchased from Fisher Scientific (Pittsburgh, PA). Ultrapure water was prepared by a Milli-Q purification system (Millipore, Bedford, MA). RESULTS AND DISCUSSION Single-Molecule Manipulation. Large DNA molecules are stretched into linear forms by the hydrodynamic frictional force

in laminar liquid flow, as shown schematically in Figure 2. This frictional force is dependent on several factors including liquid flow speed, solution viscosity, and DNA length.34 Considering T4 DNA (∼167 000 base pairs) as a long rod, we estimate that the stretching force is about 4-5 pN at a flow velocity of 0.5 mm/s in 10 mM Tris buffer.35 A key parameter is the polylysine charge density on the glass surface: at high values, DNA molecules tend to condense and fix on the surface too strongly; at low values, stretched DNA molecules often move freely and do not bind to the surface. The optimized density is ∼50 polylysine molecules/ µm2, at which T4 DNA is consistently stretched with a length variation below 5%. Recent laser trapping studies have measured the mechanical properties of single DNA molecules.23-25 The results show that the B-form DNA double helix can be stretched to its full contour length (0.34 nm/base pair) at a force of ∼5 pN. Double-helix DNA can be further stretched to 1.7 times its contour length at a force plateau of 80 pN, at which the DNA stretches and contracts at an almost constant force. To mechanically break a double-helical DNA, stretching forces as large as 500-1000 pN are required. Thus, the liquid flow forces in our experiment are sufficient for DNA stretching but not strong enough for fragmentation. However, for very large DNA molecules in the size range (34) Van Holde, K. E. Physical Biochemistry; Prentice-Hall: Englewood Cliffs, NJ, 1995. (35) The frictional force (F) was calculated according to the following equation:

F ) 6 πην

( ) 3r 2L 4

(rL/2)2/3

1/3

(3/2)

1/3

{2ln(L/r - 0.11)}

where r is the radius of DNA (∼0.5 nm), η is the solution viscosity (∼1.0 cP in 10 mM Tris buffer), ν is the liquid flow velocity, and L is the DNA chain length.

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of 1-5 million base pairs, we found that the turbulent flow or shear force can cause DNA fragmentation. Surface immobilization of the stretched DNA involves strong electrostatic interactions between the negatively charged phosphate groups on DNA and the positive charges on the modified surface. The basic double-helical structure of duplex DNA should be maintained in the immobilized state, which would allow one or two phosphate groups to contact the surface for every helical turn (see Figure 2). This geometry makes every other minor or major groove available for protein binding (i.e., not blocked by the surface). Indeed, recent studies have shown that restriction enzymes can still bind and cleave at specific sites on electrostatically immobilized DNA.36 Furthermore, the positively charged surface could mimic the in vivo environment of genomic DNA, which is bound to histone proteins via electrostatic interactions.37 Other research groups have also reported the use of positively charged surfaces to stretch genomic DNA, but the exact mechanism of stretching is still unclear.36,38 A possible explanation is that laminar liquid flow has a parabolic velocity profile and that DNA attachment occurs at the solution-glass interface where the flow velocity is zero. Since the two ends of a DNA molecule are free to move and the middle sections are restrained by the chain, the 5′- and 3′-ends should have a higher probability of contacting and attaching to the surface. Real-time video imaging reveals that as soon as a freely moving DNA molecule is attached to the surface by one end, the whole DNA is rapidly stretched and fixed on the surface in a “zippering” fashion (Figure 3). If the initial attachment occurs at a nonterminal site, the DNA is stretched and fixed in the form of a curved line. Real-Time Observation. With wide-field evanescent excitation at the glass-liquid interface, the stretched DNA molecules are observed with an intensified CCD camera at 30 frames/s. As shown in Figure 4, these molecules appear straight and continuous before laser cutting. An apparent blurring effect in the video image is caused by the CCD intensifier, which spreads diffractionlimited signals into a number of pixels. Several lines of evidence indicate that the fluorescence signals arise from single DNA molecules and not from DNA bundles. First, the observed density of single DNA molecules on the surface is consistent with the value calculated from the total number of added DNA molecules and the total surface area. Second, cross-sectional line plots show that all DNA molecules have similar intensities (Figure 4, lower part), which would not be the case if various DNA fiber bundles exist. Third, the measured physical lengths (∼59 µm) are only slightly larger than the calculated contour length for T4 DNA (56.8 µm). This difference is most likely to arise from an increase in base pair separation upon dye intercalation39 as well as a possible overstretching effect.23-25 In comparison, the thick fibrous bundles of λ-phage DNA (∼16 µm monomer length) formed in the presence of DNA-condensing polycations are several hundred micrometers in length.27 (36) (a) Meng, X.; Benson, K.; Chada, K.; Schwartz, D. C. Nat. Genet. 1995, 9, 432-438. (b) Cai, W.; Aburatani, H.; Stanton, V. P.; Housman, D. E.; Wang, Y.-K.; Schwartz, D. C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5164-5168. (37) Van Holde, K. E. Chromatin; Springer-Verlag Press: New York, 1988. (38) Bensimon, A.; Simon, A.; Chiffasudel, A.; Croquette, V.; Heslot, F.; Bensimon, D. Science 1994, 265, 2096-2098. (39) Coury, J. E.; McFail-Isom, L.; Williams, L. D.; Bottomley, L. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12283-12286.

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Figure 4. (Upper) Video-rate fluorescence images of single T4 genomic DNA molecules stretched and immobilized on a polylysinecoated glass cover slip. (Lower) Quantitative line plot of fluorescence intensities to illustrate the similarity among DNA molecules.

Photocleavage. Figure 5 shows a series of fluorescence images of a single T4 DNA molecule being cleaved with a focused laser beam at 514.5 nm. In frame B, the cutting laser beam was turned on and positioned on the stretched DNA at a specific location. After ∼100 ms, frame C showed that laser-induced scission had occurred and the stretched DNA started to recoil at the cleavage site. In frame D, the cutting laser beam was turned off to show the final gap, whose width was determined by the nearest DNA attachment sites on the surface. This process is schematically shown in Figure 6. It should be pointed out that the gap was formed by sudden DNA structural relaxation and not by gradual photobleaching of the POPO dye molecules (about 100-200) in the focused laser beam. The precision of photocleavage is fundamentally limited by optical diffraction to about half of the wavelength of the light (λ/ 2). This is because the intercalation dye is not sequence specific and strand breaks are expected to occur randomly in an irradiated segment of ∼300 nm on the stretched DNA. Substantial improvements may be achieved by using near-field optical probes40 or sequence-specific molecules such as DNA-binding proteins and synthetic oligonucleotides (triple- helix formation).41 With the use of dry surfaces to reduce the diffusion of photogenerated species, this method should allow high-resolution cleavage of large DNA molecules at selected sites. Schwartz and co-workers36 have used fluorescence microscopy and restriction enzymes for rapid optical mapping of single DNA molecules; in their approach, stretched (40) Betzig, E.; Trautman, J. K. Science 1992, 257, 189. (41) (a) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-650. (b) Best, G. C.; Dervan, P. B. J. Am. Chem. Soc. 1995, 117, 1187-1193.

Figure 5. Time-elapsed video images of a single T4 DNA molecule being cleaved by a focused laser beam. The cutting exposure time is ∼100 ms. The scattered laser light from the cutting beam is not completely removed and indicates the initial position of irradiation.

Figure 6. Schematic illustration of structural relaxation and gap formation triggered by the photocleavage of phosphodiester (P-O) bonds on double-stranded DNA.

DNA molecules are cut with an enzyme, and the order and length of DNA fragments are determined directly from fluorescence imaging. We believe that the cleavage mechanism is photochemical in nature and not photothermal. The evidence is that radical scavengers such as mercaptoethanol at high concentrations can inhibit photocleavage. This scavenger effect would not be observed for a photothermal mechanism. The active species are photogenerated via the intercalated dye and are believed to be singlet oxygen (O21) and hydroxyl (OH•) radicals. These molecules can initiate DNA strand scission by abstracting hydrogen atoms from the 5′-ribose carbon.42 Recent research in Akerman’s

group43 has shown that bis-intercalating dyes such as YOYO and POPO can induce both single-stranded and double-stranded breaks. When intercalated in duplex DNA, these dimeric dyes show enhanced activities for double-strand cleavage in an O2independent manner. Therefore, the observed DNA relaxation event could be triggered either by one double-stranded cut or by two adjacent single-stranded cuts, one on each strand. In the latter case, however, a large number of single-stranded cuts could accumulate in the irradiated region before the mechanical forces overcome the Watson-Crick hydrogen bonds between two adjacent cuts. Prospects and Applications. This work may be extended to study other photoactive molecules such as “caged” fluorescent probes.44 A short-wavelength laser can be used to activate a single fluorophore, and a long-wavelength laser probes its diffusion and photophysical behavior. The method and instrumentation also opens new possibilities for studying sequence-specific interactions of nucleic acids and proteins, which are important to many biological processes such as DNA replication, transcription, and recombination. In particular, it should be possible to observe the movement and function of single protein molecules on a stretched DNA. DNA-binding proteins of particular interest include endonucleases, gene transcription factors, and polymerases. For such complex biomolecular systems, single-molecule studies could provide fundamental insights that are not available from conventional, population-averaged measurements. Specifically, dual-beam single-molecule studies may be applied to study (i) how DNA-binding proteins find their sequence-specific site in a sea of nonspecific binding sites,45 (ii) how RNA poly(42) Sigman, D. S.; Mazumder, A.; Perrin, D. M. Chem. Rev. 1993, 93, 22952316. (43) Akerman, B.; Tuite, E. Nucleic Acids Res. 1996, 24, 1080-1090. (44) McCray, J. A.; Trentham, D. R. Annu. Rev. Biophys. Chem. 1989, 18, 239. (45) (a) Halford, S. E. Trends Biochem. Sci. 1983, 8, 455-460. (b) Rosenberg, J. M.; McClarin, J. A.; Frederick, C. A.; Wang, B.-C.; Grable, J.; Boyer, H. W.; Greene, P. Trends Biochem. Sci. 1987, 12, 395-398.

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merases move on a template DNA during active transcription,46 and (iii) how the transcription-regulatory proteins assemble and move on genomic DNA.47 A recent study by Yin et al.21 has shown that a single RNA polymerase molecule can generate a pulling force as large as 14 pN, enough to overcome the opposing force of transcription-induced DNA supercoiling (∼6 pN). High-speed optical imaging of single molecules will also extend and complement atomic force microscopy (AFM), which has been used to visualize single protein molecules bound at specific DNA sites.48,49 In conclusion, we have demonstrated the feasibility of dualbeam “pump/probe” studies at the single-molecule level. Large genomic DNA molecules are mechanically stretched by hydrodynamic flow forces and are electrostatically immobilized on polylysine-coated glass cover slips. The stretched DNA molecules (46) Nudler, E.; Goldfarb, A.; Kashlev, M. Science 1994, 265, 793-396. (47) Nikolov, D. B.; Burley, S. K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 15-22. (48) (a) Bustamante, C.; Rivetti, C. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 395-429. (b) Erie, D. A.; Yang, G.; Schultz, H. C.; Bustamante, C. Science 1994, 266, 1562-1466. (49) Allison, D. P.; Kerper, P. S.; Doktycz, M. J.; Spain, J. A.; Modrich, P.; Larimer, F. W.; Thundat, T.; Warmack, R. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8826-8829.

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are visualized by wide-field evanescent excitation and are cleaved by a tightly focused laser beam in the epi configuration. In contrast to previous laser microdissection of condensed DNA and human chromosomes, the photocleavage events in this study are triggered by as few as two phosphodiester bond cuts. In addition to improving the base pair resolution of DNA cutting, this work will have applications in gene mapping and fundamental studies of DNA-protein interactions. ACKNOWLEDGMENT We are grateful to Steven Emory for valuable discussions and to Jason Taylor for assistance in DNA stretching. This work was supported in part by the National Science Foundation (Grant CHE9610254). M.M.F. is a Harry Day Summer Research Scholar at Indiana UniversitysBloomington. S.N. acknowledges the Whitaker Foundation for a Biomedical Engineering Award and the Beckman Foundation for a Beckman Young Investigator Award. Received for review January 12, 1998. Accepted February 19, 1998. AC980040+