In the Laboratory
Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy An Undergraduate Instrumental Analysis Laboratory Experiment Takashi Ito Department of Chemistry, Kansas State University, Manhattan, KS 66506;
[email protected] It is important for science and engineering students to understand the structural, physical, and chemical properties of DNA. DNA is an important molecule in biology because it contains the genetic codes that determine the sequence of amino acid residues in proteins (1). In addition, DNA is an important analyte in many criminal investigations (2, 3) and is also used in a variety of nanotechnologies (4, 5). The properties of DNA, including its structure, charge, and hybridization, are important to understand the functions and applications of DNA. Some of these properties have been studied using modern microscopic techniques such as electron microscopy and scanning probe microscopy (1, 6). From the educational viewpoint, hands-on experience in observing the structure of DNA using such microscopes will enhance students’ understanding of the properties of DNA. It is also important to introduce modern microscopic techniques to students because they are essential tools in contemporary nanoscience research. Previously, a number of undergraduate laboratory experiments using atomic force microscopy (AFM) (7–10), electron microscopy (11), and single-molecule fluorescence microscopy (12) were reported in this Journal. These experiments aim to provide students with hands-on experience in the operation of these modern microscopes, including detailed operation procedures. As a result, they are often designed as semester-long courses for elective, upper-level students (7) and thus are not suitable for regular undergraduate lab courses in which a number of different topics must be presented to a relatively large number of students during limited course hours. The experiments reported here were developed for an advanced undergraduate lab course that covers topics taught in instrumental analysis and physical chemistry courses. The experiments have the following educational objectives: (i) to enhance students’ understanding of the chemical and physical properties of double-stranded DNA (dsDNA) molecules; (ii) to teach the principles of fluorescence microscopy and AFM through observation of dsDNA molecules; and (iii) to demonstrate controlled manipulation of molecules, which is an important concept in nanotechnology. Because of the feasibility of the experiments for undergraduate students and the short lab hours, these experiments can be easily adapted to regular undergraduate laboratory courses. Materials and Methods Linear double-stranded Lambda Phage DNA (48,502 base pairs) is used because its pure solution is commercially available (e.g., from New England Biolabs). For fluorescence microscopy observations, dsDNA molecules are labeled with a fluorescent
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intercalator YOYO-1 (1 mM dimethylsulfoxide solution; purchased from Invitrogen; λex = 491 nm, λem = 509 nm). A Nikon TE2000 inverted optical–fluorescence microscope with an oil-immersion objective (100X; NA 1.4) is used (13). AFM measurements are performed by tapping mode in air, using a Digital Instruments Multimode scanning probe microscope (with Nanoscope IIIa electronics). Hazards Tris(hydroxymethyl)aminomethane (Tris) and magnesium chloride hexahydrate cause irritation upon inhalation, skin contact, or ingestion. Dimethylsulfoxide may cause irritation upon inhalation or skin contact. YOYO-1 has not been tested for toxicity and therefore should be handled with care. Direct exposure of the eyes to the excitation light in fluorescence microscopy should be avoided. Results dsDNA molecules are observed under four different conditions. The experiments can be completed by 2–4 students in a 4-hour lab period. However, if the lab hours permit, the experiments can be redesigned to two lab periods to give students more time to operate the microscopes. dsDNA Molecules in an Aqueous Solution Fluorescently labeled dsDNA molecules in an aqueous solution loaded on a glass coverslip (0.2 mm thick) are observed using fluorescence microscopy. Prior to the experiment, the instructor explains the setup of the fluorescence microscope, including light sources and lenses. The students are then allowed to experiment with the microscope to learn how to properly position the sample stage and how to obtain good images of the dsDNA. They will be able to directly observe the shape and motions of the dsDNA molecules in the solution through the eyepiece. Beyond simple observation of the molecules, the phenomenon of photobleaching is also discussed with the students and is demonstrated by irradiating the sample for a long period of time. dsDNA Molecules Directly Deposited on Glass By removing the aqueous dsDNA solution, dsDNA molecules are deposited on the glass coverslip. These dsDNA molecules are observed using fluorescence microscopy. Higher deposition efficiency of dsDNA in the presence of Mg2+ in the dsDNA solution demonstrates that Mg2+ mediates the adsorption of negatively-charged DNA onto a negatively-charged glass surface.
Journal of Chemical Education • Vol. 85 No. 5 May 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
dsDNA Molecules Immobilized on PDMS in Their Stretched Form Several dsDNA molecules directly deposited on the glass may be immobilized in partially stretched manners, but the possibility to observe such molecules is low. These dsDNA molecules are stretched due to surface tension at the edge of the drop on a surface. An efficient and simple immobilization method that produces dsDNA molecules in an extended conformation was reported by Nakao et al. (14). In this procedure, a polydimethylsiloxane (PDMS) surface is first covered with a dsDNA solution. Stretched dsDNA is then produced on the PDMS surface by removal of the aqueous phase using a micropipet. Here, Nakao’s method is slightly modified to immobilize stretched dsDNA molecules over a wider area: a drop of a dsDNA solution (12 μg∙mL dsDNA) is slowly moved on a PDMS substrate by tilting the substrate before the aqueous phase is removed using the micropipet (Figure 1A). In contrast to dsDNA molecules observed in aqueous solution or directly deposited, most of the dsDNA molecules are immobilized on the PDMS in their stretched forms (Figure 1B). However, some of the molecules are observed to be slightly bent. Based on these observations, students can recognize that dsDNA molecules are flexible polymers. Additionally, students can easily measure the length of stretched dsDNA molecules and compare their results with the length calculated from the size of the dsDNA and the spacing between adjacent base pairs (bp; 0.34 nm∙bp) (15). The dsDNA length reported by two separate groups of three students was 12.6 ± 6.5 μm. This is similar to that obtained by the instructor, 13.3 ± 5.1 μm (see the online supplement), and slightly shorter than the value of 16.5 μm that is estimated from the size of the dsDNA (48502 bp and 0.34 nm∙bp). The shorter length might result from incomplete stretching of the dsDNA molecules or rupturing of the dsDNA molecules during the immobilization process. The apparent width of the molecules is also measured in this experiment. The students find the width to be much larger than the expected value of 2–3 nm, thus providing a direct demonstration of the diffraction-limited spatial resolution of fluorescence microscopy (16).
A
DNA PDMS
tilt the substrate move the solution
stretched DNA
glass or mica
stretched DNA
press & transfer
stretched DNA
Figure 1. (A) Procedures used to immobilize stretched dsDNA molecules on PDMS. (B) Fluorescence image (recorded using a 100× oil-immersion objective lens) of stretched Lambda Phage DNA (bright lines) immobilized on PDMS. The dsDNA molecules were labeled with YOYO-1 (dye:base pair = 1:10). The image was obtained through a glass coverslip.
Stretched dsDNA Molecules Transferred onto Glass or Mica dsDNA molecules immobilized on PDMS are transferred onto glass or mica (Figure 1A) (14). The results of the transfer process are observed using fluorescence microscopy. The students estimate the transfer efficiency by comparing the densities of dsDNA molecules on the PDMS and glass surfaces. The dsDNA molecules transferred to mica are observed using AFM. For AFM, it is necessary to use a mica substrate instead of a glass coverslip to obtain an atomically flat surface. Tapping-mode AFM is used during imaging of the DNA to minimize the force between the tip and sample. Prior to the measurements, the instructor explains the principles and experimental setup of AFM. To complete all of the experiments within 4 hours, the instructor should mount a tip into the tip holder and align the laser for the students. Figure 2 shows a typical AFM image of stretched dsDNA molecules transferred from PDMS onto mica. The height and width of each dsDNA molecule are measured from these images and compared with
Figure 2. Tapping-mode AFM image (2 µm x 2 µm) of stretched Lambda Phage DNA molecules (bright lines) transferred from PDMS onto mica.
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In the Laboratory
the actual values and the values obtained from the fluorescence images recorded earlier. From this comparison, the students learn that AFM can resolve more detailed structure as compared with fluorescence microscopy and also that the lateral resolution of AFM is limited by the size of the AFM tip. Summary This article describes a series of lab experiments that allow students to observe single dsDNA molecules. Visualization of these biologically important molecules enhances student interest in the laboratory, as supported by comments from students who have performed this experiment. In addition, the experiments are suitable for teaching the chemical and physical properties of dsDNA as well as the methods of fluorescence microscopy and AFM. While the focus here is on dsDNA imaging, the experiments can also be expanded in other directions, including metal nanowire synthesis on the stretched DNA molecules (17). These experiments would be also applicable to introductory graduate-level laboratory courses and would be especially valuable to students employing these microscopic methods in their graduate research. Acknowledgments The author thanks Daniel A. Higgins (Department of Chemistry, Kansas State University) for his suggestions. The author gratefully acknowledges financial support from Kansas State University. Literature Cited 1. Voet, D.; Voet, J. G.; Pratt, C. W. Fundamentals of Biochemistry; John Wiley and Sons: Hoboken, NJ, 2006. 2. Millard, J. T.; Pilon, A. M. J. Chem. Educ. 2003, 80, 444– 446.
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3. Jackson, D. D.; Abbey, C. S.; Nugent, D. J. Chem. Educ. 2006, 83, 774–776. 4. Liedl, T.; Sobey, T. L.; Simmel, F. C. NanoToday 2007, 2, 36–41. 5. Niemeyer, C. M. NanoToday 2007, 2, 42–52. 6. Hansma, H. G. Annu. Rev. Phys. Chem. 2001, 52, 71–92. 7. Glaunsinger, W. S.; Ramakrishna, B. L.; Garcia, A. A.; Pizziconi, V. J. Chem. Educ. 1997, 74, 310–311. 8. Aumann, K.; Muyskens, K. J. C.; Sinniah, K. J. Chem. Educ. 2003, 80, 187–193. 9. Zhong, C.-J.; Han, L.; Maye, M. M.; Luo, J.; Kariuki, N. N.; Jones, W. E., Jr. J. Chem. Educ. 2003, 80, 194–197. 10. Lehmpuhl, D. W. J. Chem. Educ. 2003, 80, 478–479. 11. Eyring, L. J. Chem. Educ. 1980, 57, 565–568. 12. Zimmermann, J.; van Dorp, A.; Renn, A. J. Chem. Educ. 2004, 81, 553–557. 13. Ito, T.; Sun, L.; Crooks, R . M. Chem. Commun. 2003, 1482–1483. 14. Nakao, H.; Gad, M.; Sugiyama, S.; Otobe, K.; Ohtani, T. J. Am. Chem. Soc. 2003, 125, 7162–7163. 15. Hagerman, P. J. Ann. Rev. Biophys. Biophys. Chem. 1988, 17, 265–286. 16. Giambattista, A.; Richardson, B. M.; Richardson, R. C. College Physics; McGraw-Hill: New York, 2004. 17. Richter, J.; Mertig, M.; Pompe, W.; Monch, I.; Schackert, H. K. Appl. Phys. Lett. 2001, 78, 536–538.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/May/abs680.html Abstract and keywords Full text (PDF) with links to cited JCE articles Supplement
Student handout
Detailed experimental procedures
Journal of Chemical Education • Vol. 85 No. 5 May 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
