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In the Laboratory

It is important or science and engineering students tounderstand the structural, physical, and chemical propertieso DNA. DNA is an important molecule in biology becauseit contains the genetic codes that determine the sequence oamino acid residues in proteins (1). In addition, DNA is animportant analyte in many criminal investigations (2, 3) and isalso used in a variety o nanotechnologies (4, 5). Te propertieso DNA, including its structure, charge, and hybridization,are important to understand the unctions and applications oDNA. Some o these properties have been studied using modernmicroscopic techniques such as electron microscopy and scan-

ning probe microscopy(1, 6). From the educational viewpoint,hands-on experience in observing the structure o DNA usingsuch microscopes will enhance students understanding o theproperties o DNA.

It is also important to introduce modern microscopictechniques to students because they are essential tools incontemporary nanoscience research. Previously, a numbero undergraduate laboratory experiments using atomic orcemicroscopy (AFM) (710), electron microscopy (11), andsingle-molecule uorescence microscopy(12) were reported inthis Journal. Tese experiments aim to provide students withhands-on experience in the operation o these modern micro-scopes, including detailed operation procedures. As a result,

they are oen designed as semester-long courses or elective,upper-level students (7) and thus are not suitable or regularundergraduate lab courses in which a number o dierent topicsmust be presented to a relatively large number o students duringlimited course hours.

he experiments reported here were developed or anadvanced undergraduate lab course that covers topics taught ininstrumental analysis and physical chemistry courses. Te experi-ments have the ollowing educational objectives: (i) to enhancestudents understanding o the chemical and physical propertieso double-stranded DNA (dsDNA) molecules; (ii) to teachthe principles o uorescence microscopy and AFM throughobservation o dsDNA molecules; and (iii) to demonstrate

controlled manipulation o molecules, which is an importantconcept in nanotechnology. Because o the easibility o theexperiments or undergraduate students and the short lab hours,these experiments can be easily adapted to regular undergraduatelaboratory courses.

Materials and Methods

Linear double-stranded Lambda Phage DNA (48,502 basepairs) is used because its pure solution is commercially available(e.g., rom New England Biolabs). For uorescence microscopyobservations, dsDNA molecules are labeled with a uorescent

intercalator YOYO-1 (1 mM dimethylsuloxide solution;purchased rom Invitrogen; ex = 491 nm, em = 509 nm). ANikon E2000 inverted opticaluorescence microscope withan oil-immersion objective (100X; NA 1.4) is used (13). AFMmeasurements are perormed by tapping mode in air, using aDigital Instruments Multimode scanning probe microscope(with Nanoscope IIIa electronics).

Hazards

ris(hydroxymethyl)aminomethane (ris) and magnesium

chloride hexahydrate cause irritation upon inhalation, skincontact, or ingestion. Dimethylsuloxide may cause irritationupon inhalation or skin contact. YOYO-1 has not been testedor toxicity and thereore should be handled with care. Directexposure o the eyes to the excitation light in uorescence mi-croscopy should be avoided.

Results

dsDNA molecules are observed under our dierent condi-tions. Te experiments can be completed by 24 students in a4-hour lab period. However, i the lab hours permit, the experi-ments 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 aqueoussolution loaded on a glass coverslip (0.2 mm thick) are observedusing uorescence microscopy. Prior to the experiment, theinstructor explains the setup o the uorescence microscope,including light sources and lenses. Te students are then allowedto experiment with the microscope to learn how to properlyposition the sample stage and how to obtain good images othe dsDNA. Tey will be able to directly observe the shapeand motions o the dsDNA molecules in the solution throughthe eyepiece. Beyond simple observation o the molecules, the

phenomenon o photobleaching is also discussed with the stu-dents and is demonstrated by irradiating the sample or a longperiod o time.

dsDNA Molecules Directly Deposited on Glass

By removing the aqueous dsDNA solution, dsDNAmolecules are deposited on the glass coverslip. Tese dsDNAmolecules are observed using uorescence microscopy. Higherdeposition eciency o dsDNA in the presence o Mg2+ in thedsDNA solution demonstrates that Mg2+ mediates the adsorp-tion o negatively-charged DNA onto a negatively-chargedglass surace.

Observation of DNA Molecules Using FluorescenceMicroscopy and Atomic Force Microscopy

An Undergraduate Instrumental Analysis Laboratory ExperimentTakashi Ito

Department of Chemistry, Kansas State University, Manhattan, KS 66506; ito@ksu.edu

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In the Laboratory

dsDNA Molecules Immobilized on PDMSin Their Stretched Form

Several dsDNA molecules directly deposited on the glassmay be immobilized in partially stretched manners, but thepossibility to observe such molecules is low. Tese dsDNA mol-ecules are stretched due to surace tension at the edge o the dropon a surace. An ecient and simple immobilization method

that produces dsDNA molecules in an extended conormationwas reported by Nakao et al. (14). In this procedure, a polydim-ethylsiloxane (PDMS) surace is frst covered with a dsDNAsolution. Stretched dsDNA is then produced on the PDMSsurace by removal o the aqueous phase using a micropipet.

Here, Nakaos method is slightly modifed to immobilizestretched dsDNA molecules over a wider area: a drop o adsDNA solution (12 gmL dsDNA) is slowly moved on aPDMS substrate by tilting the substrate beore the aqueousphase is removed using the micropipet (Figure 1A). In contrastto dsDNA molecules observed in aqueous solution or directlydeposited, most o the dsDNA molecules are immobilized onthe PDMS in their stretched orms (Figure 1B). However, some

o the molecules are observed to be slightly bent. Based on theseobservations, students can recognize that dsDNA molecules areexible polymers. Additionally, students can easily measure thelength o stretched dsDNA molecules and compare their resultswith the length calculated rom the size o the dsDNA and thespacing between adjacent base pairs (bp; 0.34 nmbp) (15).Te dsDNA length reported by two separate groups o threestudents was 12.6 6.5 m. Tis is similar to that obtained bythe instructor, 13.3 5.1 m (see the online supplement), andslightly shorter than the value o 16.5 m that is estimated romthe size o the dsDNA (48502 bp and 0.34 nmbp). Te shorterlength might result rom incomplete stretching o the dsDNAmolecules or rupturing o the dsDNA molecules during theimmobilization process. Te apparent width o the molecules isalso measured in this experiment. Te students fnd the widthto be much larger than the expected value o 23 nm, thus pro-viding a direct demonstration o the diraction-limited spatialresolution o uorescence microscopy(16).

Stretched dsDNA MoleculesTransferred onto Glass or Mica

dsDNA molecules immobilized on PDMS are transerredonto glass or mica (Figure 1A) (14). Te results o the transerprocess are observed using uorescence microscopy. Te stu-dents estimate the transer eciency by comparing the densitieso dsDNA molecules on the PDMS and glass suraces.

Te dsDNA molecules transerred to mica are observedusing AFM. For AFM, it is necessary to use a mica substrateinstead o a glass coverslip to obtain an atomically at surace.apping-mode AFM is used during imaging o the DNA tominimize the orce between the tip and sample. Prior to themeasurements, the instructor explains the principles and ex-perimental setup o AFM. o complete all o the experimentswithin 4 hours, the instructor should mount a tip into the tipholder and align the laser or the students. Figure 2 shows atypical AFM image o stretched dsDNA molecules transerredrom PDMS onto mica. Te height and width o each dsDNAmolecule are measured rom these images and compared with

Figure 2. Tapping-mode AFM image (2 m x 2 m) of stretchedLambda Phage DNA molecules (bright lines) transferred from PDMSonto mica.

tilt the substratemove the solution

stretched

DNA

press &transfer

glass or mica

stretched DNA

DNA

PDMS

stretchedDNA

Figure 1. (A) Procedures used to immobilize stretched dsDNAmolecules on PDMS. (B) Fluorescence image (recorded using a 100oil-immersion objective lens) of stretched Lambda Phage DNA (bright

lines) immobilized on PDMS. The dsDNA molecules were labeled withYOYO-1 (dye:base pair = 1:10). The image was obtained througha glass coverslip.

A

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682 Journal of Chemical Education Vol. 85 No. 5 May 2008 www.JCE.DivCHED.org Division of Chemical Education

In the Laboratory

the actual values and the values obtained rom the uorescenceimages recorded earlier. From this comparison, the studentslearn that AFM can resolve more detailed structure as comparedwith uorescence microscopy and also that the lateral resolutiono AFM is limited by the size o the AFM tip.

Summary

Tis article describes a series o lab experiments that allowstudents to observe single dsDNA molecules. Visualization othese biologically important molecules enhances student interestin the laboratory, as supported by comments rom students whohave perormed this experiment. In addition, the experimentsare suitable or teaching the chemical and physical propertieso dsDNA as well as the methods o uorescence microscopyand AFM. While the ocus here is on dsDNA imaging, theexperiments can also be expanded in other directions, includ-ing metal nanowire synthesis on the stretched DNA molecules(17). Tese experiments would be also applicable to introduc-tory graduate-level laboratory courses and would be especiallyvaluable to students employing these microscopic methods in

their graduate research.

Acknowledgments

Te author thanks Daniel A. Higgins (Department oChemistry, Kansas State University) or his suggestions. Teauthor grateully acknowledges fnancial support rom KansasState University.

Literature Cited

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3. Jackson, D. D.; Abbey, C. S.; Nugent, D.J. Chem. Educ.2006,83,774776.

4. Liedl, .; Sobey, . L.; Simmel, F. C. NanoToday2007,2,3641.

5. Niemeyer, C. M.NanoToday2007,2, 4252.6. Hansma, H. G.Annu. Rev. Phys. Chem.2001,52, 7192.7. Glaunsinger, W. S.; Ramakrishna, B. L.; Garcia, A. A.; Pizziconi,

V.J. Chem. Educ.1997,74, 310311.8. Aumann, K.; Muyskens, K. J. C.; Sinniah, K.J. Chem. Educ.2003,

80, 187193.9. Zhong, C.-J.; Han, L.; Maye, M. M.; Luo, J.; Kariuki, N. N.; Jones,

W. E., Jr.J. Chem. Educ.2003,80, 194197.10. Lehmpuhl, D. W.J. Chem. Educ.2003,80, 478479.11. Eyring, L.J. Chem. Educ.1980,57, 565568.12. Zimmermann, J.; van Dorp, A.; Renn, A.J. Chem. Educ.2004,

81, 553557.13. Ito, .; Sun, L.; Crooks, R. M. Chem. Commun. 2003,

14821483.14. Nakao, H.; Gad, M.; Sugiyama, S.; Otobe, K.; Ohtani, .J. Am.

Chem. Soc.2003,125, 71627163.15. Hagerman, P. J.Ann. Rev. Biophys. Biophys. Chem.1988, 17,

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