fingerprinting of single viral genomes

ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 337 (2005) 278–288

www.elsevier.com/locate/yabio

Fingerprinting of single viral genomes

Matthew M. Ferrisa,1, Thomas M. Yoshidab, Babetta L. Marronea, Richard A. Kellera,¤

a Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USAb Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA

Received 31 August 2004Available online 8 December 2004

Abstract

We demonstrate the use of technology developed for optical mapping to acquire DNA Wngerprints from single genomes for thepurpose of discrimination and identiWcation of bacteria and viruses. Single genome Wngerprinting (SGF) provides not only the sizebut also the order of the restriction fragments, which adds another dimension to the information that can be used for discrimination.Analysis of single organisms may eliminate the need to culture cells and thereby signiWcantly reduce analysis time. In addition, sam-ples containing mixtures of several organisms can be analyzed. For analysis, cells are embedded in an agarose matrix, lysed, and pro-cessed to yield intact DNA. The DNA is then deposited on a derivatized glass substrate. The elongated genome is digested with arestriction enzyme and stained with the intercalating dye YOYO-1. DNA is then quantitatively imaged with a Xuorescence micro-scope and the fragments are sized to an accuracy 790% by their Xuorescence intensity and contour length. Single genome Wnger-prints were obtained from pure samples of adenovirus, from bacteriophages � and T4 GT7, and from a mixture of the three viralgenomes. SGF will enable the Wngerprinting of uncultured and unampliWed samples and allow rapid identiWcation of microorgan-isms with applications in forensics, medicine, public health, and environmental microbiology.Published by Elsevier Inc.

Keywords: Single genome; Virus; DNA; Fingerprint; Fluorescence microscopy; Imaging

Restriction Wngerprinting has become a widespreadanalysis tool utilized for medical diagnostics, foodsafety, and bioforensics [1–3]. This molecular genomicstechnique creates genetic Wngerprints by sizing DNAfragments produced by enzymatic digestion of genomicDNA with a restriction endonuclease. MacrorestrictionWngerprinting is a speciWc type of Wngerprinting thatuses rare-cutting restriction enzymes to produce fewer,larger DNA fragments per genome [4]. Most commonly,gel electrophoresis methods are employed to size theDNA fragments comprising a restriction Wngerprint.Electrophoresis methods separate restriction fragmentsaccording to size-dependant electrophoretic mobility

* Corresponding author. Fax: +1 505 663 3024.E-mail address: [email protected] (R.A. Keller).

1 Present address: Particle Measuring Systems, Boulder, CO 80301,USA.

0003-2697/$ - see front matter. Published by Elsevier Inc.doi:10.1016/j.ab.2004.10.050

diVerences under an applied electric Weld while in a poly-mer matrix such as agarose. Once separated, fragmentsare stained and imaged to produce a characteristicWngerprint. Pulsed-Weld gel electrophoresis (PFGE)2 is aspeciWc example of such an electrophoresis techniquethat is used to size large (>20 kb), macrorestriction frag-ments [5].

The primary limitations of gel-based electrophoreticsizing methods are the long separation times and the rel-atively large amount of DNA required for analysis. Forexample, PFGE typically requires 7200 ng of DNA persample and approximately 20 h to produce a Wngerprint

2 Abbreviations used: SGF, single genome Wngerprinting; PFGE,pulsed-Weld gel electrophoresis; PCR, polymerase chain reaction; AP-DEMS, aminopropyldiethoxymethylsilane; S/N, signal-to-noise ratio;EMBL-EBI, European Molecular Biology Laboratory-European Bio-informatics Institute; CCD, charge-coupled device.

mailto: [email protected]

mailto: [email protected]

Fingerprinting of single viral genomes / M.M. Ferris et al. / Anal. Biochem. 337 (2005) 278–288 279

[5,6]. Despite these shortcomings, PFGE is the gold stan-dard method for macrorestriction Wngerprinting and iscurrently used by the Centers for Disease Control andPrevention in their PulseNet, National Molecular Sub-typing Network for Foodborne Disease Surveillanceprogram [7].

Several techniques have emerged as alternatives to gelelectrophoresis for DNA fragment sizing. Examples ofthese techniques include mass spectrometry [8,9], capil-lary and microchannel electrophoresis [10–15], andmicroXuidic entropic traps [16,17]. Each of these meth-ods provides notable advantages, relative to PFGE, inreducing both the amount of DNA required for analysisand the analysis time. However, none of these approacheshave demonstrated the fragment sizing range requiredfor macrorestriction Wngerprinting of bacteria.

We have developed an ultrasensitive Xow cytometricmethod that provides another alternative to gel electro-phoresis for DNA fragment sizing ([18] and referencestherein). Users of Xow cytometry have made consider-able progress in overcoming the primary limitations ofPFGE in macrorestriction Wngerprinting of bacteria.SpeciWcally, we have shown ultrasensitive Xow cytome-try to have a fragment sizing range comparable to thatof PFGE and to be capable of producing macrorestric-tion Wngerprints from as few as 5 to 10 completegenomes in only 30 min with similar precision and accu-racy [6,19]. While these milestones make Xow cytometryan attractive alternative to PFGE for samples requiringrapid Wngerprinting when very little DNA is available,the need for analytical methods that further reduce theamount of DNA required for Wngerprinting exists.

Bioforensic, medical, and environmental applicationswould beneWt from the ability to acquire Wngerprintsfrom single or very few genomes. Analysis at the singlegenome (e.g., single cell or single virus) level reduces thetime-consuming need to culture samples and enablesmixtures of organisms to be Wngerprinted without priorseparation. Single genome Wngerprinting (SGF) willprove useful for analysis of slow-growing and uncultur-able bacteria, which make up »99% of the bacteria innaturally occurring ecosystems [20].

Optical mapping [21–24], developed by DavidSchwartz and colleagues, is a Xuorescence-microscopy-based technique with the ability to analyze DNAfragments. BrieXy, optical mapping analyzes DNA mole-cules that have been deposited and stretched on amodiWed glass substrate and digested with rare-cuttingrestriction enzymes. Fluorescence microscopy and subse-quent image analysis of DNA fragments, each contain-ing multiple restriction sites, are used to produce anoptical map (i.e., consensus restriction map). Opticalmaps, generally created from >50£ coverage of thegenome of interest, were applied to whole-genome char-acterization in large-scale sequencing of several bacteria[25–31].

Unlike macrorestriction Wngerprints created byPFGE or ultrasensitive Xow cytometry, sizing of singlegenomes mounted to glass substrates provides morethan just the average restriction fragment size. Becausethe modiWed glass substrate used to mount DNA mole-cules preserves the order of each fragment within thedeposited DNA, there is an additional level of informa-tion (i.e., order of the restriction fragments in thegenome) [22]. The preservation of fragment order alsoassures that adjacent restriction fragments within amounted DNA molecule can be attributed to a singlegenome. This unique feature provides a huge advantagerelative to PFGE-, Xow-cytometry-, and polymerase-chain-reaction (PCR)-based methods for bacterial sub-typing in that it allows mixtures of organisms to beexamined without prior separation.

Here, we describe an adaptation of optical mappingtechniques to obtain ordered restriction Wngerprints ofsingle viral genomes. SGF results for pure and mixedsamples of adenovirus, bacteriophage �, and bacterio-phage T4 GT7 are compared to sequence-derived frag-ment size and order to demonstrate the accuracy andreproducibility of this method. We also present a displayformat that utilizes the fragment order informationinherent to our SGF data. Single genome Wngerprintinghas applications in bioforensic discrimination, medicaldiagnostics, and food safety. The possibility of incorpo-rating SGF into a microXuidic platform will fully realizethe beneWts of single-genome and single-cell analyses.

Materials and methods

Substrate preparation

Borosilicate cover glass (No. 1.5, 24 £ 50 mm; Corn-ing, Corning, NY) was derivatized following a modiWedprocedure developed by Cai et al. [25]. The most impor-tant part of this substrate preparation is thorough clean-ing of the glass prior to derivatization. Substrates wereWrst cleaned in freshly prepared piranha solution (4:1mixture of sulfuric acid and hydrogen peroxide; Sigma,St. Louis, MO) for approximately 20 min at 100 °C.(Caution! Piranha solution is dangerous and extremecaution should be used in its preparation, use, and stor-age). Cleaned glass substrates were rinsed with 18 M�water until the eZuent reached a pH of »7 (approxi-mately 15 min) before being modiWed by 3-aminopro-pyldiethoxymethylsilane (APDEMS; Gelest, Morrisville,PA). APDEMS modiWcation was accomplished by Wrsthydrolyzing the APDEMS in water for 1 h and thenincubating cleaned substrates in a freshly prepared solu-tion of 5.2 �M APDEMS in absolute ethanol (PharmcoProducts, BrookWeld, CT) for at least 48 h at room tem-perature. All APDEMS solutions were prepared andused in polyethylene containers. Substrates were stored

280 Fingerprinting of single viral genomes / M.M. Ferris et al. / Anal. Biochem. 337 (2005) 278–288

in the 5.2 �M APDEMS solution for no more than 2weeks before use. Immediately before use, substrateswere rinsed in absolute ethanol and allowed to air dry.

DNA, deposition, restriction, and staining

DNA samples were deposited on the silane-modiWedglass substrates for analysis. �X174 virion DNA(5386 bp; New England Biolabs, Beverly, MA), adenovi-rus DNA (35,937 bp, type 2; Invitrogen, Carlsbad, CA), �phage DNA (48,502 bp; New England Biolabs), and bac-teriophage T4 GT7 DNA (165,648 bp, Wako/NipponGene, Japan) were each diluted in an 1£ TE buVer(10 mM Tris–HCl, 1 mM EDTA at pH 8.0; Sigma) andstored at 4 °C until deposition. Approximately 10�l ofsolution (»25 ng/ml DNA) was applied to the interface ofa derivatized cover glass and a cleaned plain glass micro-scope slide. Mixtures of multiple viral genomes were pre-pared and diluted in 1£ TE buVer so that each genomewas equally represented at a Wnal DNA concentration of»25 ng/ml. The wicking action of the solution and over-night drying at room temperature provide an advancingand receding meniscus that served to elongate single mol-ecules of DNA as they adhered to the substrate. Oncedry, the cover glass was peeled oV of the slide beforedigestion with a restriction enzyme and/or staining.

Restriction endonuclease digestion of substrate-mounted DNA samples was accomplished using 10–50Units of restriction enzyme (XbaI, XhoI, PmeI, orEcoRI; New England Biolabs) in »500 �l of manufac-turer-recommended buVer for each 24 £ 50-mm coverglass. Restriction reactions were carried out for »2 h,with gentle shaking, in a humidiWed chamber maintainedat the appropriate temperature before rinsing each sub-strate with 1£ TE buVer to halt the reaction. Oncerinsed, samples were ready for staining.

Each substrate-mounted DNA sample was thenstained with 10 �l of 1£ YOYO-1 (Molecular Probes,Eugene, OR) and allowed to incubate in the dark atroom temperature for »15 min before analysis.

Imaging system

All samples were imaged using a Zeiss Axiophot Xuo-rescence microscope coupled to a cooled 12-bit CCDcamera (CE200A; Photometrics, Tucson, AZ). Thismicroscope used a 63£ oil immersion Plan NeoXuarobjective (Carl Zeiss, Germany), a 200-W Hg/Xe short-arc lamp (OptiQuip, Highland Mills, NY) for excitation,and a YOYO-1 speciWc Wlter set (41001; Chroma Tech-nology, Rockingham, VT) for spectral Wltering. Filteredexcitation irradiance, measured just prior to the objec-tive, was »500 �W/cm2. A commercial software package(ISee Imaging Systems, Raleigh, NC) was used for CCDimage acquisition and to control the microscope’s elec-tronic systems.

Image processing

Fluorescence images were analyzed using a commer-cial software package (Image-Pro Plus; Media Cybernet-ics, Silver Spring, MD). Raw CCD images were Wrstconverted to 12-bit Xoating-point images and correctedfor illumination shading eVects (i.e., inhomogeneous illu-mination). For each image, the mean background signal(»2000 counts on average) was then subtracted fromeach pixel to normalize the background and produce amean background signal of zero. Following correctionand normalization, Xuorescence images of surface-mounted DNA fragments had a typical signal-to-back-ground ratio of »500 and a signal-to-noise ratio (S/N) of715. DNA fragments of interest within images werevisually identiWed, using fragment alignment geometryand pattern repetition as selection criteria, prior to ana-lytical quantiWcation. QuantiWcation of each DNA frag-ment’s length and Xuorescence intensity requireddeWning the boundaries of the fragment. Fragmentboundaries were determined through image segmenta-tion involving thresholding to create a binary image thatwas then manipulated using a dilation Wlter to producean image mask deWning fragment boundaries. Pixel val-ues within the deWned fragment boundaries weresummed to determine the integrated Xuorescence inten-sity of each fragment.

Data analysis

Quantities extracted from processed images (e.g.,fragment length, width, and integrated Xuorescenceintensity) were exported to Microsoft Excel (Microsoft,Redmond, WA) for analysis. Sigma Plot (version 8.0;SPSS, Chicago, IL) was used for graphical and curve-Wtting analysis.

Virtual digests

Virtual (i.e., in silico) restriction digests of each viralgenome were used to provide the sequence-predictedfragment size of each restriction fragment and the orderof the fragments. These virtual digests serve as referencesto evaluate Wngerprint accuracy. Nucleotide sequencesfor each of the genomes used in this study were acquiredonline (www.ebi.ac.uk/genomes/) from the EuropeanMolecular Biology Laboratory-European Bioinformat-ics Institute (EMBL-EBI) database (Accession Nos.�X174, AF176027; Adenovirus type 2, J01917; bacterio-phage �, J02459; bacteriophage T4, AF158101). TheEMBL-EBI sequence for bacteriophage T4 was edited toaccount for the 3.256-kb deletion (between sites 165,251and 168,506) reported by the supplier of the T4 GT7sample used in this study. A commercially available soft-ware package (Kodon; Applied Maths, Austin, TX) wasused to parse each genome’s DNA sequence with the

http://www.ebi.ac.uk/genomes/

http://www.ebi.ac.uk/genomes/


known recognition sequence of each restriction endonu-clease enzyme to create ordered virtual digests.

Results and discussion

Deposition and imaging

There are several published reports of DNA samplesof various sizes being deposited onto modiWed glass sub-strates [21,32–36]. We adopted and modiWed existingmethods to mount viral genomes on APDEMS-modiWedglass substrates. Fig. 1 contains representative Xuores-cence images of multiple intact viral genomes mounted,in a stretched conformation, to such substrates afterstaining with YOYO-1. Each image also shows smallerDNA fragments produced by random shearing of geno-mic DNA prior to deposition. For the genomes used inthis study, we estimate that we deposit »50% of theDNA as intact genomes. We have also found that thesuccess rate associated with mounting intact genomes

decreases signiWcantly as genome size increases forgenomes over 50 kb. This is primarily due to shearingforces linked with preparation and deposition of thegenomic DNA. The images shown in Fig. 1 demonstrateour ability to produce modiWed glass substrates capableof binding DNA in a stretched conformation and todeposit DNA onto these substrates for single-moleculeimaging. The viral genomes used in this study rangefrom a few thousand basepairs (�X174, 5386 bp) to hun-dreds of thousands of basepairs (T4 GT7, 165,648 bp).

Each viral genome used in this study, and shown inFig. 1, was imaged multiple times (N 7 78) in multipleWelds of view to evaluate both contour length and inte-grated Xuorescence intensity for sizing substrate-mountedDNA. Following correction and normalization, extractedimage data were used to produce calibration curves (notshown) correlating contour length and integrated Xuo-rescence intensity to the known viral genome size inbasepairs. Both length and Xuorescence data were foundto be linear over the size range investigated (approxi-mately 5–165 kb) and linear regression Wts to the data

Fig. 1. Fluorescence images of uncut viral DNA [(A) �X174 (5386 bp); (B) adenovirus (35,937 bp); (C) bacteriophage � (48,502 bp); (D) bacterio-phage T4 GT7 (165,648 bp)] mounted on modiWed glass substrates. Each representative image shows Xuorescence from hydrodynamically depositedDNA stretched on the glass substrate and stained with YOYO-1 nucleic acid stain. Smaller randomly sheared DNA fragments that were codepositedwith the intact genomic DNA can also be seen. The scale bars in the upper left corner of each image represent 10 �m.


were obtained with correlation coeYcients (R2) of 0.99for both the contour length and the integrated Xuores-cence data. As expected [37], both contour length andintegrated Xuorescence intensity are good representa-tions of the relative fragment size for double-strandedDNA genomes. Furthermore, the reciprocal slope fromthe linear regression Wt to the contour length data(2.70 § 0.07 kb/�m) provides a quantitative measure ofDNA stretching on our substrates. This stretching coeY-

cient is reasonable and agrees with previously reportedliterature values (»2.3 kb/�m) of DNA stretching coeY-

cients on modiWed glass surfaces [23,32]. Our contourlengths show that our surface modiWcation and deposi-tion conditions produce a more relaxed DNA moleculethan that reported in the literature [38], reducing thechance of random, undesired fragmentation.

While the calibration data presented above demon-strate that both contour length and integrated Xuores-cence intensity can be used to size DNA molecules, itshould be noted that the data used to produce these cor-relation plots are representative only of uncut DNAsamples mounted on a single substrate. In general, it isour experience that Xuorescence intensity measurementsare preferred over contour length. This is primarily dueto the observation that contour length measurements aremore susceptible to errors associated with slight changesin both surface modiWcation and deposition conditions.In both cases, small and usually uncontrollable changescan result in signiWcant diVerences in the stretching ofDNA molecules. Additionally, Xuorescence intensity ispreferred when sizing restriction fragments rather thanintact genomic DNA for reasons that are discussedbelow. Photobleaching is one source of error that mustbe considered when using Xuorescence intensity mea-surements quantitatively. In this regard, we determined(data not shown) that photobleaching of YOYO-1-stained DNA immobilized on glass does not signiWcantlycontribute to DNA sizing errors when care was taken tominimize any sample exposure prior to quantitativeimaging. Samples were irradiated for several secondswithout signiWcant photobleaching. We did not Wnd itnecessary to use �-mercaptoethanol or other “anti-fade”agents to obtain the results reported here. For thesereasons, we believe that Xuorescence intensity measure-ments oVer advantages over contour length measure-ments and have chosen to use integrated Xuorescenceintensity measurements for sizing DNA molecules inthis study.

Macrorestriction analysis

Like optical mapping, our SGF method depends onsubstrate-mounted DNA being cleaved eYciently byrestriction endonuclease enzymes to produce a series ofordered DNA fragments that can be sized using inte-grated Xuorescence intensity after Xuorescently staining

the DNA. To size restriction fragments, the cleavage ofgenomic DNA by a restriction enzyme must produce agap in the Xuorescence image that can be used to deter-mine where one fragment ends and another starts. Thisrequires that DNA sticks tightly enough to the substrateto remain elongated but not so tightly that gaps do notform after enzymatic cleavage. Generally speaking, mod-iWed glass substrates producing DNA with an associatedstretching coeYcient in the range described above willalso have the appropriate surface chemistry to allow aDNA molecule to relax following cleavage by a restric-tion enzyme to form these gaps. However, because theinteractions of the DNA and the modiWed glass sub-strate determine whether a gap forms, careful control ofsurface preparation is necessary to successfully accom-plish substrate-based DNA fragment sizing. We gener-ally characterized and evaluated prepared surfaces usingcontact angle measurements, where the water contactangle of »30° indicates an ideal substrate.

The use of gaps formed by relaxation of surface-mounted DNA following restriction cleavage to identifyadjacent restriction fragments within a single genomehas two potential sources of error. First, two randomDNA fragments could be deposited onto a substratesuch that they appear to be the adjacent restrictionfragments. However, it is highly improbable that acomplete restriction Wngerprint, matching a potentialgenome of interest, would be created in this way. Second,random photofragmentation [39] of surface-mountedDNA can produce gaps that appear identical to thoseproduced by restriction enzyme cleavage. Gaps pro-duced by photofragmentation can alter apparent Wnger-prints by adding false restriction sites. However, we havefound that proper control of the substrate modiWcationchemistry and exposure times served to minimize photo-fragmentation.

The gaps, which are necessary to determine whererestriction fragments start and end, are also the reasonthat contour length measurements are not well suited torestriction fragment sizing. To size restriction fragmentsusing contour length, it must be assumed that the DNAuniformly relaxes after cleavage. This means that thecontour length, based on a calibration of fully stretchedDNA, must be judged from the middle of any gap.Because DNA can relax unequally following restriction,measurements from the middle of a gap often containsigniWcant error. In this respect, Xuorescence intensitymeasurements are better suited to sizing restrictionfragments.

Fig. 2A contains four representative Xuorescenceimages of bacteriophage � DNA following digestion byfour restriction enzymes. Bacteriophage � DNA wasmounted, digested with the speciWed restriction enzyme,and then stained with YOYO-1 before single-moleculeXuorescence imaging. Individual restriction fragmentswithin each digested � genome are easily identiWed by


gaps in Xuorescence within the molecule. The Xuores-cence images in Fig. 2 accurately reXect the order of therestriction fragments in the � genome. Virtual restrictionmaps, produced from the known sequence of � DNAand the known cleavage sites of each restriction enzyme,are also shown in Fig. 2A. These virtual restriction mapsare displayed as a solid black line, representing the48,502 bp of the � phage genome, with red hash marksindicating the sequence-predicted cleavage sites of theindicated restriction enzyme. Virtual fragment sizes, for� DNA, are listed above for each enzyme used in thisstudy. The restriction enzymes XbaI and XhoI both havea single cleavage site in the � genome and therefore eachproduce two restriction fragments. XbaI produces frag-ments that are 24,508 and 23,994 bp while XhoI producesfragments that are 33,498 and 15,004 bp. PmeI has twocleavage sites producing three restriction fragments(32,209, 7834, and 8459 bp) and EcoRI has Wve cleavagesites producing six restriction fragments (3530, 5804,7421, 5643, 4878, and 21,226 bp).

Fig. 2. (A) Representative single genome restriction Wngerprints ofbacteriophage � DNA for four restriction enzymes. Restrictionenzyme cleavage sites can easily be identiWed by gaps in the Xuores-cence signal. Virtual restriction maps, with the black line representing48,502 bp and the red hash marks indicating enzyme cleavage sites, arealso included for reference. The 5-�m scale bar shown in the EcoRIimage applies to all Xuorescence images. (B) SGF fragment sizes for sixindividual � genomes digested with EcoRI. Measured fragment sizesfor each of the six genomes are indicated by a color and symbol. Themean slope and correlation coeYcients (R2), obtained for linear Wts tothe SGF data, are 0.97 § 0.09 and 0.96 § 0.02, respectively. A line withunit slope and zero intercept is included in the plot for reference.

Fig. 2B contains a plot showing the single genomeWngerprints for six � genomes each digested with EcoRI.The SGF data included in this plot were collected frommultiple Welds on more than one substrate over severaldays and relate observed fragment size to sequence-pre-dicted fragment size. Due to the lack of an internal stan-dard for calibration purposes, observed fragment sizeswere converted to basepair units using a scaling coeY-cient determined from the sum of the fragment’s inte-grated Xuorescence signal and the basepair size of the �genome (48,502 bp). This is to say that the sum of restric-tion fragments for each of the six genomes shown isexactly equal to 48,502 bp. While this normalization pro-cedure is not a true calibration, it does allow relativefragment sizes from single � genomes to be compared toboth other digested � genomes and sequence-predictedfragment sizes. The mean correlation coeYcient (R2)obtained from linear regression Wts (not shown) to eachSGF data set was determined to be 0.96 § 0.02, where thestandard deviation of the measurement represents day-to-day reproducibility. The mean slope of linear Wts tothe data is 0.97 § 0.09, which indicates that our observedfragment sizes are in agreement with sequence-predictedfragment sizes. The agreement of these unaveraged, sin-gle genome Wngerprints with sequence-predicted Wnger-prints indicates that the sizing of restriction fragmentsfrom a single digested genome is capable of producing auseful genomic Wngerprint. The plot in Fig. 2B alsoshows that two of the fragments (fragments 2 and 4,which are 5804 and 5643 bp, respectively) in the EcoRIdigest � DNA are essentially unresolved when lookingonly at the measured SGF fragment sizes. However,images, which show the fragment’s relative order, clearlyallow discrimination of these fragments. This is a clearadvantage provided by SGF relative to PFGE or Xowcytometry, which cannot resolve these fragments.

Calibrated sizing

To generate a Wngerprint of a single genome in whichthe fragment sizes have not been scaled using the knowngenome size, it is necessary to calibrate the Xuorescenceintensity using an internal standard. A convenient inter-nal standard is a piece of DNA that is codeposited andcodigested with the sample DNA. Ideally, the internalstandard would produce three or more restriction frag-ments covering a range of size reasonably expected forthe sample. Since this set of conditions must be gener-ated using the same restriction enzyme as is used todigest the sample genome, there is generally some com-promise for the internal standard.

We chose to use � DNA as an internal standard forthe SGF studies presented here. SpeciWcally, we codepos-ited � DNA with T4 GT7 DNA on a modiWed glass sub-strate and codigested both genomes with the PmeIrestriction enzyme. We treated the T4 GT7 DNA as an


“unknown” in this study and Wngerprinted single T4GT7 genomes using � DNA as an internal standard sothat we could compare SGF results for the T4 GT7genomes with virtual digest results.

Fig. 3 shows a Xuorescence image, corresponding to asingle Weld of view, containing Wve � genomes (red) andtwo T4 GT7 genomes (green) that were codepositedbefore being codigested with PmeI. The � internal stan-dards are easily identiWed by comparing to the PmeI vir-tual digest of � DNA shown in Fig. 2. The integratedXuorescence signals corresponding to each of the �/PmeIfragments were determined and a calibration curve (datanot shown) using the mean fragment intensities andknown fragment sizes was constructed. A linear regres-sion Wt to this calibration data was obtained with a cor-relation coeYcient (R2) of 0.98.

Using the equation obtained from the linear regres-sion Wt to the Wve replicate internal standards shown inFig. 3 (red), we converted measured Xuorescence intensi-ties for the T4 GT7 fragments (green) to sizes in basepairunits. Upon carrying out this conversion for the two setsof PmeI-digested T4 GT7 genomes shown in Fig. 3, weobtained fragment sizes that were both independent ofeach other and directly comparable to virtual digestfragment sizes. Data points corresponding to the T4GT7 fragments from Fig. 3 are shown in Fig. 4 as green

Fig. 3. Fluorescence image showing the PmeI digestion products of amixture of � (pseudo-colored red) and T4 GT7 (pseudo-colored green)DNA. Relaxation of the DNA following restriction enzyme cleavagecreates clear gaps in the DNA. The mean observed integrated Xuores-cence intensities from the Wve � genomes were used, along with known�/PmeI fragment sizes, to create a calibration curve for sizing of the T4GT7/PmeI fragments in basepair units. Calibrated fragment sizes forthe two T4 GT7 genomes shown here are included in Fig. 4.

squares and blue triangles. These data sets are each sin-gle genome Wngerprints of individual T4 GT7 viralgenomes. Calibrated T4 GT7/PmeI fragment sizes, deter-mined from Wve additional single T4 GT7 genomes, arealso shown in Fig. 4 using various colored symbols thatare arbitrarily oVset on the y axis to facilitate viewing. Inaddition to the SGF data, Fig. 4 contains a curve thatrepresents the virtual digest of T4 GT7 DNA with PmeI.This curve is the sum of four Gaussian peaks, normal-ized by area, with centroids corresponding to the four T4GT7/PmeI virtual digest fragment sizes (68,928, 44,810,34,109, and 17,801 bp) and peak widths calculated fromthe mean coeYcient of variation determined for ourimage-based Xuorescence sizing of DNA standards(CV D 8%; N D 323). This curve represents the expectedT4 GT7/PmeI fragment size distribution from an inWnitenumber of Xuorescence-based fragment size measure-ments. The SGF data (i.e., calibrated sizing of fragmentswithin individual genomes) fall within the distribution(CV D 8%) of the sequence-predicted fragment sizes.These data show that a single genome, the bare mini-mum of a DNA sample size, can provide Wngerprintsthat are both accurate and precise. Because our SGFresult is produced from a single genome, where eachrestriction fragment was measured only once, there areno ensemble averaging eVects in the Wnal Wngerprints.An important distinction between our SGF andSchwartz’s optical mapping is that our SFG produces aWngerprint attributed to a single genome whereas theirapproach to optical mapping produces a consensus mapattributed to the average of many genomes.

Fig. 5 shows the SGF analysis of a mixture of threeviral genomes (adenovirus and bacteriophages � and T4GT7). Like the SGF data shown in Fig. 3, these threeviral genomes were codeposited and codigested with

Fig. 4. Calibrated SGF data for seven T4 GT7 genomes digested withPmeI are shown as various colored symbols that have been oVset onthe y axis for viewing. Each SGF data set was calibrated using � DNAas an internal standard as described in Fig. 3. The two T4 GT7genomes shown in Fig. 3 correspond to the green squares and blue tri-angles shown here. The curve represents the sequence-derived virtualdigest of T4 GT7 DNA with PmeI. Each SGF fragment size is within8% of the expected fragment size.


PmeI before staining with YOYO-1 and imaging. TheDNA fragments comprising single genome Wngerprintsare identiWed by false color (e.g., adenovirus in blue, �phage in red, and T4 GT7 in green). The SGF data fromthe three � genomes (red) were used to make a calibra-tion curve for sizing of the adenovirus and T4 GT7restriction fragments. The two lower plots show the cali-brated SGF fragment sizes for the adenovirus and T4GT7 Wngerprints shown in Fig. 5 and curves (con-structed as described above) representing the virtualdigests of each genome. These data demonstrate the abil-ity of our SGF method to produce accurate and preciserestriction Wngerprints from single genomes within amixture.

We have demonstrated single genome Wngerprintingof pure and mixed viral genomes. For these analyses, weselected completely digested whole genomes and ignoredboth partially digested genomes and randomly shearedDNA found in any sample. Based on the work of

Fig. 5. (A) Fluorescence image showing the PmeI digestion products(indicated with pseudo-color) of a mixture containing adenovirus(blue; N D 3), �(red; N D 3), and T4 GT7 DNA (green; N D 2). (B)Measured and known fragment sizes for bacteriophage � were used tocalibrate Xuorescence intensities and size the adenovirus and T4 GT7restriction fragments in basepair units. Observed fragment sizes foreach of the (A) T4 GT7 and (B) adenovirus genomes are shown alongwith curves representing the virtual digest with an 8% coeYcient ofvariation.

Schwartz et al. [25,40], it is clearly possible to use par-tially digested DNA and incomplete genomes to recon-struct ordered restriction maps. Like optical maps,Wngerprints created from partial genome and/or partialdigest data would represent an ensemble average result.Since it is our goal to create accurate single genomeWngerprints, we have limited our SGF analysis to fullydigested complete genomes. Despite these somewhatstringent selection criteria, many of our samples(»0.25 ng of DNA per substrate) contained hundreds ofgenomes well suited to SGF analysis.

Since our images contain many fragments, it is nec-essary to sort through a sample to identify fullydigested intact genomes. This process requires priorknowledge of the genomes of interest such as a librarycontaining relevant Wngerprints. With such a library, asample can be scanned and DNA fragment patternscompared to library Wngerprints to ascertain whether apattern in the sample is a reasonable match to one ofthe Wngerprints in the library. Our library was pro-duced using virtual digests produced from DNAsequences and samples are scanned visually for poten-tial matches to library Wngerprints. Pattern recognitionsoftware would facilitate higher throughput by auto-mating this task [30]. A library used for screening pur-pose could be produced by virtual digests of DNAsequences, optical mapping, SGF, Xow cytometry, orPFGE. Only libraries produced from sequences, SGF,or optical mapping are able to consider fragment orderin their comparisons. As presented here, our SGF tech-nique is best suited to rapid screening applications.Matching to this sort of library would allow one to saywith great certainty that an organism is present in asample if SGF results match the Wngerprint of a knownpathogen.

Sum rules could be applied to SGF data to accountfor partial digestions of single genomes. For example, aWngerprint containing fewer restriction fragments,whose size and fragment distribution patterns are simi-lar to the sum of multiple known restriction fragmentsin a known pathogen’s Wngerprint, is likely an incom-pletely digested genome of that pathogen. These sumrules also apply to single genomes containing morethan the expected number of restriction fragments (e.g.,overdigestion, random fragmentation, or photofrag-mentation) if their sum is equivalent to an expectedfragment size. Image comparison methods can takethese possibilities into account. While the certainty ofidentiWcation based on this type of information wouldbe diminished, it would still be useful. This library sys-tem and the overall eYciency of our SGF analysis alsolimit the certainty with which we can say that a patho-gen is not in a sample. Due to the possibility that anorganism was represented in the sample only by incom-plete genomes, there can never be 100% certainty that itwas not in the original sample.


SGF data display

Like optical mapping, SGF provides the relativeorder of restriction fragments in the genome. This infor-mation cannot be obtained using traditional genomeanalysis techniques such as Xow cytometry or PFGE. Totake advantage of the fragment order provided by SGF,we have developed a “bingo card” display. This datarepresentation oVers a way to both display and usethis fragment order information provided by our SGFanalysis.

This bingo card display format is a graphical repre-sentation incorporating both fragment size and order.For illustration, consider the two hypothetical genomesshown in Fig. 6A�. These two genomes (A and B) eachproduce three restriction fragments (fragment numbers1–3) that are similarly sized but distributed diVerentlywithin the genomes. A graph that does not take intoaccount fragment order is identical for both genomes(graph shown at the bottom of Fig. 6A�). Our bingo carddisplay shows the order of the restriction fragments andallows the two samples to be discriminated. For any onesample in the bingo card plot, a projection of the carddown the y axis will produce a good estimate of the con-ventional nonordered graph. The advantage of this dis-play format is that two similar fragments, which wouldnormally appear in a single unresolved band with Xowcytometry or PFGE, now are clearly separated in thebingo card array due to the knowledge of their locationsin the genome. An example of this bingo card display,for the four viral genomes digested with PmeI, is shownas Fig. 6B�. This particular example was created using 1-kb bins for fragment sizes with fragment numberscoinciding with virtual digest data. This example againillustrates an advantage of this format relative to con-ventional fragment size distribution histograms whenconsidering fragments 1 and 2 of the � Wngerprint. Thesetwo fragments both fall into the 7- to 8-kb bin andwould be unresolved in a conventional histogram. How-ever, the bingo card format indicates their spatial sepa-ration seen in Xuorescence images. We anticipate thatthe true usefulness of this display format will be realizedonly with SGF or optical mapping data more complexthan data presented here. We are developing correlationtools to compare bingo card representations of SGFdata for library matching and discrimination purposes.

Applications

The SGF data presented here are exciting not onlybecause they show ordered restriction Wngerprinting ofsingle viral genomes but also because of the adaptabilityof this technology to microXuidic platforms. A majorhurdle in developing analytical methods capable of sin-gle genome analysis is the availability of techniquescapable of handling and preparing single organisms. Use

of microXuidic platforms has long promised to accom-plish this task and has demonstrated signiWcant progresstoward this goal [41–47]. However, we are unaware of amicroXuidic platform that does not rely on oZine prepa-ration or analysis. As microXuidic (i.e., “lab on a chip”)applications progress to the point that they becomecapable of integrated analysis, with no need for oZinepreparation or analysis, the need to derive meaningfulconclusions for small data sets will become increasingly

Fig. 6. (A�) Simple schematic representation of our bingo card displayfor two genomes (A and B) with similar fragment sizes but diVerentfragment distributions. The bingo card display (middle) shows dis-crimination of genomes A and B while the conventional fragment sizehistogram cannot discriminate these genomes. (B�) Bingo card arrayfor PmeI digests of the viral genomes used in this study. Unlike a Xowcytometry histogram or PFGE image, this data display format allowsthe Wrst two fragments of the � Wngerprint to be resolved due to theirspatial separation in an SGF Xuorescence image.


important. Our SGF data show deWnitively that a plat-form capable of handling and preparing a single cell canproduce an accurate genomic Wngerprint. In principle, asample containing only a single genome is needed, butineYciencies in sample digestion, processing, and DNAmounting might require several genomes to be pro-cessed. Not withstanding, bioforensics, medical diagnos-tics, and many other real-world applications can beenvisioned for this technology especially where uncultur-able or unampliWed samples are encountered.

Conclusions

We have demonstrated reproducible restriction Wnger-printing of single viral genomes. Individual genomes weremounted to modiWed glass substrates, digested by restric-tion enzymes, stained with Xuorescent dyes, and imaged.Quantitative image analysis showed that integrated Xuo-rescence measurements scale with fragment size and,through the use of internal DNA standards, we are ableto size restriction fragments in basepair units. In additionto being able to size single DNA fragments, our singlegenome analysis method provides the order of fragmentswithin the genome. This order provides additional infor-mation, relative to that of ultrasensitive Xow cytometryor conventional PFGE results, that is useful in genomediscrimination.

Acknowledgments

The authors thank Dr. James Jett, Robbert Habber-sett, Yulin Shou, and Cheryl Lemanski for their contri-butions to this work. Funding was provided by theNational Institutes of Health (1R21 RR018337-01) andthe National Flow Cytometry Resource at Los AlamosNational Laboratory.

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