Two-dimensional strandness-dependentelectrophoresisGudmundur H Gunnarsson1,2, Bjarki Gudmundsson2, Hans G Thormar1,2, Arni Alfredsson1 & Jon J Jonsson1,3

1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, IS-101 Reykjavik, Iceland. 2BioCule Inc., IS-101 Reykjavik, Iceland.3Department of Genetics and Molecular Medicine, Landspitali-University Hospital, IS-101 Reykjavik, Iceland. Correspondence should be addressed to J.J.J. (jonjj@hi.is).

Published online 25 January 2007; doi:10.1038/nprot.2006.477

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness,

conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded

DNA and RNA .DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under

denaturing conditions (7 mol l�1 urea, 55 1C) so that all the molecules are single-stranded and separate according to length only.

2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification,

renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in

double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.

INTRODUCTIONInvestigators often work with complex nucleic acid samples inprocedures such as amplification, renaturation, cDNA synthesisand normalization, sonication, digestion, nicking, labeling andhybridization on microarrays. Such work requires methods todetermine the quality of complex nucleic acid samples and tomonitor their faithful manipulation1,2. Two sample characteristicsthat are often of interest are the length distribution and strandness(i.e., whether the molecules are single-stranded, double-stranded orcontain RNA � DNA hybrids). It is also useful to know whether thebases and backbone are intact, as well as whether the samplecontains nucleic acids of aberrant structure or conformation.

The majority of nucleic acid gel electrophoresis methods are basedon one-dimensional (1D) setups. Two-dimensional (2D) setups can,however, have considerable advantages, because nucleic acid mole-cules can be separated according to different characteristics in eachdimension, making it less likely that two nucleic acid molecules willco-migrate. Nucleic acid molecules are therefore more effectivelyseparated in 2D gel electrophoresis than in 1D gel electrophoresis,allowing for profiling of more complex samples. Although 2D gelelectrophoresis is common in proteome research, less attention hasbeen focused on applications in genome or transcriptome research.

2D-SDE was recently introduced as a simple and powerful methodto separate complex nucleic acids samples according to strandness,length and conformation3. 2D-SDE allows quantification and lengthdistribution analysis of a mixture containing single-stranded DNAfragments (ssDNA), double-stranded DNA fragments (dsDNA)and/or RNA � DNA hybrids. The presence of RNA � DNA hybridsis of interest in studying efficiency of reverse transcription in cDNAsynthesis of complex samples. RNA � DNA hybrids cannot beanalyzed as a separate fraction with 1D electrophoresis. In addition,2D-SDE can be used to analyze nicked, mismatched or damagedDNA fragments in complex samples. These various fractions can alsobe directly isolated for further study using well-established methodsfor isolation of DNA from polyacrylamide gels4,5.

2D-SDE is based on simple biophysical principles. The firstdimension is carried out in the presence of 7 mol l�1 urea toreduce secondary structures of ssDNA fragments. Despite the highconcentration of urea, dsDNA fragments and RNA � DNA hybrids

do not denature if the first dimension is performed at roomtemperature (20–24 1C). The nucleic acid fragments are separatedon the basis of strandness, length and conformation in the firstdimension. If nucleotide sequences of equal length are separatedunder these conditions, RNA � DNA hybrids migrate at the fastestvelocity, dsDNA fragments migrate at intermediate velocity andssDNA fragments are the slowest. Rigidity increases from ssDNA,dsDNA to RNA � DNA hybrids6–8. The more stiff and rod-like thefragment, the lower the frictional drag, is as it passes through tightpores of polyacrylamide gel9. A bulge is a loop structure in DNAcomprised of extra nucleotide residues in one strand of dsDNA nothaving counterparts on the other strand. A bulge causes bending indsDNA molecules, resulting in reduced migration velocity inpolyacrylamide gel5.

Before the electrophoresis in the second dimension, the gel isincubated at an elevated temperature, that is, 55 1C for 3 min. Atthis temperature and in the presence of 7 mol l�1 urea, dsDNAfragments denature in the gel and become single-stranded(Gunnarsson et al., unpublished observations). However, if theinvestigator wants to test whether RNA � DNA hybrids are present,the gels needs to be incubated at 92 1C for 3 min before the seconddimension electrophoresis6–8. The second dimension separation iscarried out at 55 1C in a direction perpendicular to that of the firstelectrophoresis step. As all the molecules are single-stranded and ofsimilar conformation; they separate only according to their length.

The relative location of molecules after 2D-SDE is thereforedependent on the nucleotide lengths of the single-strandedform and the difference between the migration velocities of eachfragment in the two electrophoretic dimensions. This makes itpossible to (1) separate mixtures containing ssDNA, dsDNA andRNA � DNA hybrids; (2) detect single-strand breaks in dsDNA;and (3) separate mispaired, unmatched or damaged dsDNA.

(1) Separation of ssDNA, dsDNA and RNA .DNA hybridsssDNA fragments in the original sample are not affected by theheat-denaturation step and their migration velocities in the firstand second dimension electrophoresis are the same. Therefore, theyform a diagonal line in the gel after 2D-SDE (Fig. 1). dsDNA

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fragments migrate relatively faster than ssDNA fragments in thefirst dimension. After denaturation, ssDNA fragments originatingfrom dsDNA are located further down in the gel than their equallylong counterparts that were single-stranded in the original sample.The difference in the relative migration velocities between ssDNAand dsDNA is length-dependent. Molecules that were double-stranded in the original sample therefore form an arc lying behind(to the left of) the line representing ssDNA.

The RNA � DNA hybrids have the highest relative migrationvelocity in the first dimension, so they form an arc lying behind thedsDNA arc. Signal imaging allows straightforward quantificationand length distribution analysis of each fraction. Efficient separa-tion is achieved for nucleic acid fragments in the size range of 50–5,000 bp (or nt). Separation of larger fragments is not efficient inpolyacrylamide electrophoresis.

An ssDNA molecule with intramolecular hybridization stableenough to withstand the denaturing effect of 7 mol l�1 urea wouldpresumably show aberrant migration and not fall on the line ofssDNA or the arc of dsDNA. The position would depend on thenature and proportion of intramolecular hybridization. We havenot observed this despite testing many different fragments; pre-sumably it occurs only with rare fragments.

(2) Detection of single-stranded breaks2D-SDE can also be used to detect single-stranded breaks (nicks) indsDNA fragments. In the first dimension, the nick-containingfragments are double-stranded (Fig. 2a). During the seconddimension separation, the nick-containing strand generates shorterstrands (at least two, one more than thenumber of nicks), which migrate in front ofthe arc of molecules representing originallyintact dsDNA fragments (Fig. 2b). Thisversion of the technique is called nick-dependent electrophoresis. Nicks can result

from incomplete replication or damage incurred in vivo or in vitro.Alternatively, nicks can be introduced with nicking enzymes withvarious specificities, for example, enzymes that nick mismatchedheteroduplexes, including single base-pair mismatch, in mutationscanning10. This application could have many interesting applica-tions, including in conjunction with TILLING mutagenesis11.

(3) Mispaired and damaged dsDNA2D-SDE can also be used to separate mispaired, unmatchedand damaged dsDNA based on their atypical conformation.Mispaired, unmatched and damaged nucleotide residues (collec-tively called lesions in this context) commonly induce bending indsDNA molecules, making their migration slower than dsDNAfragments with normal structure and conformation5. Denaturationtypically reduces or eliminates this bending. Both strands originat-ing from mispaired, unmatched or damaged dsDNA molecules(lesion strands) generally migrate in front of the arc comprising thedsDNA molecules. Their final location in the 2D-SDE gel isdependent on the nature of the original lesion and its exact locationin the molecule. In practice, lesion strands can be spread out in thegel, making them difficult to detect and isolate. If an ssDNApopulation is present in the original samples, those moleculescan in some instances co-migrate with the lesions strands con-founding analysis and isolation. However, if the investigator isolatesDNA from areas not defined by arcs of dsDNA or the line ofssDNA, one can directly isolate lesion strands in a specific manner.The detection of lesion strands is useful for the discovery of novelpolymorphisms as well as for the study of DNA damage and repair.

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Figure 1 | The principle of 2D-SDE. Separation of

dsDNA, ssDNA and RNA � DNA hybrids. (a) In the

first dimension, nucleic acid fragments are

separated according to strandness, length and

conformation. The RNA � DNA hybrids (blue)

migrate at the fastest velocity, dsDNA fragments

(green) migrate at intermediate velocity and

ssDNA fragments (red) are the slowest. Before

second dimension separation (perpendicular to the

first dimension), all fragments are denatured to

become single-stranded and thereafter separate

only according to their length. (b) ssDNA

fragments have the same migration velocity

in both separations and therefore form a

diagonal line; dsDNA forms an arc lying

behind the ssDNA; and RNA � DNA hybrids form

an arc lying behind the dsDNA arc. Note that molecules of the same length, but originating from different forms of strandness, migrate equally fast during the

second dimension. They are therefore placed in a vertical line in the gel.

2D-SDE to separate ssDNA, dsDNA and RNA•DNA hybrids

a b

1D, different migration velocities of equally longssDNA, and dsDNA and RNA•DNA hybrids

2D, same migration velocities of equally longssDNA, and dsDNA and RNA•DNA hybrids

a

2D-SDE to detect DNA nicking

1D, ds migration velocity of all DNA fragments 2D, ss migration velocity of all DNA fragments

b

Figure 2 | Separation of nick-containing DNA

from intact dsDNA using nick-dependent

electrophoresis. (a) The nick-containing fragments

are double-stranded during the first dimension

separation. (b) During the second dimension

separation, the nick-containing strands generate

(at least) two shorter strands, which migrate in

front of the arc of intact dsDNA fragments.

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Advantages of 2D-SDE over 1D electrophoresis methodsConclusions about the quality or faithfulness of complexnucleic acid manipulations are commonly drawn from lengthdistribution analysis after 1D gel electrophoresis. Often the aimis to confirm the formation of dsDNA products and to assesstheir length. Typically, agarose or polyacrylamide gel electro-phoresis under either denaturing or non-denaturing conditionsis used2,12–23. The main advantages of 1D electrophoresis areease of use and the simultaneous analysis of multiple samples.Such analysis has many limitations and can result in erro-neous conclusions about the quality of a complex nucleic acidsample:� Denaturing electrophoresis, for example, allows only the total

amount of products (single- and double-stranded combined) tobe quantified.

� Complex amplification products are often analyzed with non-denaturating gel electrophoresis and ethidium bromide (EtBr)staining. EtBr is a fluorescent intercalator with a strong pre-ference for staining dsDNA; therefore, it underestimates theamount of ssDNA products.

� In agarose gels, ssDNA products typically co-migrate with thedsDNA products. ssDNA may therefore confound estimates ofthe amount of dsDNA products.

� In polyacrylamide electrophoresis, ssDNA products have lowermigration velocity compared to its dsDNA counterparts. Thesamples having the most ssDNA products could be selected asoptimized because they give false indication of longer dsDNAproducts being present compared to samples with low amountof ssDNA products.

Advantages of 2D-SDE over biochemical and biophysicalmethods for separating single- and double-stranded productsfrom complex samplesSeveral methods have been introduced for analyzing amounts ofsingle- and double-stranded nucleic acid fragments in complex

samples. All of them have some major limitations that 2D-SDEdoes not have:� Investigators have used indirect methods based on differences in

absorbance or fluoresecent signals from various dyes. Theseapproaches are sensitive to physical conditions and subject tovarious biases in signal intensity. Such methods cannot be usedfor physical separation of the two fractions.

� Strandness-specific nucleases cause nonspecific degradation.Furthermore, only the single- or the double-stranded fractioncan be enriched as one of the fractions is degraded.

� Hydroxyapatite columns bind strongly to double-strandednucleic acid fragments, allowing their separation from single-stranded forms. Single-stranded fragments containing double-stranded regions will, however, co-elute with the double-stranded fraction.

Applications of 2D-SDE2D-SDE has been successfully applied to estimate renaturationefficiency, assess dsDNA and ssDNA content after complexPCR (Figs. 3 and 4a), monitor cDNA synthesis (Fig. 4b), detectnicked DNA fragments in complex samples (Fig. 4c) and estimatequality and in vitro damage of DNA samples3. The methodcan be used for normalization of complex cDNA samples and foroptimizing complex PCR protocols. Further, 2D-SDE is a valuabletool to validate reverse transcription, amplification and labelingefficiency before microarray analysis. With 2D-SDE, it is possibleto quickly validate if the complex nucleic acid sample is ofsufficient quality (‘‘chip worthy’’) or not, for a fraction of thechip cost. Indicators of quality would depend on the application.For cDNA synthesis in gene expression studies, the investi-gator might want to document the efficient reverse transcription

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3,000 bp

500 bp

100 bp

a b

Figure 3 | Comparison of agarose and 2D-SDE analysis of highly complex

genome representations generated with selective PCR amplification of

short restriction fragments from human genomic DNA. (a) Separation of

genome representation products in 2% agarose. Marker used was GeneRuler

100 bp DNA Ladder Plus (Fermentas). (b) 2D-SDE analysis of the genome

representation products reveals that most of the products are single-stranded.

a

c d

b

Figure 4 | 2D-SDE analysis of four different complex nucleic acid samples.

(a) Separation of dsDNA from ssDNA. (b) Investigation of RNA � DNA hybrids.

(c) Separation of nicked DNA from intact dsDNA. (d) Separation of bulge-

containing molecules from perfectly matched DNA. a–c are modified with

permission from ref. 3, copyright 2006, with permission from Elsevier.

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from RNA to dsDNA molecules that are sufficiently long to havebinding sites for T7 RNA polymerase. In amplifications, theinvestigator would typically like to see double-stranded productsconsistent with efficient PCR. Accumulation of single-strandedPCR products would suggest the preferential overamplification ofone DNA strand relative to the other or increased truncationof products, which in turn would lead to an incorrect estimationof the quantity of obtained products as probed through quantita-tive microarray analysis2.

Experimental design/considerationsThe only specific equipment required for 2D-SDE is an electro-phoresis unit for slab gel separation at 55 1C (e.g., the Multiphorsystem from GE Healthcare). Some investigators will find it moreconvenient to run the first dimension in a vertical polyacrylamidegel apparatus typically in 7 � 8 cm minigels, although thetechnique can be easily adapted to other gel sizes. Unlike many2D electrophoresis methods, the same gel matrix is used in bothdimensions. This eliminates the troublesome transfer of nucleicacid molecules over the junction between two different gel matrixesand reduces the complexity of the protocol. All reagents for 2D-SDE are inexpensive and commonly used in biosciences.

As 2D-SDE is based on biophysical principles, no enzymaticsteps are required. This increases the robustness of the method anddecreases reagent cost.

Unlabeled and labeled nucleic acid molecules are both effectivelyseparated using 2D-SDE. So far, we have tested several labelingstrategies including fluorescent-labeled primers and fluorescent-labeled dCTP incorporated with fill-in reactions. We have alsoeffectively stained 2D-SDE gel with EtBr after separation fordetection of unlabeled nucleic acid fragments.

Information about complex nucleic acid samples obtainedwith 2D-SDE cannot be readily obtained with other methods. Itis therefore a valuable tool in current biosciences, where the

emphasis is on working with complex genome or transcriptomepreparations.

MATERIALSREAGENTS.30% (wt/vol) acrylamide solution. Mix 29% acrylamide (wt/vol) and 1%N,N’-methylene-bisacrylamide (wt/vol). Use ultra-pure chemicals, forexample, PlusOne acrylamide from GE Healthcare ! CAUTION Acrylamideis a potent neurotoxin and is absorbed through the skin. The effects ofacrylamide are cumulative. Wear gloves and a mask when weighingpowdered acrylamide and methylenebisacrylamide. Wear gloves whenhandling solutions containing these chemicals. Although polyacrylamideis considered to be non-toxic, it should be handled with carebecause of the possibility that it might contain small quantities ofunpolymerized acrylamide.

.10% (wt/vol) ammonium persulfate, for example, PlusOne from GEHealthcare m CRITICAL Prepare fresh every other day and keep refrigeratedand protected from light.

.Urea (499% pure)

.N,N,N¢,N¢-tetramethylethylenediamine (electrophoresis grade, for example,from Sigma or Bio-Rad) ! CAUTION Harmful if inhaled or swallowed;corrosive and causes burns.

.5� Tris/borate/EDTA (TBE) buffer (0.45 mol l�1 Tris, 0.45 mol l�1 boricacid, 10 mmol l�1 EDTA). All chemicals should be 499% pure

.Sample-loading buffer (sucrose, glycerol and Ficoll-based loading buffershave all been successfully used)

.10 mg ml�1 EtBr (use molecular biology grade, e.g., from Sigma (cat. no.E1510)) ! CAUTION EtBr is a potent mutagen and toxic after an acuteexposure. EtBr can be absorbed through the skin; so it is important to avoidany direct contact with the chemical.

EQUIPMENT.Vertical electrophoresis apparatus; most commercially available systems are

suitable, for example, the Protean II unit (Bio-Rad) or the SE 400 verticalunit (GE Healthcare)

.Horizontal electrophoresis apparatus capable of operation atconstant 55 1C, for example, the Multiphor II Electrophoresis System(GE Healthcare)

.Power supply (4400 V or 5 W)

.EPH electrode wicks, paper 104 � 253 mm (GE Healthcare)

.Detection system for scanning nucleic acids, fluorescent scanners, forexample, Typhoon Variable Mode Imager (Amersham Biosciences), a regularUV transilluminator or other gel documentation systemsREAGENT SETUP2D-SDE mix (100 ml) Add 42 g urea (final concentration 7 mol l�1), 30 ml30% acrylamide solution (final concentration 9%) and 20 ml 5� TBE (finalconcentration 1� TBE) and fill up to 100 ml with double-distilled water.m CRITICAL Store at 4 1C protected from light. Prepare fresh every 2 weeks.

Staining solution (50 ml) Add 50 mg of EtBr (5 ml of 10 mg ml�1) to 50 ml of1� TBE solution.

Size of nucleic acid sample Approximately, 200–1,000 ng of nucleic acids issufficient for EtBr post–staining; 1/10 to 1/100 of the mentioned amount ofsample is needed when molecules are fluorescently tagged either by end labelingor incorporation. In some cases, increasing sample concentration may benecessary. m CRITICAL STEP High concentration of salts in a concentratedsample may greatly affect the quality of the separation in polyacrylamide gelelectrophoresis.

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Loading dye after1st dimension

Paper wicks

TBE buffer Electrophoresis direction

Overlaying glass plate

Figure 5 | Picture of a typical setup for horizontal electrophoresis in the

second dimension using a Multiphor apparatus.

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DNA size markers Digested DNA may be labeled with fluorescent groupsusing a Klenow fill-in reaction of overhangs (e.g., Fig. 4a,c). Incorporation oflabeled nucleotides in a reverse transcription reaction is useful in monitoringcDNA synthesis (Fig. 4b). Fluorescent-labeled primers can also be used forlabeling PCR products (Fig. 4d). Suitable size markers can be added to samplesfor direct measurement of length distribution. To allow length distribution

analysis of both single- and double-stranded fractions, an aliquot of the dsDNAsize marker may be denatured at 92 1C for 3 min and then transferred directly toice. The denatured size marker can then be mixed with untreated marker tocreate a mixture of single- and double-stranded marker fragments. It should benoted that the denatured size marker will slowly renature, resulting in increasedamounts of double-stranded fragments.

PROCEDUREPreparation of the 2D-SDE gel � TIMING 40 min1| Assemble a vertical gel sandwich using 0.75- or 1.0-mm-thick spacers. The well size of the comb can be adjusted accordingto the loading volume of the sample.

2| Take the required volume of 2D-SDE mix, add 10 ml of 10% ammonium persulfate and 1 ml of N,N,N¢,N¢-tetramethylethylene-diamine per milliliter and mix gently. The amount of 2D-SDE mix required for gel with dimension 0.1 � 7 � 8 cm is 5 ml.Immediately pour the gel and leave for at least 30 min to polymerize.’ PAUSE POINT The gel can be kept at 4 1C overnight.

3| Mount the gel sandwich in the vertical electrophoresis apparatus and fill the buffer chambers with an appropriate volume of1� TBE buffer.

4| Mix the appropriate sample volume in 1� gel loading buffer. Typical loading volumes are 2–25 ml if a 10-well comb wasused to cast the gel. Wash the second well from the left thoroughly to remove urea. Immediately, load the sample in that welland proceed to the next step.m CRITICAL STEP As soon as urea is washed from the well, it starts to accumulate again. If the sample is kept for prolongedtime in the well before electrophoresis, the quality of the separation may be reduced due to diffusion of the sample in the well.

First dimension separation � TIMING 45 min5| Run the first dimension at constant current 20 mA for 45 min at room temperature. If the sample is labeled withfluorescent groups, the first dimension separation can be checked on appropriate fluorescent scanner or gel documentationsystem.? TROUBLESHOOTING

Second dimension separation � TIMING 40 min6| While running the first dimension separation, equilibrate the horizontal gel electrophoresis plate at 55 1C. Bundle sevenpaper electrophoresis wicks and soak them in 1� TBE buffer. Fill the electrophoresis buffer chambers of the equipment with anappropriate amount of 1� TBE buffer.

7| After the first dimension separation, remove the spacers from the gel sandwich. Carefully open the sandwich. If using 7 �8 cm glass plates, rotate the free glass plate 901 and close the sandwich again. In this manner, 0.5 cm gel edges on each sideshould be accessible for connecting the paper electrophoresis wicks. If testing only for dsDNA and ssDNA, proceed directly toStep 8. If testing for the presence of RNA � DNA hybrids, a special denaturation step is recommended. After the first dimensionelectrophoresis, the gel sandwich is placed on a dry heat-block at 92 1C for 3 min. To ensure better heat distribution, a 92 1Chot aluminum heat-block cube can be placed on top of the gel sandwich.

8| Place the gel sandwich onto the horizontal temperature-controlled gel electrophoresis plate and connect the paper electro-phoresis wicks between the buffer chamber and the accessible end of the gel. Before second dimension separation, incubate thegel sandwich for 3 min at 55 1C. The setup is shown in Figure 5. Temperature control is best achieved with an external waterbath circulating 55 1C water through the plate.m CRITICAL STEP If the gel is not kept between glass plates, the buffer will rapidly evaporate from the gel, resulting in decreasedquality of second dimension separation.? TROUBLESHOOTING

9| Run the second dimension separation at constant power 5 W and 55 1C for up to 30 min. (Alternatively, if the power supplycannot deliver constant power, the run can be performed at constant current 15 mA with similar results.)? TROUBLESHOOTING

Staining and visualization � TIMING 10–15 min10| If the nucleic acids being analyzed are unlabeled, soak the gel in 50 ml of the staining solution for 10 min with slow orintermittent shaking (optional).? TROUBLESHOOTING

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11| Wash the gel with buffer or double-distilled water before visualizing the gel using a UV plate or the appropriate mode of aVariable Image Scanner.

� TIMINGPreparation of the 2D-SDE gel: 40 minFirst dimension electrophoresis: 45 minSecond dimension electrophoresis: 40 minPost-run manipulation of gel: 10–15 min

? TROUBLESHOOTINGTroubleshooting advice can be found in Table 1.

ANTICIPATED RESULTS2D-SDE offers a simple procedure to separate the fractions of ssDNA, dsDNA and RNA � DNA hybrids. Conventional 1Delectrophoresis, such as in agarose gels, will give information on the length distribution, but the fractions of dsDNA,ssDNA and RNA � DNA hybrids are not revealed. After 2D-SDE, dsDNA and RNA � DNA hybrids will form separate arcsand ssDNA will form a diagonal line (Fig. 1), allowing quantification, length distribution analysis and direct isolationof each fraction.

For comparison of 1D agarose and 2D-SDE analysis, highly complex genome representations were created through preferentialamplification of small restriction fragments from the human genome (Fig. 3). One such representation was created using BglIIdigestion and the protocol described by Lucito and Wigler24. Length distribution analyzed on agarose was in the expected rangeof 200–1,200 bp (Fig. 3a). Two distinct bands of an approximate length of 600 and 800 bp were present in the representation.These bands are the products of repetitive sequences. 2D-SDE analysis demonstrated that approximately 90% of the DNAmolecules were single-stranded fragments of various lengths (Fig. 3b), although the 1D agarose gel analysis gave promising

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TABLE 1 | Troubleshooting table.

Problem Possible reason Solution

Step 5: Nucleic acids do notmigrate into gel

High salt concentration in a concentrated sample Desalting of sample, for example, by columnpurification before concentrating the sample

Formation of large nucleic acid aggregatesin solution

Change buffer, heat sample before separation

Step 5: Low-quality separation ofbands in the sample

Diffusion of sample in well Wash all urea from well before sample loading andstart electrophoresis right after sample loading

Step 5: Trailing effect of bandsin sample

Too high amount of sample loaded on gel Decrease sample amount. The upper limits ofloading is around 5–10 mg of DNA

Step 8: Slow migration in seconddimension

Buffer evaporates from the gel Electrode wicks must be placed tightly to thetop glass plate

Step 8: No arcs or lines visible Second dimension electrophoresis in thewrong direction

Carefully determine the position of electrodes forsecond dimension electrophoresis

Step 9: Sample with known dsDNAappears as ssDNA

Sample did not (fully) denature. All molecules thatare identical in strandness in both dimensionsmigrate on the diagonal line. Wrong setting oftemperature controller

Increase heat up to 55 1C, manually control heatmeasurement

Step 9: A spot appears outside theline of ssDNA or arc of dsDNA

Consider a rare fragment with strong intramolecularhybridization

Characterize fragment. Consider running firstdimension at slightly higher temperature if spotcauses problems

Step 10: No sample visible Low amount of sample loaded on gel Increase sample amount. For EtBr staining use atleast 200–1,000 ng and for fluorescence-labeledsamples use at least 20–100 ng

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indications about the high quality of amplification products. The 2D-SDE result indicates that the PCR amplification was notefficient, at least not for the last cycle, as ideally PCR generates double-stranded products. Such products would, for instance,be difficult to clone, as T4 DNA ligase requires dsDNA.

Other good examples of anticipated results showing the effectiveness of 2D-SDE to separate dsDNA from ssDNA (Fig. 4a),RNA � DNA hybrids from dsDNA, ssDNA and RNA (Fig. 4b), nicked DNA from intact dsDNA (Fig. 4c) and mismatched DNA fromperfectly matched DNA (Fig. 4d) are described in more detail below.

Separation of dsDNA from ssDNASeparation of a highly complex mixture of ssDNA and dsDNA fragments (NdeII-digested human genomic DNA). Cy3-labeleddsDNA fragments formed an arc (green) separated from the line (red) representing Cy5-labeled heat-denatured ssDNA fragmentsin the original sample. Complete separation of ssDNA and dsDNA fragments was observed allowing for quantification, lengthdistribution analysis and isolation of both fractions.

Investigation of RNA .DNA hybridsAnalysis of products of first-strand cDNA synthesis. As a control, GeneRuler 100 bp DNA Ladder Plus (green, EtBr) was added tothe sample before electrophoresis. Most of the first-strand products (red, Cy5) migrated behind the dsDNA control as expectedfor RNA � DNA hybrids. A part of the first strand products were single-stranded.

Separation of nicked DNA from intact dsDNADetection of site-specific single-stranded breaks in 22 l-phage DNA fragments generated with BanI digestion. The DNA samplewas treated with the site-specific nicking endonuclease N.BstNBI to form site-specific single-stranded breaks at 61 sites. DNAfragments containing single-stranded breaks gave rise to shorter ssDNA fragments representing the nicked strand after thedenaturation step in 2D-SDE. The short DNA fragments from nicked strands migrated in front of the arc representing intactdsDNA fragments.

Separation of bulge-containing molecule from perfectly matched DNASeparation of two heteroduplexes (265 bp) containing nine unmatched nucleotides (a 9 nt bulge) in the wild-type strand from amixture of perfectly matched DNA fragments. A 274-bp PCR product was amplified from human genomic DNA having a 9-bpdeletion in one allele of exon 11 in the C-kit gene. The PCR resulted in the formation of two bulge-containing heteroduplexes(265 bp, with 9 nt bulge) and two homoduplexes (274 and 265 bp). The PCR product was mixed with a sample of 14 perfectlymatched DNA fragments. The two heteroduplexes (green) migrated in front of the arc representing the perfectly matched Cy5-labeled dsDNA fragments. Cy5-labeled fragments are red in the figure, or yellow if the fragments were long enough to also stainheavily with EtBr. The homoduplexes generated in the PCR (green) migrated as expected in the arc of the Cy5-labeled perfectlymatched DNA fragments.

ACKNOWLEDGMENTS This work was supported by the Icelandic Research Council,the University of Iceland Research Fund, the Science Fund of Landspitali-UniversityHospital and BioCule Inc. BioCule Inc. has applied for a patent on the method.Authors G.H.G., B.G., H.G.T. and J.J.J. own stock and B.G. and H.G.T. are currentlyemployed by BioCule. BioCule also funds research projects in J.J.J.’s researchlaboratory.

COMPETING INTERESTS STATEMENT The authors declare competing financialinterests (see the HTML version of this article for details).

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