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Pulsed-field GelElectrophoresis of DNAEdwin M Southern, University of Oxford, Oxford, UK

Conventional electrophoresis is not useful for DNA larger than 50 000 base pairs (bp). For

large DNA molecules up to 7 million bp (7 Mb) it is necessary to use pulsed-field

electrophoresis. This protocol describes how the procedure is carried out.

Introduction

The practical usefulness of conventional agarose gelelectrophoresis of DNA is limited to the separation of DNA up to 50 kb. Pulsed-field gel electrophoresis (PFGE)is capable of separating DNA molecules up to 7000 kb(7 Mb). First described by Schwartz and Cantor in 1984,the method has undergone a number of improvements and

modifications.The practical applications are many. In some lower

eukaryotes such as yeast and trypanosomes, the chromo-somes are small enough to be resolved as individual bands,making karyotyping possible; any cloned DNA sequencecan be assigned to chromosomes very easily by hybridiza-tion.

In higher eukaryotes even the smallest chromosomes aretoo large to separate by PFGE. However, restrictionnucleases, such as SfiI and NotI, which have an 8bprecognition sequence, produce fragments too large forconventional gels, so mapping with these enzymes requiresthe use of PFGE. Analysis of the large DNA fragments by

blotting (Southern, 1975) and hybridization can providemaps of large stretches of DNA, which can help inestablishing physical linkage between genetic loci, com-plementing the methods of classical genetics.

One feature of pulsed-field electrophoresis is thenecessity to prepare DNA samples avoiding all possibilityof random shearing of the DNA. There are several ways of preparing high molecular weight DNA, all of theminvolving the embedding and subsequent lysis of cells inblocks of agarose. One of the procedures is to pour themolten agarose and cell mixture on to a Petri dish, allow itto solidify, and then cut to the appropriate size of plug.However, a special plug mould (Figure 1) has severaladvantages over this method: it produces regular-shapedblocks that are reproducible and easy to handle. Inaddition, a sample well-former (comb) that matches thesize of the blocks enables easy loading of samples on to thegel. For ease of loading, the size of wells should be slightlylarger than the size of the plug after it has been cut anddigested.

Types of apparatus

For more detailed descriptions of construction of thdifferent types of apparatus, the reader is referred to thoriginal articles.

Field inversion gel electrophoresis (FIGE)

The use of field inversion to improve electrophoretiresolution led Tombs (1987) to use it for separating largDNA molecules. Extensive studies on the separatiocharacteristics of this system have been done by Carle eal. (1986). Net forward movement of DNA is achieved busing a greater part of the switching cycle for forward thafor reverse migration or a higher voltage in the forwarthan in thereverse direction. Under a givenset of switchinconditions, a range of DNA sizes is fractionated busurprisingly, some larger sizes show greater mobility thashorter fragments. To overcome this problem, a ‘ramp’ iused such that the time of the switching cycle is increase

throughout the run according to a preprogrammesequence (see Electrophoresis Conditions, below). Thsystem has the advantage that tracks are straight, and thapparatus is simple. However, the relationship betweesize and mobility is not understood, makingaccurate sizindifficult, especially for fragments that migrate close to thregion of minimum mobility, and bands are often broad

Article Contents

Secondary article

. Introduction

. Step 1: Equipment and Solutions

. Hazards

. Hints and Tips

Figure 1 A Perspex plug mould used for preparing DNA embedded in

agarose and a gel comb for making sample loading slots in the gel toaccommodate the DNA agarose plugs.

ENCYCLOPEDIA OF LIFE SCIENCES ©2002, John Wiley & Sons, Ltd. www.els.net

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Homogeneous crossed field electrophoresis

The main advantage of this form of electrophoresis is thatthe DNA migrates in straight tracks, which makes it easierto compare bands between tracks. The first apparatus toachieve this was a circular gelin a square horizontal gel box(Anand, 1986; Southern et al ., 1987). The gel is turned at

each switching cycle so that the DNA experiences a changein field angle of greater than 908. It was initially designedtotest a model of the mechanism of DNA separation(Southern et al ., 1987). In another form of homogeneousfield apparatus, the electrodes are in a hexagonal array andconnected by a series of resistors. The electric fields are at1208 to one another and since the electrode potentials areclamped to provide a near homogeneous field, thetechnique has been named contour-clamped homogeneouselectric field (CHEF) gel electrophoresis.

Choice of apparatus

The field inversion apparatus (Carle et al ., 1986; Tombs1987) is available from several suppliers including Hoeferand Acronym Pvt. Ltd. (Boronia 3155, Victoria, Austra-lia). The vertical apparatus (Gardiner et al ., 1986) isavailable from Beckman Instruments and CHEF appara-tus from Bio-Rad. The rotating gel apparatus is notcommercially available.

There are some major advantages in obtaining straighttracks across the whole width of the gel, since one can runsize markers on the two end tracks and all the remainingtracks on a gel can be used for sample analysis. Thissimplifies the accurate sizing of fragments.

Electrophoresis conditions

TherateofmigrationofDNAthroughageldependsonthegel concentration, voltage gradient, and temperature.Generally, electrophoresis is done in 0.5Â TAE or 0.5ÂTBE at a constant temperature and with a fixed voltagegradient across the electrodes. TAE buffer is preferredbecause TBE buffer can lead to subsequent problems withtransferring the DNA when blotting on to nitrocellulosefilters. The voltage gradients for optimum separationsdepend on the size range to be resolved and the gelconcentration (see Figures 2, 3 and 4). The appropriate

voltage gradient for a particular apparatus can be found inpublished articles.

Increasing temperature increases the mobility of DNA.Therefore, to make results from different gel runscomparable, it is essential to control temperature. Adetailed analysis of the effects of temperature has beenpublished (Mathew et al ., 1988). The effect of gelconcentration and pulse time is discussed in subsequentsections, and is based on experiments with the rotating gelsystem.

Effect of pulse time

Increasing the pulse time results in an increase in the sizrange of DNA molecules separated. There is, however, consequential loss of resolution i.e. smaller fragments. Acan be seen from Figure 2, at 6 V cm21 DNA molecules uto 400 kb are separated with a 20s pulse and 1000kb DNAwith a 60 s pulse. However, increasing the pulse time t100 s results in some smearing of the larger molecules. Tachieve good resolution of these larger DNA moleculethe voltage gradient must be reduced, which requires a

Figure 2 Effect of pulse time. Saccharomyces cerevisiae (X2180-1B)chromosomes and phage lambda (c1857Sam7) oligomers run on a 1.5%agarose gel in 0.5Â TAEat6Vcm21 and 208C. Thepulsetimesused we

(a)20 s, (b)60 s and(c) 100s. Thehighresolution,showingpolymorphis(possiblydue to mitotic recombination) in chromosome 3, is clearly visibin the 20 s pulse. In the 100 s pulse, chromosome 12 polymorphism

resolves as a doublet near thetop of thegel. Thegeneral band broadeninseen with this pulse time canbe reduced by reducing thevoltage gradien

Pulsed-field Gel Electrophoresis of DNA

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increased pulse time and results in an increased run time.For example, if a 1.5% agarose gel is run in 0.5 Â TAE at6 V c m21. with 100 s pulse time, the total run time on arotating gel system with a 22 cm diameter gel is 34 h; at5 V c m21, pulse time should be increased to 144 s and totalelectrophoresis time to 49 h.

Gel concentration

1.5% agarose gels are normally used for all separations up

to 1.5 Mb purely because of the ease of handling theserelatively rigid gels. Lower gel concentrations can be used(Figure 3), but the pulse time and voltage gradients shouldbe adjusted to achieve the required resolution.

Separation of very large DNA

For molecules larger than about 1 Mb, low voltagegradients have to be used in combination with low-concentration agarose gels. For example, 0.6% agarose

gels at a voltage gradient of 1.2 V cm2 1 resolve moleculein the size range of 1 Mb to 2.6 Mb. These runs are carrieout at 58C using a pulse duration between 0 and 60 minThe total duration of the run is between 1 and 2 weeksdepending on the resolution required (Figure 4).

Preparation and processing of samples

This type of electrophoretic analysis requires extra care i

the preparation of DNA of very large size as well as oadequate size markers; where possible, all solutions shoulbe autoclaved. Solutions that cannot be autoclaved shoulbe made up in glass-double-distilled, autoclaved water anthen filtered through a Millipore filter. Some of thesmethods have been described elsewhere (Anand, 1986Smith et al ., 1986; van Ommen and Verkerk, 1986).

The procedure described here is for tissue culture cellbut it can also be used for other cell types (e.g. circulatinlymphocytes).

Figure 3 Effect of agarose gel concentration. A composite gel with

agarose concentrations of (a) 1.5%, (b) 1.25%, (c) 1.0% and (d) 0.75%was run in 0.5Â TAE at 208C. The voltage gradient was 4 V cm21 with a

total run timeof 35h and a pulseduration of90 s.The samples werephagelambda (c1857Sam7)oligomers and Saccharomycescerevisiae (X2180-1B).

Figure 4 Separation of very large DNA. Saccharomyces cerevisiae

YNN318, X2180-1B and Schizosaccharomyces pombe 972 chromosomesrun on 0.7% agarose ((a) and (c)) and 0.6% agarose gels ((b) and (d)).Electrophoresis was in 0.5Â TAE at 58C and a voltage gradient of 1.2 V cm21 was used with 60 min pulses. The total run time was 7 days ((a) an

(b)) and 14 days ((c) and (d)). Chromosome 12 of YNN318 is longer thathat of X2180-1B andis separating as such. Therest of thechromosomeso

these two strains are of almost the same size, but the X2180-1B DNA isconsiderably retarded, clearly demonstrating the drag effects caused by

excessive DNA loading in these gels.


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Step 1: Equipment and Solutions

. 0.5ÂTAE (Recipe 1)

. 0.5ÂTBE (Recipe 2)

. Low gelling temperature (LGT) agarose. Sea PlaqueLGT agarose (FMC Corporation 200 E. RandolphDrive, Chicago, IL 60601, USA ) has proved to be quite

reliable; however, there are batch-to-batch variations inthe purity of LGT agarose. It may therefore be desirableto test a few batches to find one that yields the mostintact DNA in agarose plugs and is free from restrictionenzyme inhibitors. This can be done by embeddingbacteriophage lambda DNA in agarose plugs and thentesting for degradation and digestion. Alternatively,agarose solution can be purified by treatment with AG-1-X2 (200–400 mesh) or DEAE-cellulose ion exchangeresin. This can be done using 100–200 mL of 1–1.5%agarose, which can then be stored at 48C for subsequentuse. FMC corporation also produce a batch-tested,guaranteed nuclease-free grade of LGT agarose.

. 1% NDS (1) lauryl sarcosine 0.5 mol L2 1 EDTA,10mmolL2 1 Tris, pH 9.5. This solution is used

extensively in the preparation and storage of DNAembedded in agarose. To make 500 mL of this solutionadd 93 g of EDTA to approximately 350 mL of distillewater. Add 0.605g of Tris base and then add soliNaOH pellets to bring the pH above 8.0 to dissolve thEDTA (10–12 g of NaOH pellets are needed for thisDissolve 5.0 g of lauryl sarcosine in 50 mL of distille

water and add this to the first solution. Adjust the pH t9.5 using concentrated NaOH and make the volume uto 500 mL. Filter through a 0.2mm Millipore filter anstore at 48C.

. Proteinase K (Boehringer Mannheim). Dissolve in NDat a concentration of 20 mg mL2 1 just before use.

. Pronase (Boehringer Mannheim). Dissolve in NDS at concentration of 20 mg mL2 1 just before use. Pronasworks just as well as proteinase K and is much cheapeHowever, it maybe advisableto inactivatecontaminanthat may be present by autodigesting this pronassolution at 378C for 30 min before use.

. Zymolase-20T (Seikagaku Kogyo Co. Ltd, Tokyo

Japan). Dissolve in 1.2 mol L2 1

sorbitol at a concentration of 20mg mL21 just before use.

. Lyticase (Sigma Chemical Co.). Dissolve in 50%glycerol/water (v/v) to a concentration of 20 unitsmL2

and store at2 208C.. Gelatin (Oxoid Ltd). Dissolve in water at a concentra

tion of 1 mg mL2 1. Dispense 500mL aliquots intmicrocentrifuge tubes, pierce a hole in their topautoclave and store at2 208C.

. Spermidine and dithiothreitol (Sigma Chemical Co.Make 0.1 mol L21 solutions of each in sterile distillewater. Filter through a 0.2 mm Millipore filter and storfrozen at2 208C.

. Phenylmethylsulfonyl fluoride (PMSF) (Sigma Chemcal Co.). This is extremely toxic (see Hazards). Using microcentrifuge tube, make a 1.0 mol L2 1 solution iDMSO (dimethyl sulfoxide, spectrosol grade, BDHThis needs to be warmed to about 258C to produce clear solution, which can then be diluted to the workinconcentration of 0.1mmol L21.

Step 2: Procedure

1. Harvest the cells by centrifugation at 500 g for 10 min

2. Wash the cells in Dulbecco isotonic saline ansuspend the cells in 1.0 mL of the same solutionDisrupt any clumps using dispensing tips or plastitransfer pipettes.

3. Take an aliquot and count in a haemocytometer.4. Dilute the cell suspension to 5–6Â 107 cells mL2

this initial cell concentration can be reduced t3Â 107 cells mL2 1 to give sharper bands and generally improved gel resolution. When workinwith repeated DNA sequences, the cell concentratio

Recipe 2 50ÂTAE

Ingredient

Final concentra-

tion Volume/amount

Tris base 2 mol L21 242 g

Glacial acetic acid 2 mol L21 57.1 mL

EDTA so-dium (0.5-

molL2 1, pH

8.0)

50mmolL21

100 mL

Double-distilled

water to final

volume

1000 mL

1. Dissolve at room temperature. Stable for several months.2. Dilute 1:100 before use to make working concentration of

0.5ÂTAE

Recipe 1 10ÂTBE

Ingredient

Final concentra-

tion Volume/amount

Tris base 890 mmol L2 1 108 g

Boric acid 890 mmol L2 1 55 g

EDTA sodium 25 mmol L 21 9.3g

Double-distilled

water to final

volume

1000 mL

1. Dissolve at room temperature. Stable for several months.2. Dilute 1:20 before use to make working concentration of

0.5ÂTBE.


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Table 1 Hazards associated with this procedure

Acetic acid

CH3COOH

Corrosive. Causes severe burns. Glacial acetic acid is flam-

mable. Harmful if swallowed, inhaled or absorbed throug

skin. Material extremely destructive of tissues of mucous

membranes, upper respiratory tract, eyes and skin. Inha-

lation may be fatal. Used as a fixative. Solutions areirritan

Use a fume hood and wear face protection and gloves whepreparingsolutions andat alltimes when using glacial acet

acid.

Autoclave High-pressure steam autoclaves fitted with safety interlocks

shouldbe usedso asto prevent the premature opening of th

chamber and the explosion of bottles containing liquids.

Take special care using domestic-style pressure cookers.

Use tongs or thermally insulating gloves when loading or

unloading a hot autoclave chamber. When sterilizing

media, unscrew bottle caps half a turn to avoid explosion

during autoclaving. Operate autoclave following manu-

facturer’s guidelines.

Boric acid

H3BO3

Harmful by inhalation, in contact with skin and if swallowed

Irritating to eyes, respiratory system and skin. Possibleteratogen. Reproductive hazard. In case of contact with

eyes, rinse immediately with plenty of water for 15 min an

seek medical advice. In case of contact, immediately wash

skin with soap and copious amounts of water. If inhaled,

remove to fresh air. If not breathing give artificial respira

tion. If breathing is difficult, give oxygen. If swallowed,

wash out mouth with water provided person is conscious

Dimethyl sulfoxide

(DMSO)

(CH3)2SO

Harmful if swallowed. Irritating to eyes and skin. Readily

absorbed through skin. Avoid breathing vapour. Wear

protective clothing. May degrade under storage: keep onl

the minimum quantity necessary, under nitrogen if possibl

Dithiothreitol

(DTT)HSCH2(CHOH)2CH2SH

Harmful in contact with skin and if swallowed. Irritating to

eyes, respiratory system and skin.

EDTA

(diaminoethanetetraacetic acid; ethylenediaminetetraacetic

acid)

Harmful if swallowed. Irritating to eyes, respiratory system

and skin.

Electrical High vacuum equipment for glow-discharge treatment shoul

be used strictly according to the manufacturer’s instruc-

tions, with an implosion guard in place and a fully

functional high-voltage safety cut-out (under atmospheric

pressure).

Electrophoresis Great care must be exercised when using any electrophoresi

equipment, especially high-voltage or constant-current

supplies. If possible, always use commercially supplied

apparatus that has been designed and built to internationelectrical safetystandards: home-made equipment is alway

suspect in this regard. Always check that all wiring

connections are properly made and any interlocks fitted ar

secure before switching on the power supply. Always switc

off the power supply before disconnecting the apparatus.

Arrange the work area to reduce the risk of water or

reagents splashing on to the power pack, leads, cables or

chambers. Preferably use power supplies fitted with elec-

trical earth leakage detection circuitry and automatic cut-

off.


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can be further reduced to 1Â 107 cells mL2 1 for ahighly improved gel resolution.

5. Make 1% LGT agarose in Dulbecco saline. Cool to

378C and keep at this temperature.6. Stick tape (e.g. plastic electricalinsulation tape) on to

one surface of the clean sample plug mould (themould should have been cleaned by boiling in0.25molL21 HCl followed by several washes indistilled water to remove all traces of acid).

7. Warmthe cellsuspension to378C, mix equal volumesof cell suspension and 1% agarose solution anddispense into the plug mould. This mixture may bekeptat 378C while transferring it to the plug mould. Itis advisable to place the plug mould on ice whiletransferring the cell suspension. This enables theagaroseto setsoon afterit is dispensedand so reduces

the risk of cells settling during gelling.8. Leave the mould on ice for 5–10min to set the

agarose plugs completely. Remove the tape andgently push the plugs out, using a bent glass Pasteurpipette, into a Falcon tube containing NDS and1mgmL2 1 pronase (proteinase K can also be used,but pronase from Boehringer works equally well atthe same concentration and is much cheaper).

9. Leave the tube at room temperature for 20–30 minand then incubate at 508C overnight. Replace the

NDS containing pronase with fresh solution ancontinue incubation at 508C for another 24 h.

10. Rinse the plugs inNDS, twice for 2 h,to remove mos

of the pronase. Store in NDS at 48C.

The plugs are stable for several years in NDS. Each pluhas sufficient DNA for at least three separate restrictiodigests.

Hazards

The hazards associated with the chemicals/apparatus usein this protocol are detailed in Table 1.

Hints and Tips

General precautions

Some of the problems associated with partial and limirestriction enzyme digests have already beendiscussed. Italso found that methylation patterns vary substantiallbetween different cell lines and one must therefore b

Hot agarose Care should be taken to avoid burns when pouring hot

agarose.

Hydrochloric acid

(HCl)

May be fatal if inhaled, swallowed or absorbed through skin

Causes burns. Material extremely destructive of tissues of

upper respiratory tract, eyes and skin. Wear protective

clothing and gloves and use face protection when using

concentrated solutions.

Isopropanol(propan-2-ol; isopropyl alcohol)

CH3CH(OH)CH3

Highly flammable. Flash point 128C. Harmful by inhalationingestion or skin absorption. Causes severeirritation to eye

with risk of serious damage. Causes irritation to skin and

respiratory system. Repeated exposure may cause derma-

titis. Wear suitable protective clothing, face protection an

gloves. Use in fume hood or well-ventilated area away from

sources of ignition.

Phenylmethylsulfonyl fluoride

(PMSF)

C7H7FO2S

Very toxic by inhalation, skin contact and if swallowed.

Serine-protease inhibitor. Causes burns. Wear protective

clothing and gloves and use face protection. Contact with

water liberates extremely flammable gases. PMSF is often

used in solution with isopropanol or DMSO: see appro-

priate entries.

Spermidine(N -(3-aminopropyl)-1,4-butanediamine)

NH2(CH2)4NH(CH2)3NH2

Corrosive. Causes burns. Wear suitable protective clothing,gloves and use face protection.

Tris

(tris(hydroxymethyl)aminomethane; 2-amino-2-hydroxyme-

thylpropane-1,3-diol)

Irritating to eyes, respiratory system and skin.


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careful in interpreting and comparing results obtaineusing enzymes that are sensitive to methylation. Anothepoint relates to the sizing of fragments. DNA migration ipulsed-field gels is sensitive to the loading concentrationoverloading results in retardation of DNA fragmentcausing an overestimation of their size (Figure 5). Althougpreliminary work can be done more quickly using hig

DNA concentrations and thus achieving fast autoradiographic results, final, accurate sizing must always be donon gels where DNA concentration drag effects arminimal.

References

Anand R (1986) Pulsed field gel electrophoresis: A technique f

fractioning large DNA molecules. Trends in Genetics 2: 278.

Carle GF, Frank M and Olson MV(1986) Electrophoretic separation

large DNAmolecules by periodic inversionof theelectricfield.Scienc

232: 65.

Gardiner K, Lass W and Patterson D (1986) Fractionation of larg

mammalian DNA restriction fragments using vertical pulsed-fie

gradient gel electrophoresis. Somatic Cell and Molecular Genetics 1

185.

Mathew MK, Smith CL and Cantor CR (1988) High-resolutio

separation and accurate size determination in pulsed-field g

electrophoresis of DNA. 1. DNA size standards and the effect

agarose and temperature. Biochemistry 27: 9204.

Smith CL, Warburton PE, Gaal A and Cantor CR (1986) Analysis

genome organization and rearrangements by pulsed field gradient g

electrophoresis. In: Setlow JK and Hollaender K (eds) Genet

Engineering vol. 8, p. 45. New York: Plenum.

Southern EM (1975) Detection of specific sequences among DN

fragments separated by gel electrophoresis. Journal of Molecul

Biology 98: 503.

Southern EM, Anand R, Brown WRA and Fletcher DS (1987) A mod

for the separation of large DNA molecules by crossed field g

electrophoresis. Nucleic Acids Research 15: 5925.

Tombs MP (1987) Method for electrophoretic separator. Internation

patent application W0-87-00635.

van Ommen GJB and Verkerk JMH (1986) Preparation of genom

DNAin agarose plugs.In: DaviesKE (ed.)Human Genetic Diseases:

Practical Approach, p. 113. Oxford: IRL Press.

Further Reading

Chu G, Vollrath D and Davis RW (1986) Separation of large DN

molecules by contour-clamped homogenous electric fields. Scienc

234: 1582.

Schwartz DC and Cantor CR (1984) Separation of yeast chromosom

sized DNAs by pulsed field gradient gel electrophoresis.Cell 37: 67

Figure 5 Effect of DNA loading. On the left is an ethidium bromide-stained gel photograph of four tracks of Sfi I-digested human female (X:21translocation) DNA. On the right is the corresponding autoradiograph of

these same tracks probed with a human X-chromosome probe thathybridizes to four fragments of 565 kb, 690 kb, 715 kb and 840 kb. TheDNA loadings in tracks 1 to 4 were 1.5 mg, 3.0 mg, 3.0 mg and 6.0 mg.


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