Electrophoresis and Blotting                                                                                 Introductory ...
Electrophoresis and Blotting of DNAHow Gels Are Used to Resolve DNA                                                 is pla...
Electrophoresis and Blotting of DNAconcentrations of agarose ranges from 100 to 60 000 base                DNA can be load...
Electrophoresis and Blotting of DNA  The voltage applied to the electrophoresis system influ-              cannot rejoin to...
Electrophoresis and Blotting of DNA                                                                                       ...
Electrophoresis and Blotting of DNA   The specificity of the hybridization is determined by the            Detectionwash st...
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  1. 1. Electrophoresis and Blotting Introductory articleof DNA . Introduction Article ContentsSandeep Tamber, University of British Columbia, Vancouver, Canada . A Review of DNA Structure . How Gels Are Used to Resolve DNARobert EW Hancock, University of British Columbia, Vancouver, Canada . Agarose and Acrylamide Gels . Southern Blotting: Theory and PracticeDNA electrophoresis and blotting are techniques commonly used to visualize DNA. . ConclusionElectrophoresis uses gels to separate DNA molecules on the basis of size; whereas blottingallows researchers to identify specific DNA fragments on the basis of their sequences. doi: 10.1038/npg.els.0003746IntroductionGels that resolve deoxyribonucleic acid (DNA) are funda- A Review of DNA Structuremental to the study of molecular biology. Virtually every DNA is a negatively charged, double-stranded moleculemolecular biological procedure involving DNA, including composed of deoxyribonucleotide units that each containDNA purification, RFLP (restriction fragment length po- the sugar deoxyribose, phosphate and one of four bases.lymorphism), analysis of DNA polymorphisms, DNA The sugar-phosphate portions are joined to create a back-cloning, and DNA sequencing, requires the use of a gel. bone, which imparts a negative charge along the entireIndividual DNA molecules are separated on the basis of length of the molecule. Attached to each sugar unit is one ofsize in a system in which small fragments move more rap- four bases: adenine, thymine, cytosine or guanine. It is theidly than larger fragments. When a stain is added to vis- sequence, or order, of these bases that determine the ge-ualize the separated DNA molecules, a characteristic netic uniqueness of individual stretches of DNA in differ-pattern of DNA ‘bands’ is observed. The resolving power ent organisms.of these gels is much higher than can be achieved by other DNA comprises two strands of linked deoxyribonuc-DNA fractionation methods such as equilibrium density leotides that are oriented in opposite directions. The basescentrifugation. Depending on the method of preparation, on the two different strands specifically interact with eachgels can be used to discriminate between DNA molecules other through hydrogen bonds, such that adenine alwaysthat differ in length by a single nucleotide. Also, gels can be pairs with thymine and cytosine always pairs with guanine.used to detect smaller amounts of DNA (0.02 ng) than Because of this specific base pairing, the sequence of DNAalternative techniques such as UV absorbance ( $ 50 ng). is said to be complementary. If the sequence of one strand is In addition to the roles mentioned above, these gels are known, the sequence of the other strand can be deduced.the starting points for other techniques such as alkaline The cumulative effect of these base-pairing interactionsblotting. Alkaline blotting is a variation of the Southern produces a very stable double-stranded molecule known asblotting procedure first described by E. M. Southern in the double helix. High temperatures or highly alkaline so-1975. This procedure involves transferring to a membrane lutions (i.e. with a pH greater than 11) are required toDNA fragments that have been separated on a gel, and separate (denature) the two DNA strands from each other.immobilizing them, while preserving the banding pattern The two strands will re-join (renature) once the pH and/orof the gel. Specific detection methods can then be used to temperature are lowered. The temperature at which DNAidentify particular sequences of interest from a large pop- denatures is referred to as the melting temperature of theulation of DNA molecules. DNA molecule; this is also the temperature at which the This article will begin by briefly reviewing the relevant DNA renatures into a double helix. The melting temper-physical and chemical features of DNA as they relate to an ature of a double-stranded DNA molecule depends on theunderstanding of gels and blotting. Then the theoretical number of base pairs it contains. Short duplexes have lowerand practical aspects of using agarose and acrylamide gels melting temperatures than longer stretches of double-to analyse DNA will be covered. The article will conclude stranded DNA. The sequence of a DNA molecule alsowith a discussion of Southern blotting, an important tech- influences its melting temperature. Guanine-cytosinenique used in molecular biology that requires DNA to be (GÁC) base pairs are more stable than adenine-thymineseparated in gels. (AÁT) ones. Therefore, the more GÁC base pairs a DNA molecule has, the higher its melting temperature will be. NATURE ENCYCLOPEDIA OF LIFE SCIENCES / & 2004 Nature Publishing Group / www.els.net 1
  2. 2. Electrophoresis and Blotting of DNAHow Gels Are Used to Resolve DNA is placed into a chamber so that it stands vertically. Both systems have specific applications as discussed below.Separation of DNA in gels, or DNA electrophoresis, is The size of gels that can be run is variable and depends onbased on the fact that DNA is negatively charged and will the intended use of the gel. Small gels are generally used tomove in response to an electrical field. The gel in which the resolve simple mixtures of DNA as they separate DNADNA is separated is a viscous mesh that looks and acts like rapidly and require little material to prepare. However, if avery hard table jelly. The mesh comprises long interwoven large DNA sample or a highly complex mixture needs to beor chemically linked molecules that are penetrated by wa- resolved, larger gels must be used.ter-filled channels through which the DNA can pass. Anelectrical circuit is made by placing a gel between two elec-trodes in a chamber that is connected to a power supply.The chamber is then filled with electrophoresis buffer (ahigh salt solution), which completes the circuit. When a Agarose and Acrylamide Gelsconstant voltage is applied to the system, the negativelycharged ions in the buffer will migrate towards the positive The matrix or gel that the DNA migrates through consistselectrode (anode) and the positively charged ions will mi- of one of two polymers, agarose or acrylamide. Agarose isgrate to the negative electrode (cathode), creating a current a linear carbohydrate polymer extracted from seaweed.through the system. Since DNA is negatively charged, it Agarose gels are made by first dissolving the powderedmigrates towards the anode, but its migration is impeded agarose in hot electrophoresis buffer, and then pouring theby the mesh-like gel, and larger DNA molecules are more mixture on a horizontal gel casting plate. When the agaroseimpeded than smaller ones. cools it forms an intricate network of hydrogen bonds that result in a semi-solid gel. The resulting gel contains a matrix of pores through which the DNA must migrate. The size ofElectrophoresis systems the pores depends on the concentration of agarose in the gel; the higher the concentration of agarose, the smaller willThere are two major electrophoresis apparatus arrange- be the pores. This in turn will determine its ability to im-ments, horizontal and vertical. In a horizontal system, as pede the migration of DNA in an electric field. Generally,illustrated in Figure 1, the gel lies flat on a platform in a tank. agarose concentrations between 0.3% and 3% are used.In a vertical system, the gel is clamped on to a support that The size of DNA fragments that can be resolved by these Power supply Electrode (–) Well Gel box Comb Electrode (+) Gel casting trayFigure 1 A horizontal agarose gel apparatus. Wells are formed in the gel by placing a comb in the melted agarose before it hardens. The gel is then placedin a horizontal electrophoresis chamber filled with electrophoresis buffer and connected to a power supply. When voltage is applied to the system, the DNAmigrates towards the anode in a tight ‘lane’ as shown in the diagram of the resulting gel.2 NATURE ENCYCLOPEDIA OF LIFE SCIENCES / & 2004 Nature Publishing Group / www.els.net
  3. 3. Electrophoresis and Blotting of DNAconcentrations of agarose ranges from 100 to 60 000 base DNA can be loaded into a 0.5 cm wide well of a 1% ag-pairs in length. arose–TAE gel and be resolved adequately. Acrylamide is a monomer that polymerizes in the pres- Before being loaded on to a gel, the DNA is mixed with aence of free radicals. The free radicals are generated by sample buffer. This buffer consists of a viscous liquid suchammonium persulfate and are stabilized by N,N,N’,N’-tet- as glycerol, sucrose or Ficoll, and the dye bromophenolramethylethylenediamine (TEMED). N’,N’-methylene- blue. The sample buffer may contain another dye, xylenebisacrylamide is included in the polymerization reaction cyanol FF, in addition to the bromophenol blue. The pur-to covalently crosslink the long chains of polyacrylamide pose of this buffer is to make the DNA sample dense so thatinto a gel. The pore size of the resulting gel is dependent on it sinks to the bottom of the well. The dyes, in addition tothe length of the polyacrylamide chains, which is in turn colouring the sample to facilitate loading, are used todetermined by the concentration of acrylamide in the gel. monitor the progress of the electrophoresis. Both bromo-The degree of crosslinking, as determined by the N’,N’- phenol blue and xylene cyanol FF migrate in a predictablemethylenebisacrylamide concentration, also influences fashion in response to an electrical field. The migration ofpore size. Typically, acrylamide concentrations between these two dyes varies slightly under different conditions. In3.5% and 20% are used with one molecule of N’,N’’-meth- a 1% agarose–TAE gel, bromophenol blue comigratesylenebisacrylamide for every 29 molecules of acrylamide. with small DNA molecules around 500 base pairs long andSmaller DNA molecules can be resolved by acrylamide gels xylene cyanol FF typically comigrates with DNA frag-than with agarose gels and the optimal size range for sep- ments around 5 kilobase-pairs long. A disadvantage ofaration is from 1 to 2000 base pairs. these dyes is that they may mask the appearance of comi- The polymerization of acrylamide is inhibited by oxy- grating DNA, if it is present in low concentrations.gen. These gels must therefore be poured vertically betweentwo glass plates that are held apart by two plastic spacers.This process is time-consuming and technically more de- Detectionmanding than horizontal agarose gel electrophoresis.However, acrylamide gels have excellent resolving power. The dye most commonly used to stain DNA gels is et-They are used when a high degree of separation (i.e. a size hidium bromide. Ethidium bromide binds DNA betweendifference of a single base pair) is required, such as when the bases (i.e. it intercalates DNA) and fluoresces orangesequencing DNA. when exposed to ultraviolet light (260–360 nm). The lower limit of DNA detection by ethidium bromide is around 2 ng (2 Â 10 2 9 g). This level of sensitivity is adequate for mostElectrophoresis buffers purposes. However, there are more sensitive DNA binding dyes, such as SYBR green (detection limit 0.02 ng) that canTwo buffers are commonly used for DNA electrophoresis, be used if required.Tris acetate – EDTA (TAE) and Tris-borate – EDTA Gels can be stained either before or after an elect-(TBE). These buffers can be used interchangeably as they rophoresis run. To stain a gel before an electrophoresisare both good electrical conductors. Supercoiled DNA, the run, ethidium bromide is added to the melted agarose be-form that DNA adopts in nature, separates better in TAE fore it is poured on to the gel tray. This staining method isthan in TBE. Also, concentrated solutions of TAE have a the quickest and offers the advantage that the position oflonger shelf-life than concentrated TBE solutions, which the DNA can be monitored during the separation. Alter-tend to precipitate over time. A disadvantage of TAE is natively, the gel may be placed in a dilute solution of et-that its buffering capacity is not as great as that of TBE. hidium bromide for 10–30 min after the electrophoresisTherefore, when it is necessary to run gels for a long time, run.or at a high voltage, TBE should be used as the elect-rophoresis buffer. Gels made with TBE are stronger thanthose made with TAE; this feature is an advantage whenmaking low percentage gels, which tend to be fragile. Factors influencing mobility The rate of DNA migration through a gel depends on theDNA samples size and shape of the DNA molecules. A linear DNA mol- ecule will migrate at a rate that is inversely proportional toThe amount of DNA loaded on to a gel depends on the the log10 of its number of base pairs. This relationship iscomplexity of the sample. More sample must be loaded if due to the increased difficulty larger DNA molecules havethere are a large number of DNA fragments in the sample, ‘worming’ their way through the pores of the gel as well asso that each fragment can be readily detected upon stain- their greater frictional drag. Plasmids and other circularing. However, if too much DNA is loaded, the resolving DNA molecules are more compact than linear DNA mol-power of the gel will be lost and the result will be a smear of ecules, and thus they migrate through the gel at rates higherDNA on the gel. Up to 500 ng of a complex mixture of than would be expected from their molecular weights. NATURE ENCYCLOPEDIA OF LIFE SCIENCES / & 2004 Nature Publishing Group / www.els.net 3
  4. 4. Electrophoresis and Blotting of DNA The voltage applied to the electrophoresis system influ- cannot rejoin to itself. The gel is rinsed again with distilledences the rate of DNA migration, as do gel percentage and water and placed into the final treatment solution, onebuffer composition. Consideration of Ohm’s law (V 5 IR) containing a buffered salt solution (pH 7). The purpose ofhelps one understand the effect of these factors. The cur- this incubation is to neutralize the gel (i.e. bring the pHrent (or ion movement,) I in an electrical circuit is directly down to less than 9), so that the DNA will bind to theproportional to the electrical field or applied voltage V and membrane.inversely proportional to the resistance R of the system,provided by the gel. As the percentage of either agarose oracrylamide in a gel increases, the mesh of molecules in the Transferring the DNA from gel to membranegel becomes tighter, increasing the resistance of the systemand thereby impeding the movement of DNA through the The matrix to which the single-stranded DNA fragmentsgel. Gels made with TBE buffer tend to be more rigid (less from the gel are transferred is known as a membrane. Twoporous) than those made with TAE, so that DNA moves types of membrane are in common use: nitrocellulose andmore slowly through gels made with TBE than through gels nylon. Nitrocellulose was first used by Southern when hemade with TAE. developed the blotting technique. Although this membrane is adequate for most purposes, it is rather fragile and does not bind DNA less than 500 bases in length. Nylon mem- branes are much stronger than nitrocellulose membranesSouthern Blotting: Theory and Practice and will not easily break with handling even after repeated use. Also, nylon membranes can efficiently bind DNA asIf the identity of a specific DNA fragment on a gel must be short as 50 bases in length, and can bind up to 5 times moredetermined, it is necessary to transfer the DNA on to an DNA per cm2 than can nitrocellulose. Another advantagealternative medium while maintaining the separation or of nylon membranes is that they can bind DNA covalentlybanding pattern of the gel. This transfer step is required when they are subjected to either UV crosslinking or al-because, if a DNA gel is left to sit for an extended period, kaline transfer. DNA is immobilized to nitrocellulosethe DNA will diffuse out of the bands. The transfer process membranes by baking the membrane in an oven, but theis commonly referred to as Southern blotting. Basically, attachment is noncovalent and thus not as strong as it isthis procedure begins with running an agarose DNA gel. with nylon membranes. It is important to have a strongThe DNA on the gel undergoes certain treatments to sep- association between the DNA and the membrane so thatarate it to its single-stranded form and is then transferred the loss of DNA from the membrane is minimized duringon to a membrane with the ability to bind DNA strongly. the subsequent wash steps. Despite all of the advantagesAfter the DNA has been immobilized on to the membrane, associated with nylon membranes, they have not com-it is reacted with a probe (labelled pieces of single-stranded pletely replaced nitrocellulose membranes because theyDNA of known sequence). Since the two strands of DNA tend to demonstrate a high background noise to signalare complementary, the unknown DNA fragments that ratio after they have been stained. DNA sequences of in-bind (hybridize) to the probe will have a sequence the same terest may therefore be masked by the high level of back-as or similar to the probe’s. ground staining or radioactivity. The blotting or transfer procedure has remained essen-Gel pretreatment tially unchanged since it was first described in 1975 and is shown in Figure 2. Basically, the gel is placed on top of aPrior to the transfer step, the DNA in the gel must undergo piece of filter paper lying on a support in a tray containingsome treatment steps to ensure the complete transfer of the transfer buffer (a high salt solution). The membrane isDNA from the gel to the membrane. First the gel is placed placed on top of the gel, followed by more filter paper, ain a glass tray containing a solution of hydrochloric acid so large pile of paper towels, and finally a heavy object such asthat a process known as depurination can occur. The acid a text book.reacts with and removes some of the adenine and guanine The transfer of DNA from the gel to the membrane oc-bases from the DNA molecule. The gel is washed with curs by capillary action. The transfer buffer is drawn updistilled water and placed into another glass tray contain- through the filter paper, the gel, the membrane, and finallying a solution of sodium hydroxide, a strong base that up to the stack of paper towels. Because DNA gels are quiteserves two purposes. The first is to break the DNA back- porous, as the transfer buffer migrates to the paper towels,bone where it has been depurinated. This is important be- the DNA is carried out of the gel and blotted on to thecause smaller DNA molecules transfer more efficiently membrane. The high salt concentration in the transferthan larger ones. The second role of sodium hydroxide is to buffer promotes the binding of DNA to the membrane.promote the denaturation (i.e. unzipping) of the double- Generally, the transfer procedure takes approximately 18 hstranded DNA molecules such that the DNA becomes to complete. If the DNA fragments are less than 1000 basesingle-stranded and, after transfer and immobilization, pairs long, the transfer may take as little as 1–2 h.4 NATURE ENCYCLOPEDIA OF LIFE SCIENCES / & 2004 Nature Publishing Group / www.els.net
  5. 5. Electrophoresis and Blotting of DNA Weight Buffer Paper towels Membrane Filter paper wick Gel SupportFigure 2 The Southern transfer apparatus. As the buffer travels up the filter paper wick through the layers of filter paper, gel, membrane and paper towels,the DNA is deposited from the gel to the membrane. The weight (say, a textbook) ensures that all of the layers remain in close contact during the transferprocess. There have been several modifications of the original of all hybridization procedures is to maximize the signalblotting procedure described by Southern. These modified from the probe binding to the target DNA on the mem-procedures attempt to decrease the transfer time and in- brane, while minimizing its nonspecific binding to thecrease the efficiency of transfer. One procedure involves membrane and nontarget DNA (background).setting the transfer apparatus upside down so the gel is on Before hybridization is carried out, the membrane isthe top. This downward capillary transfer aided by gravity, treated with a blocking agent to prevent nonspecific asso-in addition to being faster than the conventional proce- ciation of probe with the membrane. A variety of poly-dure, does not place excessive pressure on the gel, and thus meric inert agents, such as skim milk powder, Denhardt’sthere is no possibility of crushing it. Another modification, reagent (a mixture of Ficoll, polyvinylpyrrolidone and bo-electroblotting, involves using an electrophoresis appara- vine serum albumin), denatured fragmented salmon spermtus to mobilize the DNA from the gel on to the membrane. DNA or heparin can be used to bind the unused DNAVacuum blotting uses vacuum pressure to draw the trans- binding sites on the membrane. Skim milk powder andfer buffer through the gel. Denhardt’s reagent are two most commonly used blocking Alkaline blotting involves the use of a positively charged agents. Skim milk powder is the cheapest and easiest to use,nylon membrane and sodium hydroxide in the transfer whereas Denhardt’s reagent can more effectively blockbuffer. With this modified procedure, the DNA denatures nylon membranes.and binds covalently to the membrane as it is being trans- The basic hybridization procedure involves placing theferred from the gel, thus minimizing DNA loss during the membrane in a plastic bag or hybridization tube with thetransfer. With this procedure the transfer process only labelled probe in a small volume of a high salt solution.takes approximately 2 h and the denaturing gel pretreat- Keeping the volume of the hybridization solution lowment step is optional. serves to increase DNA concentration, while the high salt content promotes DNA binding. The specificity of the hy- bridization can be increased by adding formamide (whichDNA hybridization disrupts hydrogen bonds) to the buffer. After the hybrid- ization reaction, the unbound and nonspecifically boundTo understand DNA hybridization, it is important to re- probe is removed from the membrane via a series of wash-member that the DNA immobilized on the membrane is es. As the aim of the hybridization process is to encouragesingle-stranded and that any introduced single-stranded hybrid formation between the probe and target DNA whilenucleic acid (i.e. the probe) will bind to this immobilized destabilizing all other hybrids, the actual hybridizationDNA. The stability of the resulting double-stranded mol- procedure must be tailored to the specific probe and targetecule depends on the degree of similarity between the two DNA. The variations made on this basic procedure aim tostrands (i.e. the number of complementary base pairs), increase both the specificity and the amount of probetemperature, ionic strength, and the presence of chemicals bound to the target DNA.that disrupt hydrogen bonds, such as formamide. The goal NATURE ENCYCLOPEDIA OF LIFE SCIENCES / & 2004 Nature Publishing Group / www.els.net 5
  6. 6. Electrophoresis and Blotting of DNA The specificity of the hybridization is determined by the Detectionwash steps, which vary with respect to temperature and saltconcentration. The wash steps are carried out in a step-wise In the past, probes were usually labelled with radioactivemanner. First a low-temperature, high-salt wash is done to phosphorus and the resulting labelled Southern blots wereremove unbound probe. Then more stringent washes at imaged on X-ray film. Today, however, there are morehigher temperatures, with less concentrated salt solutions, convenient and safer labels. The most common compoundare done to remove probe bound nonspecifically to the used to label probes is digoxigenin (DIG). The DIG label ismembrane and that bound to nontarget DNA via short bound to the probe and can be detected with commerciallyregions of complementarity. In theory, DNA hybrids con- available antibodies that have an enzyme attached to them.sisting of the probe and target DNA should have no or few The enzyme can be one of a variety of enzymes that con-mismatched base pairs and thus a higher melting temper- verts a substrate to yield a coloured product that can beature than any other type of hybrid formed. Therefore, the visualized on the membrane. This method of detection ishigher temperatures of the latter wash steps should melt very sensitive. The lower limit of DNA detection is 0.1 pgany nonspecific probe–nontarget DNA hybrids and leave (10 2 13 g). Another advantage of the DIG system is that thethe probe–target DNA hybrids intact. Similarly, since salt antibodies are highly specific for the DIG label, thus therestabilizes DNA binding, decreasing its concentration in the is less background noise associated with it.latter wash solutions will destabilize any mismatched hy-brids, leaving the probe bound only to the target DNA. The strength of the signal depends on the amount of Conclusionprobe that is bound to the target DNA. This interactiondepends on the extent of sequence similarity between the DNA gels are a fundamental methodology in moleculartarget DNA and probe, the hybridization time, the DNA biology. Together with alkaline blotting, they provide sci-concentration and the probe length. Although longer hy- entists with powerful and specific methods for analysingbridization times lead to a stronger signal, they also lead to unknown DNA molecules. The methods described abovehigher nonspecific probe binding. Hybridization times are provide the basis for many practical procedures and aretherefore generally kept as short as possible. The rate and much used in forensic science, disease diagnosis, discoveryextent of hybridization increase with the DNA concentra- of the genetic basis of disease, and investigation of pop-tion. However, it is difficult to determine or predict what ulation health and epidemiology.the concentration of the DNA immobilized on the mem-brane will be, which is why it is important to keep thehybridization volume low. While more probe can be used, Further Readinggenerally this is not done because it may also lead to a high Ausubel FM, Brent R, Kingston RE et al. (eds) (1993) Current Protocolslevel of nonspecific association of probe with the mem- in Molecular Biology. New York: Wiley.brane. Inert polymers such as dextran or PEG (polyeth- Old R and Primrose S (1994) Principles of Gene Manipulation, An In-ylene glycol) form interlocking meshes with the probe and troduction to Genetic Engineering, 5th edn. London: Blackwell Scien-can be used to increase the effective probe concentration. tific. Sambrook J, Tritsch EF and Maniatis T (1989) Molecular Cloning. AProbes are usually selected to be around 200 bases in length Laboratory Manual. New York: Cold Spring Harbor Laboratory.to give good signals. The signal given off by smaller probes Southern EM (2000) Blotting at 25. Trends in Biochemical Sciencesis not as strong, and longer probes may be difficult to wash 25(12): 585–588.away if they associate nonspecifically with the immobilized Westermeier R (2001) Electrophoresis in Practice, 3rd edn. New York:DNA. Wiley.6 NATURE ENCYCLOPEDIA OF LIFE SCIENCES / & 2004 Nature Publishing Group / www.els.net