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Electrophoresis and Blotting of DNA

  1. Sandeep Tamber,
  2. Robert EW Hancock

Published Online: 11 MAR 2004

DOI: 10.1038/npg.els.0003746

eLS

eLS

How to Cite

Tamber, S. and Hancock, R. E. 2004. Electrophoresis and Blotting of DNA. eLS. .

Author Information

  1. University of British Columbia, Vancouver, Canada

Publication History

  1. Published Online: 11 MAR 2004

This is not the most recent version of the article. View current version (8 DEC 2013)

Introduction

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading

Gels that resolve deoxyribonucleic acid (DNA) are fundamental to the study of molecular biology. Virtually every molecular biological procedure involving DNA, including DNA purification, RFLP (restriction fragment length polymorphism), analysis of DNA polymorphisms, DNA cloning, and DNA sequencing, requires the use of a gel. Individual DNA molecules are separated on the basis of size in a system in which small fragments move more rapidly than larger fragments. When a stain is added to visualize the separated DNA molecules, a characteristic pattern of DNA ‘bands’ is observed. The resolving power of these gels is much higher than can be achieved by other DNA fractionation methods such as equilibrium density centrifugation. Depending on the method of preparation, gels can be used to discriminate between DNA molecules that differ in length by a single nucleotide. Also, gels can be used to detect smaller amounts of DNA (0.02 ng) than alternative techniques such as UV absorbance (∼50 ng).

In addition to the roles mentioned above, these gels are the starting points for other techniques such as alkaline blotting. Alkaline blotting is a variation of the Southern blotting procedure first described by E. M. Southern in 1975. This procedure involves transferring to a membrane DNA fragments that have been separated on a gel, and immobilizing them, while preserving the banding pattern of the gel. Specific detection methods can then be used to identify particular sequences of interest from a large population of DNA molecules.

This article will begin by briefly reviewing the relevant physical and chemical features of DNA as they relate to an understanding of gels and blotting. Then the theoretical and practical aspects of using agarose and acrylamide gels to analyse DNA will be covered. The article will conclude with a discussion of Southern blotting, an important technique used in molecular biology that requires DNA to be separated in gels.

A Review of DNA Structure

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading

DNA is a negatively charged, double-stranded molecule composed of deoxyribonucleotide units that each contain the sugar deoxyribose, phosphate and one of four bases. The sugar-phosphate portions are joined to create a backbone, which imparts a negative charge along the entire length of the molecule. Attached to each sugar unit is one of four bases: adenine, thymine, cytosine or guanine. It is the sequence, or order, of these bases that determine the genetic uniqueness of individual stretches of DNA in different organisms.

DNA comprises two strands of linked deoxyribonucleotides that are oriented in opposite directions. The bases on the two different strands specifically interact with each other through hydrogen bonds, such that adenine always pairs with thymine and cytosine always pairs with guanine. Because of this specific base pairing, the sequence of DNA is said to be complementary. If the sequence of one strand is known, the sequence of the other strand can be deduced.

The cumulative effect of these base-pairing interactions produces a very stable double-stranded molecule known as the double helix. High temperatures or highly alkaline solutions (i.e. with a pH greater than 11) are required to separate (denature) the two DNA strands from each other. The two strands will re-join (renature) once the pH and/or temperature are lowered. The temperature at which DNA denatures is referred to as the melting temperature of the DNA molecule; this is also the temperature at which the DNA renatures into a double helix. The melting temperature of a double-stranded DNA molecule depends on the number of base pairs it contains. Short duplexes have lower melting temperatures than longer stretches of double-stranded DNA. The sequence of a DNA molecule also influences its melting temperature. Guanine-cytosine (G•C) base pairs are more stable than adenine-thymine (A•T) ones. Therefore, the more G•C base pairs a DNA molecule has, the higher its melting temperature will be.

How Gels Are Used to Resolve DNA

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading

Separation of DNA in gels, or DNA electrophoresis, is based on the fact that DNA is negatively charged and will move in response to an electrical field. The gel in which the DNA is separated is a viscous mesh that looks and acts like very hard table jelly. The mesh comprises long interwoven or chemically linked molecules that are penetrated by water-filled channels through which the DNA can pass. An electrical circuit is made by placing a gel between two electrodes in a chamber that is connected to a power supply. The chamber is then filled with electrophoresis buffer (a high salt solution), which completes the circuit. When a constant voltage is applied to the system, the negatively charged ions in the buffer will migrate towards the positive electrode (anode) and the positively charged ions will migrate to the negative electrode (cathode), creating a current through the system. Since DNA is negatively charged, it migrates towards the anode, but its migration is impeded by the mesh-like gel, and larger DNA molecules are more impeded than smaller ones.

Electrophoresis systems

There are two major electrophoresis apparatus arrangements, horizontal and vertical. In a horizontal system, as illustrated in Figure 1, the gel lies flat on a platform in a tank. In a vertical system, the gel is clamped on to a support that is placed into a chamber so that it stands vertically. Both systems have specific applications as discussed below.

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Figure 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 placed in a horizontal electrophoresis chamber filled with electrophoresis buffer and connected to a power supply. When voltage is applied to the system, the DNA migrates towards the anode in a tight ‘lane’ as shown in the diagram of the resulting gel.

The size of gels that can be run is variable and depends on the intended use of the gel. Small gels are generally used to resolve simple mixtures of DNA as they separate DNA rapidly and require little material to prepare. However, if a large DNA sample or a highly complex mixture needs to be resolved, larger gels must be used.

Agarose and Acrylamide Gels

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading

The matrix or gel that the DNA migrates through consists of one of two polymers, agarose or acrylamide. Agarose is a linear carbohydrate polymer extracted from seaweed. Agarose gels are made by first dissolving the powdered agarose in hot electrophoresis buffer, and then pouring the mixture on a horizontal gel casting plate. When the agarose 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 of the pores depends on the concentration of agarose in the gel; the higher the concentration of agarose, the smaller will be the pores. This in turn will determine its ability to impede the migration of DNA in an electric field. Generally, agarose concentrations between 0.3% and 3% are used. The size of DNA fragments that can be resolved by these concentrations of agarose ranges from 100 to 60 000 base pairs in length.

Acrylamide is a monomer that polymerizes in the presence of free radicals. The free radicals are generated by ammonium persulfate and are stabilized by N,N,N′,N′-tetramethylethylenediamine (TEMED). N′,N′-methylenebisacrylamide is included in the polymerization reaction to covalently crosslink the long chains of polyacrylamide into a gel. The pore size of the resulting gel is dependent on the length of the polyacrylamide chains, which is in turn determined by the concentration of acrylamide in the gel. The degree of crosslinking, as determined by the N′,N′-methylenebisacrylamide concentration, also influences pore size. Typically, acrylamide concentrations between 3.5% and 20% are used with one molecule of N′, N′-methylenebisacrylamide for every 29 molecules of acrylamide. Smaller DNA molecules can be resolved by acrylamide gels than with agarose gels and the optimal size range for separation is from 1 to 2000 base pairs.

The polymerization of acrylamide is inhibited by oxygen. These gels must therefore be poured vertically between two glass plates that are held apart by two plastic spacers. This process is time-consuming and technically more demanding than horizontal agarose gel electrophoresis. However, acrylamide gels have excellent resolving power. They are used when a high degree of separation (i.e. a size difference of a single base pair) is required, such as when sequencing DNA.

Electrophoresis buffers

Two buffers are commonly used for DNA electrophoresis, Tris acetate – EDTA (TAE) and Tris-borate – EDTA (TBE). These buffers can be used interchangeably as they are both good electrical conductors. Supercoiled DNA, the form that DNA adopts in nature, separates better in TAE than in TBE. Also, concentrated solutions of TAE have a longer shelf-life than concentrated TBE solutions, which tend to precipitate over time. A disadvantage of TAE is that its buffering capacity is not as great as that of TBE. Therefore, when it is necessary to run gels for a long time, or at a high voltage, TBE should be used as the electrophoresis buffer. Gels made with TBE are stronger than those made with TAE; this feature is an advantage when making low percentage gels, which tend to be fragile.

DNA samples

The amount of DNA loaded on to a gel depends on the complexity of the sample. More sample must be loaded if there are a large number of DNA fragments in the sample, so that each fragment can be readily detected upon staining. However, if too much DNA is loaded, the resolving power of the gel will be lost and the result will be a smear of DNA on the gel. Up to 500 ng of a complex mixture of DNA can be loaded into a 0.5 cm wide well of a 1% agarose–TAE gel and be resolved adequately.

Before being loaded on to a gel, the DNA is mixed with a sample buffer. This buffer consists of a viscous liquid such as glycerol, sucrose or Ficoll, and the dye bromophenol blue. The sample buffer may contain another dye, xylene cyanol FF, in addition to the bromophenol blue. The purpose of this buffer is to make the DNA sample dense so that it sinks to the bottom of the well. The dyes, in addition to colouring the sample to facilitate loading, are used to monitor the progress of the electrophoresis. Both bromophenol blue and xylene cyanol FF migrate in a predictable fashion in response to an electrical field. The migration of these two dyes varies slightly under different conditions. In a 1% agarose–TAE gel, bromophenol blue comigrates with small DNA molecules around 500 base pairs long and xylene cyanol FF typically comigrates with DNA fragments around 5 kilobase-pairs long. A disadvantage of these dyes is that they may mask the appearance of comigrating DNA, if it is present in low concentrations.

Detection

The dye most commonly used to stain DNA gels is ethidium bromide. Ethidium bromide binds DNA between the bases (i.e. it intercalates DNA) and fluoresces orange when exposed to ultraviolet light (260–360 nm). The lower limit of DNA detection by ethidium bromide is around 2 ng (2 × 10−9 g). This level of sensitivity is adequate for most purposes. However, there are more sensitive DNA binding dyes, such as SYBR green (detection limit 0.02 ng) that can be used if required.

Gels can be stained either before or after an electrophoresis run. To stain a gel before an electrophoresis run, ethidium bromide is added to the melted agarose before it is poured on to the gel tray. This staining method is the quickest and offers the advantage that the position of the DNA can be monitored during the separation. Alternatively, the gel may be placed in a dilute solution of ethidium bromide for 10–30 min after the electrophoresis run.

Factors influencing mobility

The rate of DNA migration through a gel depends on the size and shape of the DNA molecules. A linear DNA molecule will migrate at a rate that is inversely proportional to the log10 of its number of base pairs. This relationship is due to the increased difficulty larger DNA molecules have ‘worming’ their way through the pores of the gel as well as their greater frictional drag. Plasmids and other circular DNA molecules are more compact than linear DNA molecules, and thus they migrate through the gel at rates higher than would be expected from their molecular weights.

The voltage applied to the electrophoresis system influences the rate of DNA migration, as do gel percentage and buffer composition. Consideration of Ohm's law (V = IR) helps one understand the effect of these factors. The current (or ion movement,) I in an electrical circuit is directly proportional to the electrical field or applied voltage V and inversely proportional to the resistance R of the system, provided by the gel. As the percentage of either agarose or acrylamide in a gel increases, the mesh of molecules in the gel becomes tighter, increasing the resistance of the system and thereby impeding the movement of DNA through the gel. Gels made with TBE buffer tend to be more rigid (less porous) than those made with TAE, so that DNA moves more slowly through gels made with TBE than through gels made with TAE.

Southern Blotting: Theory and Practice

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading

If the identity of a specific DNA fragment on a gel must be determined, it is necessary to transfer the DNA on to an alternative medium while maintaining the separation or banding pattern of the gel. This transfer step is required because, if a DNA gel is left to sit for an extended period, the DNA will diffuse out of the bands. The transfer process is commonly referred to as Southern blotting. Basically, this procedure begins with running an agarose DNA gel. The DNA on the gel undergoes certain treatments to separate it to its single-stranded form and is then transferred on to a membrane with the ability to bind DNA strongly. After the DNA has been immobilized on to the membrane, it is reacted with a probe (labelled pieces of single-stranded DNA of known sequence). Since the two strands of DNA are complementary, the unknown DNA fragments that bind (hybridize) to the probe will have a sequence the same as or similar to the probe's.

Gel pretreatment

Prior to the transfer step, the DNA in the gel must undergo some treatment steps to ensure the complete transfer of the DNA from the gel to the membrane. First the gel is placed in a glass tray containing a solution of hydrochloric acid so that a process known as depurination can occur. The acid reacts with and removes some of the adenine and guanine bases from the DNA molecule. The gel is washed with distilled water and placed into another glass tray containing a solution of sodium hydroxide, a strong base that serves two purposes. The first is to break the DNA backbone where it has been depurinated. This is important because smaller DNA molecules transfer more efficiently than larger ones. The second role of sodium hydroxide is to promote the denaturation (i.e. unzipping) of the double-stranded DNA molecules such that the DNA becomes single-stranded and, after transfer and immobilization, cannot rejoin to itself. The gel is rinsed again with distilled water and placed into the final treatment solution, one containing a buffered salt solution (pH 7). The purpose of this incubation is to neutralize the gel (i.e. bring the pH down to less than 9), so that the DNA will bind to the membrane.

Transferring the DNA from gel to membrane

The matrix to which the single-stranded DNA fragments from the gel are transferred is known as a membrane. Two types of membrane are in common use: nitrocellulose and nylon. Nitrocellulose was first used by Southern when he 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 membranes are much stronger than nitrocellulose membranes and will not easily break with handling even after repeated use. Also, nylon membranes can efficiently bind DNA as short as 50 bases in length, and can bind up to 5 times more DNA per cm2 than can nitrocellulose. Another advantage of nylon membranes is that they can bind DNA covalently when they are subjected to either UV crosslinking or alkaline transfer. DNA is immobilized to nitrocellulose membranes by baking the membrane in an oven, but the attachment is noncovalent and thus not as strong as it is with nylon membranes. It is important to have a strong association between the DNA and the membrane so that the loss of DNA from the membrane is minimized during the subsequent wash steps. Despite all of the advantages associated with nylon membranes, they have not completely replaced nitrocellulose membranes because they tend to demonstrate a high background noise to signal ratio after they have been stained. DNA sequences of interest may therefore be masked by the high level of background staining or radioactivity.

The blotting or transfer procedure has remained essentially unchanged since it was first described in 1975 and is shown in Figure 2. Basically, the gel is placed on top of a piece of filter paper lying on a support in a tray containing transfer buffer (a high salt solution). The membrane is placed on top of the gel, followed by more filter paper, a large pile of paper towels, and finally a heavy object such as a text book.

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Figure 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 transfer process.

The transfer of DNA from the gel to the membrane occurs by capillary action. The transfer buffer is drawn up through the filter paper, the gel, the membrane, and finally up to the stack of paper towels. Because DNA gels are quite porous, as the transfer buffer migrates to the paper towels, the DNA is carried out of the gel and blotted on to the membrane. The high salt concentration in the transfer buffer promotes the binding of DNA to the membrane. Generally, the transfer procedure takes approximately 18 h to complete. If the DNA fragments are less than 1000 base pairs long, the transfer may take as little as 1–2 h.

There have been several modifications of the original blotting procedure described by Southern. These modified procedures attempt to decrease the transfer time and increase the efficiency of transfer. One procedure involves setting the transfer apparatus upside down so the gel is on the top. This downward capillary transfer aided by gravity, in addition to being faster than the conventional procedure, does not place excessive pressure on the gel, and thus there is no possibility of crushing it. Another modification, electroblotting, involves using an electrophoresis apparatus to mobilize the DNA from the gel on to the membrane. Vacuum blotting uses vacuum pressure to draw the transfer buffer through the gel.

Alkaline blotting involves the use of a positively charged nylon membrane and sodium hydroxide in the transfer buffer. With this modified procedure, the DNA denatures and binds covalently to the membrane as it is being transferred from the gel, thus minimizing DNA loss during the transfer. With this procedure the transfer process only takes approximately 2 h and the denaturing gel pretreatment step is optional.

DNA hybridization

To understand DNA hybridization, it is important to remember that the DNA immobilized on the membrane is single-stranded and that any introduced single-stranded nucleic acid (i.e. the probe) will bind to this immobilized DNA. The stability of the resulting double-stranded molecule depends on the degree of similarity between the two strands (i.e. the number of complementary base pairs), temperature, ionic strength, and the presence of chemicals that disrupt hydrogen bonds, such as formamide. The goal of all hybridization procedures is to maximize the signal from the probe binding to the target DNA on the membrane, while minimizing its nonspecific binding to the membrane and nontarget DNA (background).

Before hybridization is carried out, the membrane is treated with a blocking agent to prevent nonspecific association of probe with the membrane. A variety of polymeric inert agents, such as skim milk powder, Denhardt's reagent (a mixture of Ficoll, polyvinylpyrrolidone and bovine serum albumin), denatured fragmented salmon sperm DNA or heparin can be used to bind the unused DNA binding sites on the membrane. Skim milk powder and Denhardt's reagent are two most commonly used blocking agents. Skim milk powder is the cheapest and easiest to use, whereas Denhardt's reagent can more effectively block nylon membranes.

The basic hybridization procedure involves placing the membrane in a plastic bag or hybridization tube with the labelled probe in a small volume of a high salt solution. Keeping the volume of the hybridization solution low serves to increase DNA concentration, while the high salt content promotes DNA binding. The specificity of the hybridization can be increased by adding formamide (which disrupts hydrogen bonds) to the buffer. After the hybridization reaction, the unbound and nonspecifically bound probe is removed from the membrane via a series of washes. As the aim of the hybridization process is to encourage hybrid formation between the probe and target DNA while destabilizing all other hybrids, the actual hybridization procedure must be tailored to the specific probe and target DNA. The variations made on this basic procedure aim to increase both the specificity and the amount of probe bound to the target DNA.

The specificity of the hybridization is determined by the wash steps, which vary with respect to temperature and salt concentration. The wash steps are carried out in a step-wise manner. First a low-temperature, high-salt wash is done to remove unbound probe. Then more stringent washes at higher temperatures, with less concentrated salt solutions, are done to remove probe bound nonspecifically to the membrane and that bound to nontarget DNA via short regions of complementarity. In theory, DNA hybrids consisting of the probe and target DNA should have no or few mismatched base pairs and thus a higher melting temperature than any other type of hybrid formed. Therefore, the higher temperatures of the latter wash steps should melt any nonspecific probe–nontarget DNA hybrids and leave the probe–target DNA hybrids intact. Similarly, since salt stabilizes DNA binding, decreasing its concentration in the latter wash solutions will destabilize any mismatched hybrids, leaving the probe bound only to the target DNA.

The strength of the signal depends on the amount of probe that is bound to the target DNA. This interaction depends on the extent of sequence similarity between the target DNA and probe, the hybridization time, the DNA concentration and the probe length. Although longer hybridization times lead to a stronger signal, they also lead to higher nonspecific probe binding. Hybridization times are therefore generally kept as short as possible. The rate and extent of hybridization increase with the DNA concentration. However, it is difficult to determine or predict what the concentration of the DNA immobilized on the membrane will be, which is why it is important to keep the hybridization volume low. While more probe can be used, generally this is not done because it may also lead to a high level of nonspecific association of probe with the membrane. Inert polymers such as dextran or PEG (polyethylene glycol) form interlocking meshes with the probe and can be used to increase the effective probe concentration. Probes are usually selected to be around 200 bases in length to give good signals. The signal given off by smaller probes is not as strong, and longer probes may be difficult to wash away if they associate nonspecifically with the immobilized DNA.

Detection

In the past, probes were usually labelled with radioactive phosphorus and the resulting labelled Southern blots were imaged on X-ray film. Today, however, there are more convenient and safer labels. The most common compound used to label probes is digoxigenin (DIG). The DIG label is bound to the probe and can be detected with commercially available antibodies that have an enzyme attached to them. The enzyme can be one of a variety of enzymes that converts a substrate to yield a coloured product that can be visualized on the membrane. This method of detection is very sensitive. The lower limit of DNA detection is 0.1 pg (10−13 g). Another advantage of the DIG system is that the antibodies are highly specific for the DIG label, thus there is less background noise associated with it.

Conclusion

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading

DNA gels are a fundamental methodology in molecular biology. Together with alkaline blotting, they provide scientists with powerful and specific methods for analysing unknown DNA molecules. The methods described above provide the basis for many practical procedures and are much used in forensic science, disease diagnosis, discovery of the genetic basis of disease, and investigation of population health and epidemiology.

Glossary
Denaturation

The disruption of hydrogen bonds and van der Waals interactions, by heat or by chemical means, leading to a loss of secondary structure in a macromolecule.

Electrophoresis

The separation of charged molecules on the basis of their migration rates in an electric field.

Hybridization

Base pairing between single-stranded nucleic acids (either DNA or RNA) to yield double-stranded nucleic acid molecules.

Stringency

The degree of specificity (i.e. base mismatch) desired in a hybridization reaction. The stringency of a hybridization reaction is determined by the wash conditions (mild wash conditions for low stringency/many base mismatches and harsh wash conditions for high stringency/no or few base mismatches).

Further Reading

  1. Top of page
  2. Introduction
  3. A Review of DNA Structure
  4. How Gels Are Used to Resolve DNA
  5. Agarose and Acrylamide Gels
  6. Southern Blotting: Theory and Practice
  7. Conclusion
  8. Further Reading
  • Ausubel FM, Brent R, Kingston RE et al. (eds) (1993) Current Protocols in Molecular Biology. New York: Wiley.
  • Old R and Primrose S (1994) Principles of Gene Manipulation, An Introduction to Genetic Engineering, 5th edn. London: Blackwell Scientific.
  • Sambrook J, Tritsch EF and Maniatis T (1989) Molecular Cloning. A Laboratory Manual. New York: Cold Spring Harbor Laboratory.
  • Southern EM (2000) Blotting at 25. Trends in Biochemical Sciences 25(12): 585588.
  • Westermeier R (2001) Electrophoresis in Practice, 3rd edn. New York: Wiley.