Ncbe Briefing Gel Electrophoresis

No. 1 | JANUARY 2017

NCBE BRIEFING

GEL ELECTROPHORESIS Gel electrophoresis is a key technique in modern biology that features in all of the new A-Level Biology specifications in England. It is a way of separating DNA, RNA or proteins based on their size and/or the electrical charge on the molecules.

DNA gel electrophoresis Visualising DNA For DNA gel electrophoresis, a gel is cast from After electrophoresis, the DNA is visualised. In agarose, dissolved in buffer solution. Agarose is a very research laboratories, a fluorescent dye will have pure (and expensive) form of agar, which is obtained been incorporated into the agarose gel before it from seaweed. At one end of the slab of gel are several was cast. After the gel has been ‘run’ it is illuminated small wells, made by the teeth of a comb that was with ultraviolet (UV) light and the dye, which binds to placed in the molten agarose before it set. A buffer DNA, shows up as bright fluorescent bands. Ethidium solution is poured over the gel, so that it fills the wells bromide was until recently the most commonly used and makes contact with the electrodes at each end of DNA stain. Ethidium bromide has similar dimensions the gel. Ions in the buffer solution conduct electricity. to a base pair in DNA. When ethidium bromide binds to DNA, it slips between adjacent base pairs and The test samples (DNA fragments) are mixed with a stretches the double helix. This causes errors when small volume of loading dye. This dye is dissolved in a the DNA is replicated. dense sugar solution, so that when it is added to the wells, it sinks to the bottom, taking the DNA sample with it. An electrical potential is applied across the gel. Phosphate groups give the DNA fragments a negative electrical charge, so that the DNA migrates through the gel towards the positive electrode. Small DNA fragments move quickly through the porous gel; larger molecules travel more slowly. In this way the pieces of DNA are separated by size. The loading dye also moves through the gel, so that the progress of the electrophoresis can be seen (the DNA itself Above: Intercalation of ethidium bromide between two adjacent bases in a DNA molecule. is invisible). Short-wavelength UV light is itself harmful and ethidium bromide’s breakdown products are thought to be potent mutagens and carcinogens. Ethidium bromide should therefore not be used in schools*. For reasons of safety and because UV light of this wavelength causes unwanted mutations in the DNA being studied, several alternative stains are now often used in research labs. These include SYBRsafe® and GelRed®, which although they are thought to be safer than ethidium bromide, are far more expensive [Ethidium bromide costs £4.50 per mL compared with £133 per mL for SYBRsafe® and £200 per mL for www.ncbe.reading.ac.uk 1 Copyright © NCBE, University of Reading, 2017 NCBE BRIEFING

GelRed® (2016 prices).] An additional advantage of N some of these compounds is that they will fluoresce Azure A + (H C) N NH.CH in blue, rather than harmful UV light. 3 2 S 3 Cl-

In schools, safer, cheaper dye solutions are used N Azure B to stain the entire gel, including the DNA, after + (H3C)2N S NH2 electrophoresis. Suitable stains include Azure A and Cl- Azure B, Toluidine blue O and Nile blue sulphate. This type of stain is not thought to intercalate within the N CH3

DNA double helix, but instead binds ionically to the + Toluidine blue O (H3C)2N S NH2 negatively-charged phosphate groups of the DNA. Cl-

O NH2 (CH2CH3)2N Such dyes are not as sensitive as ethidium bromide + Nile blue sulphate 1 SO -2 and the newer fluorescent dyes, and some of them 2 4 N may colour the gel heavily. Consequently, prolonged ‘destaining’ in water may be necessary before the Above: Some dyes that are thought to bind ionically DNA bands can be seen. Methylene blue, which is to DNA. sometimes used for staining DNA on agarose gels in schools, is far from ideal, as it requires destaining and it fades rapidly after use. HOW MUTAGENIC IS Although these alternatives to ethidium bromide ETHIDIUM BROMIDE? are thought to be relatively safe, they have not been intensively studied for long-term effects and the In recent years there has been some mechanisms by which they bind to DNA are not fully controversy about the dangers of ethidium understood. As with all laboratory chemicals, suitable bromide. The compound is used as a drug safety precautions should be exercised when handling for treating cattle with trypanosomiasis any dyes, particularly when they are in dry, powdered (African sleeping sickness) at far greater form. concentrations than are used in the lab. The cattle do not seem to suffer any adverse effects, but since the animals are usually Viewing gels slaughtered after a few years, any long-term harm would not be noticed. When ethidium Gels stained with a blue dye such as Toluidine blue O bromide is metabolised in the liver, the can be viewed in daylight. A smartphone with a white compounds produced are highly mutagenic. background light (some ‘torch’ apps are suitable) can It is probably correct to say that ethidium be used as a ‘lightbox‘. Alternatively, LED lightboxes bromide is not as harmful as some people sold for tracing can be used. A yellow-coloured filter think it is, but it should still be handled with may help to enhance the contrast when photographing care and disposed of correctly. The relevant gels that have been stained with blue dyes. safety regulations state that it MUST NOT be used in UK schools*. Storing stained gels

Gels stained with Toluidine blue O or Azure A can be H2N NH2 stored refrigerated in a plastic bag to prevent them + from drying out. Provided they are not exposed to N Br - light, gels kept like this will not fade for many months. Ethidium C2H5 bromide * See: www.ncbe.reading.ac.uk/SAFETY/dnasafety. html www.ncbe.reading.ac.uk 2 Copyright © NCBE, University of Reading, 2017 No. 1 | JANUARY 2017

EFFECT OF VOLTAGE

Fragment What’s the best voltage to use? size (kb) At low voltages, movement of linear DNA is proportional to the voltage 23.13 applied. As the voltage is increased, the mobility of the higher molecular mass fragments is increased differentially (the larger fragments tend to 9.42 ‘catch up’ with the smaller ones). Hence the effective range of separation 6.56 is decreased as the voltage is increased. For the best resolution, 0.8% 4.36 agarose gels should be run at no more than 5 V per cm (as determined by the distance between the electrodes). The NCBE electrophoresis 2.32 equipment, which is designed to work at 36 V, has a distance between the 2.03 electrodes of ~85 mm, which is close to the optimum.

0.56 Calculating the resolution of a gel For λ DNA digested by HindIII (shown on the left), the resolution can be 5 V / cm 20 V / cm calculated by dividing the distance between the 23 and 2 kb fragments by Good Poor the total distance travelled by the 2 kb fragment. separation separation

EFFECT OF GEL CONCENTRATION

Gel concentration also affects the movement of DNA fragments. There is a linear relationship between the logarithm of the mobility of the DNA and the gel concentration. By altering the agarose concentration it is possible to control the range of sizes of fragments that can be separated by electrophoresis. The example on the left shows λ DNA digested by HindIII. The optimum gel concentration for separating these λ DNA fragments is ~0.8% (w/v), which is the concentration suggested in the NCBE’s Lambda protocol module (see page 6).

For larger or smaller DNA fragments, a different agarose concentration may be better. For instance, to show chloroplast DNA fragments of between ~500 and several thousand base-pairs, such as those produced by the NCBE’s PCR and plant evolution module, an agarose concentration of 1.5% (w/v) is recommended. The table below shows the concentration of agarose needed for separating DNA fragments of different sizes.

0.5 1.0 1.5 2.0 Gel concentration (%) Agarose Separation range Relative Above: Even a small difference (% w/v) (kb) gel strength in gel concentration can have a 0.3 60–5 Very weak significant effect on the quality of the results you see. For that 0.6 200–1 Weak reason, it is important to make 0.7 10–0.8 Moderate up agarose solutions accurately, 0.9 7–0.5 Moderate using buffer, not water. Don’t try to weigh out small amounts of 1.2 6–0.4 Strong agarose, make up a large volume: 1.5 4–0.2 Strong it will keep indefinitely in a sealed container. 2.0 3–0.1 Strong

Polyacrylamide gels and protein It is also possible to separate proteins using a special electrophoresis type of agarose, but in contrast to the procedure using polyacryamide, with agarose the proteins are To separate proteins by electrophoresis, gels cast separated by electrical charge only (not charge and from polyacrylamide are sometimes used. Before size). This is because the pores within the agarose proteins are run on a gel, they are treated with a strong gel are relatively large and the proteins can easily pass detergent, sodium dodecyl sulphate (SDS). This, through them. coupled with heating, causes the tightly-folded protein molecules to unfold and become linear, so that they As with DNA, the proteins on the gel are stained will move through the gel according to their sizes, not with an appropriate dye. Dyes originally developed the way in which they are folded. The SDS also binds to for textiles such as Coomassie blue (which bind the proteins, giving them an overall negative charge, to proteins like wool) are often used. De-staining so that they move towards to positive electrode. This (often with water) is then necessary to remove the type of electrophoresis is called SDS-PAGE (SDS- background stain from the gel before the protein polyacrylamide gel electrophoresis). bands can be seen.

SAFETY OF POLYACRYLAMIDE GELS Polyacrylamide gels must not be cast in a school, as the two materials used to make them (acrylamide and bis-acrylamide) are neurotoxins. Safe, pre-cast polyacrylamide gels may be purchased, but it is important to check their shelf-life, as they can seldom be stored for more than 12 months.

Restriction enzymes Whole genomic DNA is too big to run on a gel. Typically, one or more restriction enzymes (restriction endonucleases) are used to cut the DNA molecules into smaller fragments before electrophoresis. Such enzymes are produced by bacteria as a defence against ‘foreign’ nucleic acids e.g., from invading bacteriophages. These enzymes bind to specific sequences of bases in double- stranded DNA and cut the DNA, either directly at the sites they 'recognise' and bind to, or at another position in the DNA molecule. Small differences in DNA sequences that can be detected by the action of such enzymes are called ‘restriction fragment length polymorphisms’ (RFLPs). These are often Above: Restriction enzyme BamHI bound to double- stranded DNA. This view is looking down the axis of the used as genetic markers when they occur near to DNA molecule (ball-and-stick model, in the centre of genes of interest that are difficult to detect directly. the image). The restriction enzyme is shown in ‘cartoon‘ format, with b-pleated sheets in yellow and a-helicies Restriction DNA base pair in magenta. enzyme name ‘recognition’ site Source microorganism STRAIN (5'a3') This image uses data from: Newman, M., et al (1995) BamHI Structure of BamHI endonuclease bound to DNA: G$GATCC Bacillus amyloliquefaciens H partial folding and unfolding on DNA binding. Science EcoRI 269, 656–663 [Protein Data Bank ID: 1BHM]. G$A AT TC Escherichia coli RY13 HindIII The software used to produce the image was UCSF A $AGCTT Haemophilus inﬂuenzae Rd Chimera and VMD, which can be obtained from: www. cgl.ucsf.edu/chimera/ and: www.ks.uiuc.edu/ Above: Some examples of restriction enzymes. Research/vmd/ respectively. www.ncbe.reading.ac.uk 4 Copyright © NCBE, University of Reading, 2017 No. 1 | JANUARY 2017

NCBE ELECTROPHORESIS PRODUCTS

The NCBE’s award-winning electrophoresis To power the electrophoresis equipment, we supply equipment is probably the world’s the most a 36 V MAINS TRANSFORMER. This is a safe, fast cost-effective system for gel electrophoresis. and economical alternative to the batteries that More than half a million sets have been provided some people have used in the past. to schools since 1992. The NCBE’s prototype electrophoresis kit is now in the Science Museum Finally, all of the consumable items (agarose, DNA, in London. enzymes etc.) are provided in MODULES. The modules’ contents vary, but they usually include The NCBE equipment uses very little agarose and sufficient materials for 16 students or working buffer, making it economical to run. groups to carry out the practical work. Full details are given on the NCBE web site: www.ncbe.reading. There are three parts to the NCBE’s electrophoresis ac.uk/electrophoresis. system. All of the re-usable items (gel tanks, combs etc.) come in a BASE UNIT. The base unit contains All of the module contents will keep, if stored eight sets of equipment. correctly, for at least a year.

How do I decide what I need? 36 volt mains transformer Decide how many base units you need, according to This transformer is a safe, cost-effective your class and/or working group sizes. Remember and environmentally-friendly alternative to that the base unit contains eight sets of hardware. batteries. With the connector provided, a single Next, choose which module(s) you’re interested transformer can power four NCBE gel tanks. in. Again, you’ll need to order the correct number for your class size(s). The modules also act as ‘refill packs’, although you can also buy most items individually.

Electrophoresis base unit This pack contains eight sets of the items required for gel electrophoresis.

8 NCBE gel tanks; 8 4-toothed combs; 8 6-toothed combs; 8 pairs of red and black wires with crocodile clips; 8 microsyringe dispensing units (without tips). At 36 volts, the ideal voltage for the NCBE electrophoresis equipment, a 0.8% agarose gel will take two hours to run: gels made with a greater concentration of agarose may take slightly longer.

Individual replacement items The page overleaf describes the ‘modules’ of consumable items for gel electrophoresis that the NCBE currently supplies. All of the individual items from these modules are also available separately.

NCBE ELECTROPHORESIS MODULES

The lambda protocol sequences of bases in this DNA and cut it at precise locations. Three different restriction enzymes are This practical exercise has become a classic for provided in this module: BamHI, HindIII and EcoRI. demonstrating the action of different restriction After treatment with the individual enzymes, enzymes on DNA. the lambda DNA fragments are separated by gel electrophoresis. Once the gel has been run, the The bacteriophage lambda (λ) has double- DNA is stained to reveal distinctive patterns of stranded DNA which is 48,502 base-pairs in length. bands which correspond to fragments of different Different restriction enzymes ‘recognise’ specific sizes.

Nature’s dice a sex-linked or an autosomal recessive condition. Students treat the DNA samples provided Genetics is often difficult for students to with a restriction enzyme and run them on understand. This innovative practical work uses electrophoresis gels. The results from the class modern DNA technology to help students learn are pooled so that the pattern of inheritance may about classical Mendelian inheritance. be determined. This activity is a novel practical way of reinforcing learning about Mendelian This exercise provides a practical simulation inheritance, the use of restriction enzymes and gel of genetic screening, centred on a fictitious electrophoresis. It presents an ideal opportunity extended family with 24 members. The DNA to stimulate discussion about genetic counselling, samples can be distributed by the teacher so that confidentiality of genetic information and other students can investigate the inheritance of either ethical concerns.

The PCR and plant evolution extensively in medicine, in molecular genetics and in pure research. This practical kit provides This module allows students to amplify chloroplast materials for the simple extraction of chloroplast DNA using the polymerase chain reaction (PCR). DNA from plant tissue, its amplification by the The length of the fragments produced can be used PCR, and gel electrophoresis of the PCR product. to infer evolutionary relationships. Students can use plants of their choice and identify possible evolutionary relationships between The polymerase chain reaction (PCR) is one of different species. This mirrors the molecular the most important and powerful methods in methods used in modern plant taxonomy. This molecular biology. It enables millions of copies of activity presents an ideal opportunity for open- specific DNA sequences to be made easily and ended investigations by individual students or quickly. The technique and variations of it are used groups.

Protein power! Small samples of protein-containing foods (e.g., fish or nuts) are mixed with Laemmli buffer. This The NCBE’s electrophoresis equipment can be linearizes the proteins and gives them a negative used to analyse proteins as well as DNA. You do, electrical charge. The samples are separated by however, need a special type of agarose (which is electrophoresis, then the gel is stained and de- supplied in the box) to carry out this work. stained to reveal the protein bands.

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