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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 agarose, dissolved in buffer solution. Agarose is a very pure (and expensive) form of agar, which is obtained from seaweed. At one end of the slab of gel are several small wells, made by the teeth of a comb that was placed in the molten agarose before it set. A buffer solution is poured over the gel, so that it fills the wells and makes contact with the electrodes at each end of the gel. Ions in the buffer solution conduct electricity. After electrophoresis, the DNA is visualised. In research laboratories, a fluorescent dye will have been incorporated into the agarose gel before it was cast. After the gel has been ‘run’ it is illuminated with ultraviolet (UV) light and the dye, which binds to DNA, shows up as bright fluorescent bands. Ethidium bromide was until recently the most commonly used DNA stain. Ethidium bromide has similar dimensions to a base pair in DNA. When ethidium bromide binds to DNA, it slips between adjacent base pairs and stretches the double helix. This causes errors when the DNA is replicated. The test samples (DNA fragments) are mixed with a small volume of loading dye. This dye is dissolved in a 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 is invisible). Above: Intercalation of ethidium bromide between two adjacent bases in a DNA molecule. 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 some of these compounds is that they will fluoresce in blue, rather than harmful UV light. In schools, safer, cheaper dye solutions are used to stain the entire gel, including the DNA, after electrophoresis. Suitable stains include Azure A and Azure B, Toluidine blue O and Nile blue sulphate. This type of stain is not thought to intercalate within the DNA double helix, but instead binds ionically to the negatively-charged phosphate groups of the DNA. Such dyes are not as sensitive as ethidium bromide and the newer fluorescent dyes, and some of them may colour the gel heavily. Consequently, prolonged ‘destaining’ in water may be necessary before the DNA bands can be seen. Methylene blue, which is sometimes used for staining DNA on agarose gels in schools, is far from ideal, as it requires destaining and it fades rapidly after use. Although these alternatives to ethidium bromide are thought to be relatively safe, they have not been intensively studied for long-term effects and the mechanisms by which they bind to DNA are not fully understood. As with all laboratory chemicals, suitable safety precautions should be exercised when handling any dyes, particularly when they are in dry, powdered form. Viewing gels Gels stained with a blue dye such as Toluidine blue O can be viewed in daylight. A smartphone with a white background light (some ‘torch’ apps are suitable) can be used as a ‘lightbox‘. Alternatively, LED lightboxes sold for tracing can be used. A yellow-coloured filter may help to enhance the contrast when photographing gels that have been stained with blue dyes. Azure A Azure B Toluidine blue O Nile blue sulphate Above: Some dyes that are thought to bind ionically to DNA. HOW MUTAGENIC IS ETHIDIUM BROMIDE? In recent years there has been some controversy about the dangers of ethidium bromide. The compound is used as a drug for treating cattle with trypanosomiasis (African sleeping sickness) at far greater concentrations than are used in the lab. The cattle do not seem to suffer any adverse effects, but since the animals are usually slaughtered after a few years, any long-term harm would not be noticed. When ethidium bromide is metabolised in the liver, the compounds produced are highly mutagenic. It is probably correct to say that ethidium bromide is not as harmful as some people think it is, but it should still be handled with care and disposed of correctly. The relevant 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 stored refrigerated in a plastic bag to prevent them from drying out. Provided they are not exposed to light, gels kept like this will not fade for many months. * See: www.ncbe.reading.ac.uk/SAFETY/dnasafety. html www.ncbe.reading.ac.uk Ethidium bromide 2 Copyright © NCBE, University of Reading, 2017 No. 1 | JANUARY 2017 EFFECT OF VOLTAGE Fragment size (kb) What’s the best voltage to use? At low voltages, movement of linear DNA is proportional to the voltage applied. As the voltage is increased, the mobility of the higher molecular mass fragments is increased differentially (the larger fragments tend to ‘catch up’ with the smaller ones). Hence the effective range of separation is decreased as the voltage is increased. For the best resolution, 0.8% agarose gels should be run at no more than 5 V per cm (as determined by the distance between the electrodes). The NCBE electrophoresis equipment, which is designed to work at 36 V, has a distance between the electrodes of ~85 mm, which is close to the optimum. 5 V / cm Good separation 20 V / cm Poor separation Calculating the resolution of a gel For λ DNA digested by HindIII (shown on the left), the resolution can be calculated by dividing the distance between the 23 and 2 kb fragments by the total distance travelled by the 2 kb fragment. 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 (%) Above: Even a small difference in gel concentration can have a significant effect on the quality of the results you see. For that reason, it is important to make up agarose solutions accurately, using buffer, not water. Don’t try to weigh out small amounts of agarose, make up a large volume: it will keep indefinitely in a sealed container. Copyright © NCBE, University of Reading, 2017 Agarose (% w/v) Separation range (kb) Relative gel strength 0.3 60–5 Very weak 0.6 200–1 Weak 3 0.7 10–0.8 Moderate 0.9 7–0.5 Moderate 1.2 6–0.4 Strong 1.5 4–0.2 Strong 2.0 3–0.1 Strong www.ncbe.reading.ac.uk NCBE BRIEFING Polyacrylamide gelsand protein electrophoresis To separate proteins by electrophoresis, gels cast from polyacrylamide are sometimes used. Before proteins are run on a gel, they are treated with a strong detergent, sodium dodecyl sulphate (SDS). This, coupled with heating, causes the tightly-folded protein molecules to unfold and become linear, so that they will move through the gel according to their sizes, not the way in which they are folded. The SDS also binds to the proteins, giving them an overall negative charge, so that they move towards to positive electrode. This type of electrophoresis is called SDS-PAGE (SDSpolyacrylamide gel electrophoresis). It is also possible to separate proteins using a special type of agarose, but in contrast to the procedure using polyacryamide, with agarose the proteins are separated by electrical charge only (not charge and size). This is because the pores within the agarose gel are relatively large and the proteins can easily pass through them. As with DNA, the proteins on the gel are stained with an appropriate dye. Dyes originally developed for textiles such as Coomassie blue (which bind to proteins like wool) are often used. De-staining (often with water) is then necessary to remove the background stain from the gel before the protein 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 doublestranded 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 used as genetic markers when they occur near to genes of interest that are difficult to detect directly. Restriction enzyme name Source microorganism STRAIN DNA base pair ‘recognition’ site (5'a3') BamHI Bacillus amyloliquefaciens H G$GATCC EcoRI Escherichia coli RY13 G$AATTC HindIII Haemophilus influenzae Rd A $AGCTT Above: Some examples of restriction enzymes. www.ncbe.reading.ac.uk Above: Restriction enzyme BamHI bound to doublestranded DNA. This view is looking down the axis of the DNA molecule (ball-and-stick model, in the centre of the image). The restriction enzyme is shown in ‘cartoon‘ format, with b-pleated sheets in yellow and a-helicies in magenta. This image uses data from: Newman, M., et al (1995) Structure of BamHI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269, 656–663 [Protein Data Bank ID: 1BHM]. The software used to produce the image was UCSF Chimera and VMD, which can be obtained from: www. cgl.ucsf.edu/chimera/ and: www.ks.uiuc.edu/ Research/vmd/ respectively. 4 Copyright © NCBE, University of Reading, 2017 No. 1 | JANUARY 2017 NCBE ELECTROPHORESIS PRODUCTS The NCBE’s award-winning electrophoresis equipment is probably the world’s the most cost-effective system for gel electrophoresis. More than half a million sets have been provided to schools since 1992. The NCBE’s prototype electrophoresis kit is now in the Science Museum in London. The NCBE equipment uses very little agarose and buffer, making it economical to run. There are three parts to the NCBE’s electrophoresis system. All of the re-usable items (gel tanks, combs etc.) come in a BASE UNIT. The base unit contains eight sets of equipment. To power the electrophoresis equipment, we supply a 36 V MAINS TRANSFORMER. This is a safe, fast and economical alternative to the batteries that some people have used in the past. Finally, all of the consumable items (agarose, DNA, enzymes etc.) are provided in MODULES. The modules’ contents vary, but they usually include sufficient materials for 16 students or working groups to carry out the practical work. Full details are given on the NCBE web site: www.ncbe.reading. ac.uk/electrophoresis. All of the module contents will keep, if stored 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 your class and/or working group sizes. Remember that the base unit contains eight sets of hardware. Next, choose which module(s) you’re interested 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. This transformer is a safe, cost-effective and environmentally-friendly alternative to batteries. With the connector provided, a single transformer can power four NCBE gel tanks. 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. Copyright © NCBE, University of Reading, 2017 5 www.ncbe.reading.ac.uk NCBE BRIEFING NCBE ELECTROPHORESIS MODULES The lambda protocol This practical exercise has become a classic for demonstrating the action of different restriction enzymes on DNA. The bacteriophage lambda (λ) has doublestranded DNA which is 48,502 base-pairs in length. Different restriction enzymes ‘recognise’ specific Nature’s dice Genetics is often difficult for students to understand. This innovative practical work uses modern DNA technology to help students learn about classical Mendelian inheritance. This exercise provides a practical simulation of genetic screening, centred on a fictitious extended family with 24 members. The DNA samples can be distributed by the teacher so that students can investigate the inheritance of either The PCR and plant evolution This module allows students to amplify chloroplast DNA using the polymerase chain reaction (PCR). The length of the fragments produced can be used to infer evolutionary relationships. The polymerase chain reaction (PCR) is one of the most important and powerful methods in molecular biology. It enables millions of copies of specific DNA sequences to be made easily and quickly. The technique and variations of it are used Protein power! The NCBE’s electrophoresis equipment can be used to analyse proteins as well as DNA. You do, however, need a special type of agarose (which is supplied in the box) to carry out this work. sequences of bases in this DNA and cut it at precise locations. Three different restriction enzymes are provided in this module: BamHI, HindIII and EcoRI. After treatment with the individual enzymes, the lambda DNA fragments are separated by gel electrophoresis. Once the gel has been run, the DNA is stained to reveal distinctive patterns of bands which correspond to fragments of different sizes. a sex-linked or an autosomal recessive condition. Students treat the DNA samples provided with a restriction enzyme and run them on electrophoresis gels. The results from the class are pooled so that the pattern of inheritance may be determined. This activity is a novel practical way of reinforcing learning about Mendelian inheritance, the use of restriction enzymes and gel electrophoresis. It presents an ideal opportunity to stimulate discussion about genetic counselling, confidentiality of genetic information and other ethical concerns. extensively in medicine, in molecular genetics and in pure research. This practical kit provides materials for the simple extraction of chloroplast DNA from plant tissue, its amplification by the PCR, and gel electrophoresis of the PCR product. Students can use plants of their choice and identify possible evolutionary relationships between different species. This mirrors the molecular methods used in modern plant taxonomy. This activity presents an ideal opportunity for openended investigations by individual students or groups. Small samples of protein-containing foods (e.g., fish or nuts) are mixed with Laemmli buffer. This linearizes the proteins and gives them a negative electrical charge. The samples are separated by electrophoresis, then the gel is stained and destained to reveal the protein bands. National Centre for Biotechnology Education, University of Reading, 2 Earley Gate, Reading RG6 6AU. United Kingdom Tel: + 44 (0) 118 9873743. Fax: + 44 (0) 118 9750140. eMail: [email protected] Web: www.ncbe.reading.ac.uk www.ncbe.reading.ac.uk 6