(classroom kit), A (refill) (classroom kit), A (refill) Exploring Electrophoresis. Nature s Dice:

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1 CAROLINA Autosomal Recessive Trait Sex-Linked Trait (classroom kit), A (refill) (classroom kit), A (refill) Exploring Electrophoresis Nature s Dice: A genetic screening simulation Female Male TEACHER S MANUAL

2 Contacting Carolina Biological Carolina Biological welcomes comments from teachers and students using this kit. Carolina Biological Supply Company Biotechnology Department 2700 York Road Burlington, NC Telephone: Fax: Acknowledgments The activity described in this booklet and the accompanying Student s Guide is based upon one developed by John Schollar (National Centre for Biotechnology Education, The University of Reading, UK) and John Watson (Ecole Europienne de Luxembourg). The original version was published as Unit 18: The EIBE Family under the auspices of the European Initiative for Biotechnology Education ( The gel electrophoresis apparatus in this kit was originally developed by Dean Madden and John Schollar at the NCBE. This booklet and the associated Student s Guide were written and illustrated by Dean Madden, based on the text of the original EIBE publication Dean Madden Printed in USA

3 Exploring Electrophoresis: Nature s Dice Background The Student s Guide in this kit describes a practical, laboratory-based simulation of genetic screening centered around a fictitious family. It aims to enliven the teaching of genetics and to integrate theory, technology, and some ethical issues in one activity. (Figure 1), and, from these samples, DNA has been extracted and amplified using the polymerase chain reaction (PCR). At the single locus being investigated, there are two different alleles (i.e., types of DNA) possible. Female Students are asked to determine the mode of inheritance of a genetic condition by analyzing DNA samples. Restriction enzymes are used to cut the DNA into fragments and these fragments are separated by gel electrophoresis. The DNA is then visualized by staining and the results are analyzed. Male The topics covered are classic Mendelian inheritance (autosomal and sexlinked traits). the use of restriction enzymes to cut DNA and help identify genetic variation. DNA gel electrophoresis. genetic screening. This activity can be used to introduce some of the issues that arise from genetic screening and to emphasize the importance of genetic counseling. Selection of a Condition There are two ways in which this exercise may be presented. It can be used to illustrate a true medical condition and its inheritance. The use of a restriction enzyme to identify the altered DNA has parallels with techniques used to identify actual medical conditions. For example, sickle-cell anemia can result from a single base-pair change (point mutation), which can be detected by restriction enzyme analysis much like the analysis performed in this exercise. Alternatively, if teachers do not wish to tackle a real condition, a fictitious trait (such as diligence or laziness ) may be chosen. There are advantages and disadvantages to both approaches. Using a fictitious condition might be considered to be making light of serious medical issues. In addition, it could lead students to assume that complex traits are inherited in a Mendelian fashion and may not make students sufficiently aware of multifactorial conditions and environmental influences. In choosing which approach to take, keep in mind that this activity is intended as part of a teaching program and it aims to introduce issues which can then be explored further. The Story (to present to students) A particular gene is being investigated. Blood samples have been taken from 2 members of a large family 1 Figure Someone who is homozygous for the dominant allele (genotype DD) will have only DNA of type D. A person who is homozygous for the recessive allele giving rise to the condition chosen by students or teachers (genotype dd) will have only DNA of type d. Heterozygous individuals (genotype Dd) will have DNA of both types. Amplification of the DNA region of interest using PCR creates fragments of the same size for both alleles, in this case 6,500 base-pairs. The samples from each family member contain the amplified DNA. The task is to detect which forms of DNA are present in each sample by cutting the DNA with a restriction enzyme (BamHl) and examining the resulting DNA fragments by means of gel electrophoresis. The difference in the DNA sequence of the alleles D and d is such that, in the d allele, there is a base sequence that can be recognized and cut by the restriction enzyme. In contrast, the D allele has no restriction site for BamHl and will not be cut by the enzyme. Thus, after treatment of the amplified DNA with restriction enzyme and electrophoresis, the following results may be seen: Genotype # of bands Size DD or AA 1 6,500 bp dd or aa 2,000 bp & 2,500 bp Dd or Aa 6,500 bp,,000 bp, & 2,500 bp Note: the alleles A and a are used in the simulation of a sex-linked condition. A DNA marker (consisting of fragments of known sizes) is run alongside the samples to confirm the sizes of the fragments Teacher s Manual

4 Exploring Electrophoresis: Nature s Dice The Truth About the DNA Samples (for the teacher) Human DNA has not, of course, been used in this genomic screening simulation. Instead, three bacterial plasmids of different sizes have been used to produce preparations that will give one, two, or three bands on the gel following digestion with BamHl. Plasmids are double-stranded, circular pieces of DNA, but a plasmid preparation may contain the plasmid in several different states: relaxed (one strand broken); linear (both strands broken); or supercoiled (twisted tightly). The different forms run in an agarose gel at different rates, thus producing several bands for a single plasmid. During this simulation, each plasmid is treated with a restriction enzyme. The plasmids chosen have only a single site for the enzyme, so that after treatment the circular DNA will form a linear fragment that will give a single band after electrophoresis. The simulated samples will contain a combination of plasmids of different sizes that will produce the patterns you observe following digestion. Organization Before the Laboratory Session Prepare the agarose gel (a 0.8% solution) by melting the agarose in TBE buffer. Add 1.2 g agarose powder to 150 ml of 1 TBE buffer. Before the lab, melt the agarose and keep it molten at C (e.g., in a water bath) until it is required. If you use the 6-toothed comb, five gel boxes will provide sufficient wells for the 2 samples plus a lambda marker on each gel. Digestion of the DNA Samples and Casting of Agarose Gels Students place the DNA samples in the enzyme tubes, mix, then immediately incubate them for 0 5 minutes at 7 C. Please note that students need to share the microsyringes. Make sure that they change tips between digests and before loading each digest on the gel. During incubation, the agarose gels can be cast. The digested DNA can be frozen at this stage, for loading and running on a different day. Prepared gels can be stored for several days, provided they are not allowed to dry out. To store the gels, place a little TBE buffer in the gel boxes, place them in a plastic bag and store in a refrigerator. Loading and Running the Gels Loading takes ~10 minutes. At 5 volts, it takes ~60 minutes to run the gels. After running, the gels should be stained immediately, because the DNA fragments will diffuse in the gel if the gels are left to sit. Staining the Gel Staining takes no more than 10 minutes, with bands normally becoming visible after 20 minutes. Destain the gel with distilled water. Change the water several times over the course of 0 minutes. Destaining overnight in a small volume of water will make the bands even clearer. Students will require a photocopy of Figure 1 on which to record the class s results. Materials Included in the classroom kits Perishables: 2 Tubes containing DNA to simulate family genotypes 5 HindIII lambda marker Room Temperature Storage: strips of 8 tubes of dried restriction enzymes agarose, 1.2 g carbon fiber sheet, 11 cm 10 cm CarolinaBLU final stain concentrate, 50 ml 5 combs, 6-well 5 pairs leads with alligator clips 5 tubes 10 loading dye, 100 µl 5 gel boxes 8 microsyringes 72 tips TBE buffer, 20, 50 ml Teacher s Manual (includes blackline masters for figures 1, 2, and ) 5 Student Guides Interpretation of Results Autosomal Recessive Inheritance (Kit ) Figure 2 shows the results for the inheritance of an autosomal recessive condition. Several points can be made while discussing these results with students: The mother (8) is homozygous for the dominant allele, while the father (9) is homozygous for the recessive allele. One would therefore predict that all their children will be heterozygous. Teacher s Manual

5 Exploring Electrophoresis: Nature s Dice Family members 12 and 1, being heterozygous, will produce gametes carrying either dominant or recessive alleles (in a 50:50 ratio). The children from 12 and 1 show only two of the three possible genotypes for this locus. This should lead to a discussion about probability and reality (avoiding possible misconceptions that every fourth child must be of a particular genotype if the previous three were of another type). Figure 2 shows the number of DNA bands to be expected on the electrophoresis gels indicating an autosomal recessive condition. Figure shows the number of DNA bands to be expected on the electrophoresis gels indicating a sex-linked condition. Female Male Female 1 Figure Male 1 2 family display suggests that they have received no genetic material from their father with respect to the gene linked to the condition Laboratory Safety DNA and Enzymes 1 15 Figure There is a deliberate problem posed by the analysis of the gels. Individuals 5 and 6 have two children, one of whom turns out not to follow the expected pedigree. As the mother (6) is heterozygous and the father (5) is homozygous for the dominant allele, one child (15) presents no problems; however, child 1 is unexpectedly found to be homozygous for the recessive allele. Students usually suggest several possible explanations: The child may be adopted, i.e., the parents are not her biological parents. There was a mix-up in the samples and the test should be repeated. There was an unexpected mutation (after some thought the students may realize that this must have occurred during the formation of the father s sperm). Finally, some students will, no doubt, suggest that the mother has had an affair with an unknown man. Sex-Linked Inheritance (Kit ) Figure is a family tree that illustrates the inheritance of a sex-linked recessive condition. It has been designed to ask the question, Is the inheritance of the condition a normal recessive inheritance pattern or is it sex linked? Students have to examine the gel electrophoresis results and the family tree and make deductions about the mode of inheritance The tip-off for the students that this is a sex-linked condition should be that the inheritance pattern of the alleles that many of the males in the The naturally-occurring bacterial DNA and restriction enzymes provided with this kit are safe to use. DNA of human origin has not been used. Living organisms are not used at all, so there is no need to follow strict aseptic techniques. However, cleanliness is important to prevent cross-contamination and to ensure success. For example, DNases present in sweat on your fingertips will degrade DNA. Spilled enzymes or DNA solution should be wiped up promptly. Agarose Gel If a microwave oven is used to melt the agarose gel, ensure that the gel is placed in an unsealed container. If a microwave oven is not available, a boiling water bath or hotplate may be used instead. The gel must be swirled occasionally as it melts to prevent charring. The use of a Bunsen burner to melt agarose is not recommended. Warning! Hot, molten agarose can scald, so it must be handled with care. It is advisable to let the molten agarose cool until it is comfortable to handle before pouring the gel. Electrode Tissue (Carbon Fiber) The carbon fiber electrode material may release small fibers, which can cause minor skin irritation if you handle the tissue a lot. It is a wise precaution to wear protective gloves if you find the tissue unpleasant to handle. However, the fibers released are too large to enter the lungs, so it is not necessary to wear a face mask. In addition, the fibers are soluble in body fluids and are completely biodegradable. 5 Teacher s Manual

6 Exploring Electrophoresis: Nature s Dice Electrical Supply Warning! The gel electrophoresis equipment was designed to be used at low voltages (5 volts) with drycell batteries. Under no circumstances should this voltage be exceeded, as the live electrical components are not isolated from the user. Serious or lethal electrical shock could result were the equipment to be connected directly to a power supply that has not been specifically designed for use with this equipment. DNA Stain (CarolinaBLU ) When used as directed, CarolinaBLU presents no serious safety hazard, although care should be taken to prevent splashes on the skin or in the eyes (e.g., wear protective gloves and safety glasses when handling the stain). Used stain can be diluted with water and washed down the drain. Electrophoresis Buffer Solution (TRIS-Borate-EDTA, i.e., TBE) When diluted and used as directed, this buffer presents no serious safety hazards. Used buffer can be washed down the drain. Loading Dye (Bromophenol blue/sucrose) When used as directed, this loading dye presents no serious safety hazards. Used loading dye can be washed down the drain. Reuse of Materials Electrophoresis Buffer Solution (TBE) The TBE buffer solution can be retained and reused several times if desired. Eventually the buffering capacity is reduced due to electrolysis and the buffer does not work well, but at the low voltages encountered with this equipment this should not be evident for some time. CarolinaBLU The CarolinaBLU stain may also be reused. After many uses, its effectiveness will be reduced as it is gradually diluted with buffer solution washed from the gel. With old stain you may find it necessary to soak the gel for longer than the minutes that is suggested in the student s guide. Agarose Gel Agarose is very expensive. Damaged, unused gel (e.g., from a poorly cast gel), may be remelted and used again. Do not attempt to reuse agarose that you have already used for electrophoresis, however. Agarose is not like nutrient agar; nothing will grow on it once it has been made up, so you can prepare a large batch and just use what you need. It can be remelted and allowed to set several times, although care should be taken to prevent excessive evaporation of water during reheating or the percentage of your gel will increase and the DNA bands will not separate as well. Boiling is required to melt the agarose, but once molten it can be maintained in that state at C. Prepared agarose gel should be stored in an airtight container (e.g., a screw-topped bottle) to prevent desiccation. Tips and Tubes Both the tips and tubes are autoclavable, and may be washed, sterilized, and reused if you wish. However, although this may seem desirable to prevent waste, we do not recommend it. Even minute residual amounts of detergent on the plastic may inhibit the delicate (and expensive) restriction enzymes. It is better to recycle the waste plastic (polypropylene) than to reuse it. Storage of Reagents DNA The DNA should be stored in a freezer at 20 C. For short periods (e.g., for 1 or 2 days) it can be kept in a refrigerator at 5 C. Restriction Enzymes The enzymes must be stored at room temperature and be tightly sealed in the foil pouches provided. The desiccant packet should be kept in each foil pouch, since excess moisture can quickly lead to a reduction in the activity of the dried enzymes. Note: It does not matter if the caps come off the enzyme tubes during storage, as long as the pouch is adequately sealed. Electrophoresis Buffer Solution (TRIS-Borate-EDTA, TBE) TBE buffer concentrate and diluted buffer solution should be stored at room temperature. We strongly recommend that you dilute all of the TBE concentrate when you receive it and then store the dilute solution until it is needed. Do not attempt to make up small volumes of solution as errors will result, and dust entering the concentrate will provide nuclei around which crystals can form. If crystals do come out of solution in large quantities, you will need to purchase (or make up) new TBE concentrate. CarolinaBLU Both the concentrate and diluted stain may be stored at room temperature. 6 Teacher s Manual

7 Exploring Electrophoresis: Nature s Dice Hints and Tips Using Syringes Students ability to dispense precise volumes using the syringes will improve with experience. It is therefore a good idea to practice transferring liquids (e.g., food coloring) before starting work with expensive DNA. Practice gels may be cast from cheap agar (use a 1% solution) rather than agarose. Some extra microsyringe tips are provided in the kit for this purpose. Note: For your convenience, you may also purchase Practice Pipetting Stations from Carolina Biological Supply Company (Catalog #RN ). DNA Solutions Should the DNA solution become scattered inside the tubes, tap them repeatedly on a hard surface to return the liquid to the bottom of the tubes. A centrifuge may also be used to spin the DNA down inside the tubes. Melting Agarose Ensure that the container used to prepare the agarose gel is clean. Tiny flecks of dust will not affect the way the gel runs, but they can prove a nuisance when you are trying to see faint bands in the gel. For convenience, dissolve and melt the agarose, using a microwave oven. Less than a minute at full power in a 90-W oven is sufficient for 100 ml of gel. The container (flask or beaker) used to hold the molten agarose must not be sealed, but covered lightly with plastic wrap that has been punctured with one or two small holes. Swirl the gel halfway through the heating cycle to ensure that it is thoroughly mixed and to prevent boiling over. Agarose gel can be prepared using a hotplate. If this is done, the gel must be swirled as it melts, to prevent charring. Better still, stand the container of agarose in a saucepan of boiling water. The use of a Bunsen burner to melt agarose is not recommended. Once melted, the gel may be kept in a molten state by standing the container in a water bath at C until the gel is needed. Take care when handling the molten gel; it will be very hot, and can scald. The gel should be allowed to cool to C before it is poured. Batteries Dry cells (the alkaline or zinc-carbon types) are suitable for use with this equipment. Several runs may be obtained from one battery. Gel Takes Too Long to Run With two new or freshly-charged 9-V batteries, the loading dye should move about 5 mm per hour. At a lower voltage, the loading dye will move more slowly. If, after the first 20 minutes, the loading dye does not seem to have moved and bubbles are not visible at the cathode, check the electrical contacts between the batteries and the electrodes. Ensure that there is enough buffer above the gel to cover the plastic ridges at each end of the tank, but not so much that most of the current passes through the buffer solution rather than the gel. Remember that during long runs (e.g., overnight) or in a warm environment liquid may evaporate from the buffer. The tank should be covered with the 6-toothed comb to reduce such evaporation. Corrosion of Alligator Clips The alligator clip used at the anode will slowly corrode due to electrolysis. This can discolor the buffer solution and gel, but will not interfere with the electrophoresis. Eventually, you will need to replace the clip. Viewing the Stained Gel The bands are seen most easily by holding the gel at an angle against a well-lit surface. Several authors suggest that a yellow or orange filter improves the contrast between stained bands and the background. While this may be true if you are taking photographs, filters can make it more difficult to spot very faint bands. A magnifying lens can be useful; flat fresnel lenses the size of a credit card (sold for use with atlases) are ideal. If the contrast between the background and the DNA bands is strong, the gel may be viewed using an overhead projector. Troubleshooting There Is No DNA on the Gel There are several possible causes here. The wells may have been punctured when loading the gel (this should be clearly visible). In this case, the DNA samples will have run along under the gel rather than through it. Alternatively, the DNA samples may not have been mixed properly with the loading dye. Finally, has the gel been left to run for too long? If the loading dye has moved off the end of the gel, the DNA may have done so too, leaving no samples in the gel. 7 Teacher s Manual

8 Exploring Electrophoresis: Nature s Dice The DNA Bands Are Very Faint Are you using the stain correctly? Once you have applied the CarolinaBLU to the gel, poured off the stain, then rinsed off the excess, take care not to leave any water on the gel. If necessary, wipe over the gel gently with a fingertip to disperse beads of water. The blue stain that starts in the upper layer of the gel can redissolve even in small drops of water left on the surface. Drawing away the stain into water drops on the surface of the gel prevents it from passing down through the gel to stain the nucleic acid. The Bands on the Gel Are Very Close Together and Are Difficult to Tell Apart There are several possible causes of this. The buffer may have evaporated excessively while the gel was running (slowing the electrophoresis down). Another possibility is simply that the gel has not been run for long enough, leaving the DNA fragments bunched together close to the wells. Alternatively, if you have remelted the agarose you used to make the gel several times, you may be using a more concentrated gel than usual, which can result in DNA fragments not separating as quickly or as well Storing Stained Gels Unlike the bands on gels stained with methylene blue, those stained by CarolinaBLU should not fade for many months, provided that the gel is stored out of the light and in distilled or deionized water. We recommend that gels are stored, tightly-wrapped in a plastic bag, in a refrigerator, with a small amount of distilled water. Further Resources Kingston, H. M ABC of Clinical Genetics. Second edition. BMJ Publishing Group. ISBN: Ridley, M Genome. The Autobiography of a Species in 2 Chapters. HarperCollins. ISBN: Cavalli-Sforza, L. L Genes, Peoples and Languages. Penguin Books. ISBN: Web Sites Chromosome 11 (A simulated genetic counseling exercise) Blackett Family DNA activity (Analysis of DNA fingerprinting data) /introduction.html Understanding gene testing The Human Genome project - Exploring our molecular selves The Human Genome - A guide to online information resources Online Mendelian inheritance in man 8 Teacher s Manual GeneCards (Weizmann Institute of Science) bioinfo.weizmann.ac.il/cards/index.html GeneTests - GeneClinics Human Genome Organisation Nature: Genome gateway The Wellcome Trust Nufield Council on Bioethics (UK) The Dolan DNA Learning Center (Cold Spring Harbor Laboratory)

9 Nature s Dice Fill in the results (that is the number of bands in the gel) on this diagram. Female Male 1 2 Figure Carolina Biological Supply Company

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11 Nature s Dice Showing the number of DNA bands to be expected on the electrophoresis gels Female Male Carolina Biological Supply Company Figure 2

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13 Nature s Dice Showing the number of DNA bands to be expected on the electrophoresis gels Female Male 1 2 Figure Carolina Biological Supply Company

14 To order call: (US and Canada) (International) For technical help call: Carolina Biological Supply Company 2700 York Road, Burlington, North Carolina CB