Molecular Bio Lab Introduction to basis of technique DNA is the basic chemical building block of life. It is made of up four types of nucleotides: cytosine (C), guanine (G), thymine (T), adenine (A). These nucleotides are arranged in various combinations to encode sequences of genetic information. Many DNA sequences are unique to specific species and even to individuals. Because DNA has distinct sequences it can be used to differentiate or identify organisms. There are several methods of distinguishing different DNA sequences. One method, called DNA sequencing, actually allows scientists to determine the exact order of each nucleotide in a DNA molecule. This process if very precise, but is more expensive and takes a longer time to complete than other techniques. A simple method of comparing DNA from different sources is to cut or digest it with restriction enzymes. Restriction enzymes can be thought of as molecular scissors. They are proteins that cleave DNA only where certain combinations of nucleotides occur. In Table 1, there are several restriction enzymes listed along with their recognition sequences. The site where the enzyme cuts is indicated by arrows. Table 1. Restriction enzymes and their recognition sequences Restriction Recognition Enzyme Sequence (site of cutting) EcoR I Msc I Hind III Sma I If you look at Table 2 you will see two DNA sequences. Imagine that we digest both these pieces of DNA with the restriction enzyme Msc I. Draw a line through the sequences where they would be cut. Table 2. Examples of DNA sequences that may be digested with Msc I DNA Sequence Number of fragments after digestion 1 ATGCGTGGCCAGCTGGGCCATGGCCAGGTTCGTC TACGCACCGGTCGACCCGGTACCGGTCCAAGCAG 2 TAGCCGGATTGCTGGCCATCGTTGCGGGAAATTTT ATCGGCCTAACGACCGGTAGCAACGCCCTTTAAAA You should see that the cuts would generate 3 small pieces in the first strand of DNA and 2 larger pieces in the second strand of DNA. So even if we didn’t know the sequence of the DNA, we would know that the digestion gave us three small pieces from one DNA source and two large pieces from the other DNA source. Because the digestion yielded different sizes and numbers of fragments, we know the sequences of the DNA strands are not the same. But, as you’ll see when you look at the DNA samples provided, DNA dissolves in solution and appears to the naked eye like water in the bottom of a tube. How can we actually see the three little fragments and the two larger ones? They can’t be seen with the eye, or even a microscope, but we can visualize them using agarose gel electrophoresis and a fluorescent dye that binds to DNA. Agarose is a gelatin-like substance that is purified from seaweed. Polymers of agarose form a tiny mesh network when they solidify into a gel. Agarose gels can be thought of as molecular sieves. When you shake sand through a sieve, the smallest pieces come out first and the larger pieces remain in the sieve. Just like sand, when you push DNA through an agarose gel, the smallest pieces will move faster through the gel than the larger pieces. We can push DNA through the gel by applying an electrical current. The top of the gel is negatively charged (cathode) and the bottom of the gel is positively charged (anode). DNA molecules are negatively charged because of phosphates in the backbone of the structure. The DNA is loaded into a small chamber called a well at the top of the gel. When the electrical current is applied the negatively charged DNA will move towards the positive charge at the bottom of the gel with the smaller fragments moving faster than the large fragments. The electrical current is then stopped and the DNA is stuck in the gel. The fragments of DNA can be seen as distinct bands when the gel is viewed with ultra violet (UV) light that excites the fluorescent dye in the gel. A band is not just a single piece of DNA, but a collection of pieces that are all the same size. An example of an agarose gel with DNA bands being illuminated with UV light is shown in Figure 1. Draw an arrow to represent the direction that the DNA moves in the gel. Are the smallest DNA fragments found at the top or the bottom of this gel? Figure 1. Sample of an agarose gel Copper electrode (-) Copper electrode (+) In order to apply an electrical current, the gel inserted into an apparatus with control buttons and an adaptor cord that is inserted into an electrical outlet. A diagram of the E-gel Base is shown in Figure 2. Figure 2. Diagram of E-gel Base Normally, a DNA ladder is also run in the gel with your DNA samples. The ladder has a pool of fragments of known sizes and two colored dyes. One of the dyes is small and will migrate quickly through the gel at the same rate as the smallest pieces of DNA. The other dye is large and will migrate slowly through the gel at the same rate as the largest pieces of DNA. The two dyes in the ladder will allow you to make sure that your DNA fragments are fully separated, but the small fragments don’t run off the bottom of the gel. You can also compare your DNA bands to those of the ladder and estimate the size of your DNA fragments. Double-stranded DNA is measured in pairs of nucleotides called base pairs. Figure 3, on the following page, shows in image of the DNA ladder that you will use in this lab. Look again at table 2, above each DNA sequence write the number of base pairs found in the fragments of digested DNA. Figure 3. Image of DNA ladder and size of DNA fragments in each band (numbers indicate base pairs of DNA) This method of comparing DNA is less expensive and faster than DNA sequencing. Rapid identification of DNA is important in a variety of scientific fields, like forensics and epidemiology. You’ve probably heard of forensic science, where molecular biology can be used to analyze evidence found at the scene of a crime. Epidemiology is a lesser known field of science that studies the cause and distribution of disease in a population to control spread of disease. When the cause is identified, it is called the etiological agent of the disease. For example, this type of DNA analysis could be used to determine the source of a food poisoning outbreak. Food poisoning is often caused by ingestion of a food contaminated with bacteria. The bacteria colonize the intestine and infect the person who consumed the food causing nausea diarrhea, abdominal pain, head ache, vomiting, gastroenteritis, fever, or fatigue. In some cases the illness may result in death. It is critical that scientists identify the contaminated food as quickly as possible to prevent the spread of disease. Today we would like you to help solve an epidemiological study. Food Poisoning Lab A food poisoning outbreak has occurred in your town. Yesterday 45 people were admitted into the local hospital because they were experiencing symptoms of severe food infection including vomiting, abdominal pain, and diarrhea. The clinical lab at the hospital determined that the people were infected by the bacteria Salmonella enterica. A questionnaire was given to all of the patients asking where and what they had eaten in the last 48 hours. All of the patients had attended a school picnic the previous day. The remaining food was stored in a refrigerator at the school. You are a microbiologist with the department of health and we need you to find out as soon as possible which of the food was contaminated to prevent further illness. Table 3 below shows the results of the questionnaire and indicates what percentage of infected patients ate each type of food. Table 3. Percentage of patients that consumed each food from the picnic. Food at the picnic % of infected patients who consumed the food Spinach salad 57% Potato salad 25% Sliced tomatoes 37% Green beans 98% Peanuts 15% Hot dogs 53% Based on the results of the questionnaire, what is your hypothesis for which food likely contains bacteria that caused the infection and it the etiological agent of the outbreak? DNA from S. enterica as well as DNA from the bacterial culture of the food has been purified from cell samples for you by your assistant. You will find these tubes in your kit labeled S (pre-cut Salmonella DNA) and G (DNA from bacteria found in green beans). The Salmonella DNA has already been digested with Sma I. You will need to digest DNA from the bacteria found in green beans with the same restriction enzyme. You will then analyze the size of DNA fragments produced in the digestion using agarose gel electrophoresis. By comparing the bands of the Salmonella DNA to those of the bacteria cultured from the food, you should be able to determine if the green beans contain Salmonella and were the cause of the food poisoning outbreak. You should also be able to determine the approximate size of each fragment relative to the DNA ladder. If your hypothesis is correct, how will the bands of the predigested Salmonella DNA compare to the DNA isolated from bacteria in green beans that you digest? Materials This lab should be done in pairs. Before getting started, please check that you and your partner have the following materials: E-gel base E-gel Pipette Box of pipette tips Tube labeled S (Pre-cut Salmonella DNA) Tube labeled G (DNA from bacteria found in green beans) Tube labeled L (DNA ladder) Tube labeled D (Loading dye) Tube labeled B (Restriction enzyme buffer) Tube labeled W (Sterile water) Beaker (for disposal of used pipette tips) Empty 1.5 ml tube labeled U Tube rack Lab gloves DNA digestion The first step of the lab is to digest the DNA purified from the bacteria growing in the green beans. Follow the procedure below: 1. Put on your gloves to protect your samples from your DNA 2. Get an empty 1.5 ml tube and label the top with your initials and U (for unknown) 3. To the empty tube add 14 µl of sterile water (from the W tube) 4. Add 2 µl of restriction enzyme buffer (from the B tube) 5. Add 3 µl of DNA from bacteria found in the green beans (from the G tube) 6. At the front of the room there is an ice bucket that contains a tube labeled Sma I, bring your tube over to the ice bucket and add 1 µl of the Sma I restriction enzyme 7. Mix the reaction components by pipetting up and down in the tube 8. Set the tube into your tube rack and incubate the reaction at room temperature for 10 minutes 9. While the reaction is incubating set up your E-gel Gel electrophoresis The next step is to analyze the fragments produced in the digestion reaction. You will compare your reaction to undigested DNA, the Salmonella DNA that has already been digested with Sma I, and the DNA ladder. Using undigested DNA is a negative control. It allows you to see that the DNA was not cut prior to your digestion. Using the pre-cut Salmonella DNA is a positive control. It allows you to see what your DNA digest should look like if the bacteria contaminating the green beans were also Salmonella. The DNA ladder will allow you to estimate the size of all the DNA bands in the gel. To carry out this analysis, follow the procedure below: 1. Plug in your E-gel Base 2. Open the package containing the E-gel and insert it into the base following the diagram in Figure 2 and the demonstration from your instructor 3. Check that a red light comes on at the top of the gel 4. Hold down the 15 min. button until you hear two short beeps 5. Check that the light is now blinking green 6. After about 1 min., the gel will start beeping and a red light will blink 7. Once your restriction digestion is complete, add 2 µl of loading dye to the tube 8. Make a dilution of the remaining DNA from tube G by adding 14 µl of sterile water to the tube. This is the undigested negative control DNA sample. 9. Add 2 µl of loading dye to the diluted DNA in tube G 10. Add 2 µl of loading dye to the pre-cut Salmonella DNA in tube S 11. Your samples are now ready to load into your gel 12. Gently, remove the plastic comb that is in the wells at the top of the gel 13. Following the demonstration by your instructor, each partner should practice loading the gel with loading dye alone 14. Once you are comfortable, load 20 µl of your DNA samples (Tubes U, G, and S) and 10 µl of ladder (TUBE L) into the gel into the lane numbers corresponding to the chart below 1 2 3 4 5 6 7 8 9 10 11 12 practice practice Ladder Your Undigested Pre-cut (TUBE digestion DNA Salmonella L) reaction (TUBE G) DNA (TUBE (TUBE S) U) 15. After you gel is loaded, press the 30 min. button again and the light should turn green 16. The gel will beep when it has completed the run 17. Remove your gel from the E-gel base and look at it on the Safe-Imager light box 18. Take a picture of the gel if possible and estimate the size of the DNA fragments Based on your results, did Salmonella cause the food poisoning outbreak? How do you know?
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