DNA Fingerprinting By Restriction Enzyme Digestion of DNA

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1 Introduction DNA Fingerprinting By Restriction Enzyme Digestion of DNA In this exercise you will be learning how variation in DNA sequence can be used to distinguish between individual DNA molecules and therefore between individual organisms. You will be using restriction endonuclease enzymes that recognize and cleave DNA sequences followed by agarose gel electrophoresis to resolve the resulting DNA fragments. You will then attempt to identify unique and distinguishable patterns of DNA fragment sizes to determine the relatedness of the samples. Restriction digestion and DNA fingerprinting helped usher in the molecular age of biological research. Very little DNA work is done without a restriction digestion step somewhere in the process. It is used in forensics in criminal cases and in paternity testing. It is used by epidemiologists to identify individual strains of infections microorganisms and identify sources of infection. In the laboratory, it is used for genetic mapping and for cloning. Learning Objectives Conceptual 1. That the uniqueness of genetic profiles can be exploited to distinguish individual organisms. 2. That restriction endonucleases are enzymes that cleave DNA at specificallyrecognized target sequences. 3. That agarose gel electrophoresis is the use of a solid matrix and electrical current to separate DNA molecules by size. Practical 1. To gain experience performing enzyme reactions and agarose gel electrophoresis. 2. To become familiar with the use of common laboratory equipment and to conduct enzyme reactions and agarose gel electrophoresis, which will include pipettors, balances, water baths, electrophoresis gel boxes, ultraviolet transilluminator. 3. To learn the proper handling procedures for enzymes and DNA and biohazardous wastes. Underlying Science Restriction enzymes Restriction enzymes are endonucleases that recognize specific sequences of typically 4 to 8 base pair regions of DNA and then cleave the phosphodiester bonds between the nucleotides. Restriction enzymes may cut anywhere within the recognition sequence, but a given enzyme will always cut between the same two nucleotides (see Table 1).

2 TABLE 1. The cleavage of DNA by restriction enzymes. Target sequence and the resultant fragments are shown. Note that EcoRI and SalI produce sticky ends with overhanging nucleotides while DraI and HincII produce blunt ends. Pu = any purine (adenine or guanine); Py = any pyrimidine (thymine or cytosine). Restriction Enzyme EcoRI SalI DraI HincII Recognition Sequence GAATTC CTTAAG GTCGAC CAGCTG TTTAAA AAATTT GTPyPuAC CAPuPyTG Resultant fragments G AATTC CTTAA G G TCGAC CAGCT G TTT AAA AAA TTT GTPy PuAC CAPu PyTG Many restriction enzyme sites (including those shown above) are palindromes that is, reading the upper strand from 5 to 3 is the same as reading the lower strand from 5 to 3. Why would a bacterium want to make an enzyme that cleaves DNA? Restriction enzymes probably evolved as a bacterial defense against bacteriophages (viruses that infect bacteria). In fact restriction enzymes were discovered through research into bacterial resistance to bacteriophage; their discovery enabled the first manipulations of DNA, and the development of molecular biology itself. The name restriction enzyme comes from the enzyme s function of restricting access to the cell. Viral DNA invading a bacterial cell defended by these enzymes will be digested into small, non-functional pieces. A bacterium protects its own DNA from digestion using other enzymes that modify nucleotides in the recognition site. For example, E.coli makes the restriction enzyme EcoRI and the methylating enzyme EcoRI methylase. The methylase modifies EcoRI sites in the bacteria s own genome to prevent it from being digested (See below). m GAATTC CTTAAG m Host induced methylation of DNA. Methylation of the adenines one residue away from the cleavage site protects host DNA from being cleaved. The m indicates methylation. Even though DNA sequence is not random. Genome size stretches of DNA can, statistically, be treated as such. Therefore we can use simple mathematical statistics to estimate the average size fragment obtained by digestion with a particular enzyme using

3 the information about its target site. This is useful in selecting the correct enzyme(s) for the particular DNA target and application. For a "random" DNA sequence, the average distance between restriction sites (targets) is: (length of recognized target) 4 There is an exception for targets with degenerate positions: 2 (# of degenerate positions) x 4 (# of non-degenerate positions ) The basis of DNA fingerprint by restriction analysis is pretty straightforward. Since different organisms (or indeed, different individuals) have variations in their genetic code, restriction target sites will be located in different positions in their genomes. Restriction digestion at those sites with therefore generate DNA fragment sizes that are unique to that organism or individual. The DNA(s) in question is first cut with one or more restriction enzymes and then the resulting fragment sizes (often referred to as RFLP-Restriction Fragment Length Polymorphism) are visualized and analyzed by Agarose Gel Electrophoresis. This technique can be applied to almost any DNA sample from whole genomes to individual genes. Electrophoresis Agarose gel electrophoresis is a way to separate DNA fragments by their sizes and visualize them. The gel consists of a buffer solution (which maintains the proper ph and salt concentration) and contains between 0.5 to 2.5% agarose, a polysaccharide derived from red algae. The agarose forms a porous matrix; the more agarose present, the tighter this matrix is. To separate DNA fragments, you place the digested samples in the wells of the agarose gel and apply an electrical current across the gel. Since DNA is negatively charged at neutral ph (due to its phosphate backbone) it will move toward the positive (red) electrode (See below). The DNA must slip through the pores in the agarose matrix as it moves toward the positive electrode. Smaller DNA fragments can fit through the pores easier than larger fragments. As a result, small fragments of DNA migrate through the gel faster than larger DNA fragments thus alowing the separation of DNA fragments by size. Negatively charged DNA fragments migrate toward the positive electrode during agarose electrophoresis.

4 The agarose gel also contains a dye (ethidium bromide) that fluoresces orange when illuminated with UV light. Ethidium bromide inserts (intercalates) between the base pairs of the DNA present in the gel. Because ethidium bromide fluorescence is greater when it is intercalated, the location of DNA fragments in the gel can be observed when the gel is illuminated with UV light. A DNA size standard that consists of a set of DNA fragments of known sizes is also run on the gel to determine the sizes of the fragments in your sample. The migration of DNA through an agarose gel is not linear with respect to size. There is a logarithmic relationship between the molecular weight of a DNA fragment and the distance traveled through the gel. The molecular size of an unknown linear piece of DNA can be estimated by comparison of the distance that it travels with that of the standards, but a more accurate estimate can be found by graphing the size of the standards (in base pairs) on a log scale vs. the distance traveled (in mm) on a linear scale and using this graph to determine the size of the unknown piece(s) of DNA based on the distance of their migration. Laboratory Exercise In the first part of the laboratory exercise, you will be digesting Lambda DNA samples with a restriction enzyme. In the second part of the exercise, you will run the samples on an agarose gel to determine if there are any of the samples from campus match the bioterror hoax material. Exercise Scenario The FBI is recruiting on campus. They have a need of professionals from every field imaginable including the sciences, and, in particular, molecular forensic science. The advances in molecular biology have been used to solve a variety of crimes and missing persons cases. The FBI seeks to attract only the best and brightest individuals and has contacted the honors biology program and specifically requested that we include the following molecular forensic exercise in the 159H course. We agreed to have this years class participate in an actual investigation related to a possible bioterrorism case. Dr. Quackenbush of the FBI has provided us with DNA extracted from a suspicious substance found in a suitcase which was left unattended at the Detroit Metro Airport. The substance was a white power but upon microscopic examination the material did not appear to be cellular or spore like. After examination by electron microscopy the material was determined to be viral in origin. Further examination showed that this material is a bacteriophage called Lambda. The material is normally considered harmless to humans and other cases have taken priority over what appears to have been a hoax. However such a hoax is not taken lightly, and the FBI believes that they may be able to trace the origin of material by determining the genetic profile of the particular Lambda found at Detroit Metro Airport. They suspect this was staged by evil scientists at that hotbed of terrorist activities the University of Michigan! They want to know if the airport strain matches the genetic profile of Lambda used by evil researchers at U of M. By identifying a possible origin of the hoax material the FBI will gain new leads in their investigation. Materials

5 Restriction Enzyme Digest Eppendorf centrifuge tubes (sometimes called Ep tubes) Racks for holding the Ep tubes Ice buckets Vortex mixer Hind III (restriction enzyme) Restriction enzyme buffer at ten fold (10X) the proper concentration needed for the reaction 37 C water bath Sample DNA extracted from the seized bacteriaphage particals, and DNA extracted from bacteriaphage samples taken from several labs at U of M. DNA Sample 1: Detroit Metro Airport material DNA Sample 2: wildtype phage Lambda DNA Sample 3: evil U of M scientist #1 DNA Sample 4: evil U of M scientist #2 DNA Sample 5: evil U of M scientist #3 Electrophoresis 1x TAE gel running buffer Agarose 2.5 mg/ml ethidium bromide (Note: This compound is a known mutagen and suspected carcinogen. You must handle this material cautiously.) DNA size markers (1 KB ladder) Electrophoresis power supply Gel loading buffer (25% ficoll, 0.05% bromphenol blue) Latex Gloves Polaroid camera and film Plastic bags to store gels in Safety glasses Submarine gel kit, including tank tray and combs. Ultraviolet transilluminator Methods Restriction Enzyme Reaction You will be performing the restriction enzyme digests in a volume of 20ul. These digests are performed by enzymes that function under specific conditions of salt concentration and ph. Therefore the reactions are performed in a buffered solution. Restriction enzymes are supplied with concentrated buffer to make this buffered solution. You will be digesting 10 ul of DNA (about 200ng) in a 20 ul volume you will need to add 10X buffer, water, DNA sample and enzyme in the proper proportions to make the final buffer concentration 1X. The following amounts should be used for a single reaction, and they should be mixed in this order.

6 7 ul H2O 2 ul 10X buffer 10 ul DNA 1 ul Hind III (10 U/ ul) 20 ul You will be performing four reactions so you will make a reaction cocktail instead of mixing each reaction individually, and you will make enough cocktail for one extra reaction to be sure that you have enough cocktail for each reaction. Single reaction Cocktail for 6 reactions 7 ul H2O 42 ul H2O 2 ul 10X buffer 12 ul 10X buffer 10 ul DNA Do not add the experimental reagents to the cocktail 1 ul Hind III 6 ul Hind III Mix the cocktail by gently vortexing. 1. Obtain tubes containing 10 µl of DNA from each of the samples. 2. Use a Sharpie to label each of five sample tubes so that you may identify the DNA sample. Include your group name or number and section number on each tube so everyone knows whose experiment it is. 3. Add 10 µl of the reaction cocktail to each 10 ul DNA sample. 4. Mix by gently vortexing. 5. Place reactions in the 37 C water bath for about 1 hour. 6. After the incubation add 5 ul loading buffer to each sample to prepare them for electrophoresis. Electrophoresis Make an agarose gel: 1. Put on protective gloves and set up your gel box. Ask for help if you are unsure how to set up the apparatus (See below). Set the gel deck in the center of the electrophoresis tank. Slide the gel casting dams down the V grooves of the electrophoresis tank. Insert the comb into the comb position slot closest to the negative (black) electrode. Make sure the surface of the gel deck is level. 2. Weigh out 0.25 g of agarose and add to 25 ml of TAE buffer (final conc. 1% agaose) in a 125 ml flask, mix 3. Heat in microwave until JUST boiling (Set microwave for 1 min and watch the flask carefully). GENTLY swirl--without creating bubbles--and check to see if agarose is completely dissolved. Continue heating as necessary. 4. Put the flask in the 50 C water bath to cool. 5. Put on protective gloves and add 1 µl of 2.5 mg/ml ethidium bromide to the agarose mixture. GENLTY swirl without introducing bubbles. CAUTION: Ethidium bromide is a mutagen, wear latex gloves whenever you work with it and dispose of all ethidiumcontaminated materials as instrucyed in class.

7 6. Pour the molten agarose solution into the apparatus. Ensure that the agarose is evenly distributed over the surface of the gel deck and remove any air bubbles by pushing them to the end of the gel with a pipet tip. 7. Allow the agarose to cool until it is solidified (about min). The agarose will change from clear to translucent when solidified. 8. Remove the gel casting dams and add enough TAE buffer to submerge the gel under 1 to 2 mm of buffer. 9. Remove the comb: Gently wiggle the comb to free the teeth from the gel. Slightly lift up on one side of the comb, then the other. 10. Rinse the comb and the gel dams in dh 2 O and place them to dry. Running the gel: 1. Add 2 µl of 10X loading buffer to each of your restriction digests and gently vortex. 2. Spin the tube for 2 sec in a microcentrifuge to collect the liquid at the bottom of the tube. 3. Pipet 20 µl of the DNA molecular weight standard into the first well of the gel. 4. Load all 22 µl of your restriction digests mixed with loading dye into the following wells. Be sure to write down the order of your samples in your notebook. 5. After you have loaded your digests on the gel dispose if the Ep tubes in the Biohazardous waste container. Your used pipette tips belong in the biohazardous waste too. 6. Close the lid of the gel box and connect the apparatus to a power supply. Check with your instructors to be sure you have the polarity correct. 7. Set the voltage of the power supply to 100 volts and turn it on. Tiny bubbles will rise from the electrode wires in the gel box when it is properly connected. 8. Electrophorese the DNA at 100 volts until the blue dye-front migrates about 2/3 of the way through the gel (approximately 40 to 45 min). 9. Disconnect the gel from the power supply 10. Put on latex gloves, Turn on the nearest water faucet and leave the water running. 11. Using the spatula provided, lift the gel off of the gel deck and place it in a plastic baggie. Handle the gel with care so that the agarose does not tear. 12. Rinse off your gloves under the running faucet and dry them with paper towels BEFORE YOU SEAL THE BAG OR TOUCH ANYTHING ELSE. 13. Dispose of the paper towel as directed in class. 14. View the gel on the UV transilluminator. CAUTION: UV can damage the retina, ALWAYS wear safety glasses!! The TA will show you how to photograph your gel. 15. Dispose of the electrophoresis buffer and the gel as directed. 16. After you have run and photographed your gel you will carefully (i.e. wearing gloves and with out spilling) dispose to the EtBr-contaminated running buffer in the EtBr liquid waste container and your gel in the EtBr solid waste container (open the baggie and slide the gel into the container, then dispose of the baggie in the container as well). 17. Rinse out the electrophoresis apparatus with tap water followed by distilled water and place it upside down on paper towels to dry. 18. Any solid materials that have come in contact with the gel or the running buffer (pipette tips, paper towels, etc.) should be disposed of in the solid EtBr waste container.

8 Gloves should be rinsed under running water, dried with a paper towel and then both gloves and towels placed in the solid EtBr waste. 19. DON T FORGET TO ALWAYS WASH YOUR HANDS BEFORE LEAVING LAB. Controls and Variables Controls For any experimental design, proper controls--both positive and negative should be included to ensure your results are valid and interpretable. We will talk at length about experimental design over the course of the semester. Briefly POSITIVE CONTROLS confirm that your assay (whatever it may be) is working properly and show you what a positive result actually looks like (not always straightforward). NEGATIVE CONTROLS confirm that you are not getting FALSE positive results and show you what a negative result looks like (also not always straightforward). In the case of today s electrophoresis gel, the DNA size marker (1 KB ladder) serves as a positive control. It is used to demonstrate that small fragments do indeed migrate faster than large ones, it confirms that you set up the apparatus correctly and that the DNA was moving through the gel as it should and it confirms that the EtBr staining worked properly. It also can be used to determine the sizes of your unknown fragments and thus, when graphed, can serve for the generation of a standard curve for DNA sizes vs. migration distance. In general, standard curves can be considered positive controls. The empty gel lanes (since you didn t use them all) serve as negative controls--making sure that the bands you see are from the loaded sample and not simply some gel artifact (false positive). For budgetary and scientific reasons, we made a number of assumptions about the restriction digestions that allowed us to forego controls in that part of the exercise (BAD experimental design on our part). We assumed that 1) the digestion reactions worked we had all the conditions right; 2) the sample DNA templates were intact--not digested or degraded prior to our digestions with Hind III. Variables Wewill be concerned with three types of variables in experimental design. MANIPULATED (also called Treatment or Independent ) VARIABLE: This is the parameter you vary or manipulate between treatments. It is what you are testing the effects of. Whenever possible, you try to make all other parameters constant between treatments. RESPONSE (also called Dependent ) VARIABLE: Basically the results that you measure and record. CONTROLLED VARIABLES: All parameters that you keep constant between treatments. Ideally, all parameters other than the manipulated variable. Questions to be addressed in your lab notebook. Make a conclusion about the source of the airport strain based upon the results of your analyses. Explain why.

9 Include a copy of your gel photograph in your notebook. Select one of your unknown lanes, indicate on the photo which lane you selected and estimate the sizes of your DNA fragments in that lane. Instead of setting up five reaction cocktails, you made one master cocktail and dispensed it into five aliquots. What is the advantage of setting up your experiment this way? Think and discuss in terms of experimental design and variables as discussed above. We made some assumptions in running the restriction digestions. Suggest some possible positive and negative controls that could (should!) have been included to test those assumptions.