DNA fingerprinting. Figure 1: Diagrammatic representation of the DNA fingerprinting to be done during this lab experiment.

Transcription

1 DNA fingerprinting BACKGROUND DNA fingerprinting requires the use of several biotechnological techniques, and can vary among different DNA fingerprinting protocols. One way to make a DNA fingerprint involves the use of Restriction Enzyme Digestion, Polymerase Chain Reaction (PCR) and Agarose Gel Electrophoresis (Figure 1). In order to understand DNA fingerprinting as in its entirety, it is important to first understand each of these constitutive parts. Figure 1: Diagrammatic representation of the DNA fingerprinting to be done during this lab experiment. Restriction Enzyme discovery led to the awarding of the Nobel Prize to Daniel Nathans, Werner Arber and Hamilton Smith in These restriction endonucleases, as they were first called, are part of an innate bacterial defense system against viral attack which cuts apart (digests) viral DNA into smaller pieces and are named for the bacterial species from which they were first isolated. For instance EcoRI (note italics) was isolated from Escherichia coli and has a recognition site of GAATTC and its complement (Figure 2). Along any given piece of DNA wherever this specific sequence of bases is found, EcoRI will cleave (or cut) the DNA into two pieces. In vivo, this leads to non-functional viral DNA that is incapable of causing a viral infection. In vitro, these enzymes can be used to identify and cleave molecules of DNA at these specific restriction sites. Because DNA from any two individuals is unique, the digestion of every individual s DNA (be it a bacterial or human cell) will result in a unique set of restriction fragments (pieces of DNA produced by digestion with a given restriction enzyme). These restriction fragments can be compared between individuals to identify restriction fragment length polymorphisms (RFLPs). Although the name sounds complicated, it is descriptive; polymorphisms (poly- many; -morphos- shapes) of different lengths are created when DNA is digested by a given restriction enzyme. These RFLPs are the basis of one type of DNA fingerprint.

2 Figure 2: Recognition site of EcoRI is shaded, the actual cut in the DNA strand is indicated by the placement of the arrows. Restriction digest results in two DNA molecule with complementary single-stranded sticky ends. The discussion above describes the digestion of a single molecule of DNA with a single restriction enzyme. This is a situation that, practically speaking, is worthless as even large molecules of DNA require the use of an electron micrograph to visualize them. In order to easily see the RFLPs that result from a restriction digest, it is necessary to start the process with millions of identical copies of DNA. That s where PCR comes into play. Polymerase chain reaction (PCR) as a technique was considered so important to the field of molecular biology that its discoverer, Kary Mullis, was awarded the Nobel Prize in Fundamentally, PCR is a process whereby a researcher may easily and inexpensively produce millions of copies of DNA from a single molecule of DNA. PCR utilizes a cycling system with three basic steps (Figure 3). 1. Denaturing: temperatures near 95 C are sufficient to denature doublestranded DNA molecules into single strands, exposing the complementary bases. 2. Annealing: temperatures near 60 C allow for the binding of primers to a complementary section of the now-single-stranded template DNA. 3. Elongating: temperatures near 72 C allow for the synthesis of new DNA strands by DNA polymerase at the site of the 3 end of the primer molecule. Figure 3: Diagrammatic representation of the steps in a PCR cycle. Note that at the completion of the three cycles (temperature profiles) the number of DNA molecules has doubled. The use of primers is essential during PCR because DNA polymerase will only bind to the 3 - OH end of an existing DNA molecule. In vivo, this 3 -OH is supplied by RNA polymerase whereas during PCR it is supplied by primer DNA. Primers are short, single-stranded pieces

3 of DNA that have been engineered to bind to a specific portion of the target DNA genome. They are typically ~25bp in length and are specific enough to ensure that they will only bind to one location on the template DNA. The annealing temperature of the PCR reaction is determined by how well the target and primer DNA complementarily bind- the closer the match between the two, the higher the annealing temperature (the range is typically C). At the foundation of today s PCR reactions is a thermostable DNA polymerase isolated from Thermus aquaticus. Prior to 1983, researchers were performing a type of PCR but it was cumbersome and expensive because mesophilic DNA polymerases could not withstand the high temperatures required during the PCR reaction cycle. For instance, it was denatured along with the DNA in the denaturing step, and operated at an optimal temperature of 37 C- much lower than the annealing temperature of the reaction- which led to non-specific binding of the primer DNA to non-target areas of the template DNA. In addition to these criteria, PCR also requires dntps (free nucleotide triphosphates to be incorporated into the new DNA molecule), and a liquid buffer to ensure ph and other aspects of the environment are kept constant. Template DNA, primers, dntps and buffer are all combined in a small tube and are placed into a thermocycler- a machine that will cycle the reaction through the temperature profile listed above for a specific number of cycles. At the end of every cycle the number of target DNA molecules doubles and one molecule can be amplified into a million in just 20 cycles (that s about 40 minutes!). As stated above, this concept of amplifying DNA is important to many different areas of biotechnology and molecular biology because one molecule of DNA is too small to be seen without an electron microscope- and is therefore functionally invisible. PCR accomplishes the task of producing millions of copies, and restriction enzyme digests create the RFLPs that will genetically fingerprint different individuals. Agarose Gel Electrophoresis is the technique that will be used to visualize the millions of RFLP copies generated during PCR and subsequent restriction enzyme digestion. Agarose Gel Electrophoresis is a technique that uses electricity to move negatively charged DNA molecules through an agar-containing gel. This technique separates molecules of DNA based on the length of time it takes for the DNA molecule to migrate through the agar (a gelatin-like substance much like the agar Petri dishes are made with). The term electrophoresis literally means to carry with electricity, and that is exactly what it does. Although all DNA molecules (due to their net negative charge) are attracted to, and thus move toward a positive electrode, their movement is impeded by the agarose matrix. This means that larger pieces take a longer time to move through the matrix. The end result is that all of the DNA subjected to Agarose Gel Electrophoresis will be separated based on the size of the molecules, and different length RFLPs will be seen as unique bands. The DNA is then visualized using the fluorescent chemical ethidium bromide (a powerful carcinogen and mutagen) which intercalates (positions between) base pairs in a molecule of DNA (Figure 4). When ethidium bromide is excited by ultraviolet light, it emits orange (visible) light. The relative size of these separated and fluorescing fragments is then determined by comparing them to a molecular standard of DNA pieces (e.g., molecular marker, ladder) whose size is known, just like measuring length against a ruler.

4 Figure 4: Left: chemical structure of ethidium bromide; right: ethidium bromide intercalated between basepairs of DNA molecule. INTRODUCTION In this experiment, you will use DNA Fingerprinting to identify the source of a food poisoning endemic using a universal primer for a ~1500bp portion of the 16S rdna gene. You will receive four samples: environmental sample 1; environmental sample 2, environmental sample 3, and a patient stool sample. Prior to your receiving them, these samples have been processed in the following manner: Samples have been collected from the environments (i.e., restaurants, patient). Samples have been cultured to enrich for the presence of any bacterial contaminants. The genomic DNA has been extracted from the bacterial samples and PCR has been used to amplify the 16S subunit of the ribosomal DNA (a specific region of the chromosome). You will receive an eppendorf tube with the PCR product, and you will perform a restriction digest of each of your four DNA samples using two restriction enzymes: EcoRI (mentioned above) and HindIII (derived from Haemophilus influenzae). Once you have performed the digests, you will run the samples on a 1% agarose gel in order to visualize the banding patterns that make up the DNA fingerprint. Finally, you will determine the source of the food poisoning endemic by comparing the DNA fingerprint of the patient stool sample to the three environmental samples. LAB EXERCISE I. PCR Protocol (These steps are done for you, they are included here only to show you how your sample has been processed) 1. Enrich environmental & patient samples in broth media. 2. Streak broth for isolation of likely Gram negative bacterial pathogen on EMB and MacConkey agar. 3. Transfer a large colony of organisms from each sample to 1.5ml eppendorf tube containing 250µl sterile water. 4. Vortex the samples until the colony is broken up and all the cells have been resuspended. 5. Boil the tubes 10 minutes to lyse the bacterial cells.

5 6. Place the tubes at room temperature to cool. 7. Add a 5µl aliquot of each sample (four total) to separate 500µl eppendorf tubes. 8. To each of these four tubes add 20µl PCR Master Mix (containing buffer, dntps, water, Taq polymerase and primers). 9. Load samples into the thermocycler and run at the following settings: 1. step 1: 95 C for 15 minute (initial denaturation) 2. step 2: 94 C for 1 minute (denaturing step) 3. step 3: 60 C for 1 minute (annealing step) 4. step 4: 72 C for 1 minute (elongation step) 5. step 5: repeat steps 2 4 for 32 cycles 6. step 6: 72 C for 10 minutes (final elongation step) 7. step 7: 4 C until machine is turned off (simulates cooling until further use) II. Restriction Enzyme Digest Class supplies Team supplies 37 C water bath 4 tubes containing 1 each of the samples: env 1, env 2, env 3, patient 4 eppendorf tubes 1 tube Digest Master Mix Pipet Pipet tips 1. Add 5µl of each of the four PCR products above to separate 500µl eppendorf tubes. 2. Add 15µl of Digest Master Mix (containing buffer, restriction enzymes and water) to each tube. 3. Incubate the tubes in a 37 C water bath for 1 hour. (Perform Agarose Gel Electrophoresis Set-up during this incubation.) III. Agarose Gel Electrophoresis Supplies Microwave Weigh station Per group: Electrophoresis apparatus and gel tray Agar Weigh paper 250 ml flask with 50 ml sterile SB buffer 1 bottle SB buffer Pipets Pipet tips Restriction digests from above

6 Protocol 1. Assemble your electrophoresis apparatus & gel tray according to the directions given to you by your instructor. 2. Prepare a 1% agarose gel: 1. Add 0.5g agarose to 50ml SB buffer in a 250ml flask. 2. Microwave the solution until all of the agarose has melted (~45 sec). 3. Allow the gel to cool slightly (until the bottom of the glass flask is not too hot to touch). 4. Your instructor will add 4µl ethidium bromide to the gel. EtBr is carcinogenic- do not allow it to touch your skin. 5. Swirl the agarose solution gently to mix it and pour it into the gel tray & comb. 6. Allow the gel to cool until it solidifies. 3. Once the gel is cool transfer it to an electrophoresis chamber. 4. Fill the chamber with enough SB buffer to cover the gel. 5. Remove the comb. 6. Load the gel with your samples according to the directions given to you by your instructor. 1. Add 5µl loading buffer to each of your four samples, load the entire samples into separate wells of the gel. 2. Load 20µl of the molecular marker next to your samples. 7. Run the gel at V for minutes (until the colored dye has migrated ¾ of the way through the gel). 8. Visualize gel & capture image photographically.

7 DATA AND OBSERVATIONS 1. Measure the distance traveled for each band for both the molecular marker and each of the four samples. 2. Graph the molecular weight (in basepairs) vs. the distance traveled for each of the bands in your molecular marker. 3. Calculate the size of each of the fragments of your four samples, based on the graph constructed above. 4. Determine which environmental sample is the likely source of the bacterial food poisoning. References 1. Madigan, M., J. M. Martinko, P. V. Dunlap and D. P. Clark. Brock s Biology of Microorganisms, 11 th Edition. Benjamin Cummings Tortora, G. J., B. R. Funke and C. L. Case. Microbiology An Introduction, 9th Edition. Addison-Wesley Sambrook, J., E.F. Fritsch and T. Maniatis. Molecular Cloning: A Laboratory Manual, 2 nd Edition. Cold Spring Harbor Laboratory Press