USING RESTRICTION ENZYMES TO IDENTIFY SIMILAR SPECIES How many species do you see? ___________ ___________ There are a lot of organisms that look very similar to one another but are different species. However, there are also individuals that look very different but are the same species. So how can we tell two similar species apart without being an expert in every organism’s morphology? We will learn a genetic technique to differentiate between 2 different species using DNA. For example, the following two species of fish are very closely related and difficult to differentiate morphologically. Write down some observations that you make about the difference between these two species Exercise I: An example restriction enzyme digest using foam noodles. Imagine that we have identified two organisms morphologically, pink and blue. We then extracted DNA and amplified a specific gene (represented by a foam noodle). This gene will then be “digested” or exposed to the enzyme BamHI for a period of time. Each group of 3 students will have a gene from an individual (6 total in the class), will identify restriction sites on their gene (foam noodle), and make the cuts using a plastic knife. Write in the blank the forward (3’-5’) and reverse (5’-3’) sequence of nucleotides for the restriction site for the SmaI enzyme. 5’- C C C G G G -3’ 3’- G G G C C C -5’ Each member of your group will have a specific job. One person will be the nucleotide counter, who will identify the specific sequence of the enzyme restriction site and will count the number of nucleotides for each segment. Another person will need to physically cut the noodle using a plastic knife once either the teacher or the GK12 fellow have approved your restriction sites. And the recorder will write down the morphological identification of their individual (pink or blue) and the size and number of their fragments. Select a member of your group to do each job and write their names below. _________________________ : nucleotide counter _________________________ : noodle cutter _________________________ : data recorder How many SmaI restriction sites were on your gene? _______ How many fragments of DNA were left after the gene was digested? ______ How many base pairs are in each fragment? 1. _____ 2. _____ Once the gene is digest please clean up scrap foam from you bench. Exercise II: Separating restriction fragments using gel Your gene should have separated into 3 fragments. If it didn’t, ask me to help you find the other restriction site(s). Each student will take one of the 3 fragments produced by your restriction enzyme digest and as a group move to the front of the classroom. There will be 6 groups, each going into a different “well.” In this hypothetical scenario, the room will be filled with an imaginary gel matrix. The gel matrix will mean the larger/longer pieces of DNA bump into more of the gel molecules, making them run slower through the gel matrix. If your piece of DNA is larger than 40 base pairs you’ll only be allowed to take a step every 4 seconds. If your DNA fragment is between 39-30 base pairs you can take a step every 3 seconds. If your DNA fragment is less than 29-20 base pairs you can take a step every 2 seconds. If your DNA fragment is less than 19 base pairs you can take a step every 1 seconds. Wait tell I turn on the machine (I will say “go”) after a few seconds we will stop to see the banding pattern on the gel. Remember, because of the phosphate in the DNA the molecule is negatively charged so we will apply a positive charge to attract the DNA molecules on the opposite side of the classroom and a negative charge behind the DNA (the chalk board) to repel the DNA molecules. Restriction enzymes or restriction endonucleases are proteins produced by bacteria to prevent or restrict invasion by foreign DNA. They act as DNA scissors, cutting the foreign DNA into pieces so that it cannot function. Restriction enzymes recognize and “cut” DNA strands at specific sequences called restriction sites. Each different restriction enzyme has its own site that is a very specific sequence of nucleotides. Example restriction sites and cutting patterns for specific enzymes: EcoRI 5'- G A A T T C -3' 3'- C T T A A G -5' BamHI 5'- G G A T C C -3' 3'- C C T A G G -5' AluI 5'- A G C T -3' 3'- T C G A -5' Under appropriate conditions (salt concentration, pH, and temperature), a restriction enzyme will cleave a piece of DNA into a series of fragments. The number and sizes of the fragments depend on the number and location of restriction sites for that enzyme in the given DNA. Evolution is the change in allele frequencies in a population over time. Remember that an allele is an alternate form of a gene, meaning that one gene can have variations in sequence patterns called alleles. And, between two species these sequences can vary dramatically. So, there may be variations in the locations of restriction sites between two species. This variation in restriction site location could potentially lead to size variations in fragments after exposure to a restriction enzyme that we can use to identify different species. Now I want each group to restart this procedure by using toothpicks and tape to put the DNA molecules back together. Then look at the other restriction sites provided in the explanation to see if another enzyme will restrict this gene in both species. Write all enzymes that can digest your DNA molecule below. __________________________________________________________ __________________________________________________________ Compare your list of enzymes with your classmates for the next 5 minutes and see if there are any proteins that will restrict this gene in both species. If there are other enzymes that will restrict this gene in both species, lets run another gel (using the same rules as before) to see if it can be useful in determining species identifications. Did all the morphological identifications match the DNA identifications using SmaI? If not how did they differ and how can you tell that the identifications are wrong? No, there was a pink that showed a genetic identity of a blue. Were there other enzymes that cut your fragment? List your morphological identification, genetic identification, and the enzymes that restricted your gene. Pink: EcoRI, AluI Blue: BamHI, AluI If there were a second enzyme that could digest both species would it work for species identification? Please explain why or why not. AluI, no, both species had the same fragment sizes and thus banding patterns AluI BamHI EcoRI SmaI Blue 50, 25, 25 2 medium fragments, 1 small fragments GGAACCGCCCGGGCAACTTAAGCAGCTCACGGGATCCTTGTATTGGATCC Pink 10, 20, 70 2 small fragments, 1 large fragment GGATCCGCACGGGCAACTTAAGCAGCTCCCGGGATCCTTGTATTGGATAC