3) Now, let's calculate our PCR yield: how many copies of the cytochrome b gene we may have in each sample following PCR (if all went according to plan). We performed 35 cycles of PCR on our bushmeat samples. Initial denaturation at 94 °C for 4 min and with a final extension at 72 °C for 10 minutes. The equation to calculate the final number of DNA strands created by PCR = N2^n, where N = the original number of DNA molecules to be copied and n = the # of PCR cycles.
4) Add these calculations for each sample to your spreadsheet. |
Bushmeat is legal in some African countries but is illegal in Kenya. Once bushmeat as been processed, it is indistinguishable from domestic meat. Therefore DNA analysis is required to determined if the meat sold, labeled as beef, pork, goat or lamb, is actually wildlife meat. We will be testing several samples to ascertain the species of origin.
Please read over the summary below from a report entitled "Lifting the Siege: Securing Kenya's Wildlife."
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Where did our meat samples originate? |
Our samples are from the Taita Taveta district of southeastern Kenya, which includes Kenya's largest national park system, Tsavo East and Tsavo West National Parks. Specifically, our samples are from the Kasigau area between the two parks, located on the trailing edge of the Eastern Arc Mountains. The Kasigau landscape is dominated by Mt. Kasigau, which the Titata people settled around to serve as a water catchment. The area also serves as a migration corridor between Tsavo East and West National Parks and is rife with human wildlife conflict.
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Bushmeat Processing: Using aseptic techniques, the bushmeat samples are cut into approximately 1 cc sections. They labeled and stored in ethanol at -20 degrees Celsius. Special care is taken to ensure no cross-contamination or human contamination occurs. Samples are then carried back or shipped to the WKU biotechnology Center. -----> This has already been done. |
This process is similar to the extraction we completed with the strawberry. However it involves many more steps and results in a cleaner product with far less protein. We use special "kits" as pictured to streamline the process. Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. Once extraction is complete, the DNA sample is tested to ensure an adequate amount of intact DNA was extracted from the sample. -----> This process has already been done with our meat samples. In Lab 10, you will practice extracting DNA from a strawberry to better understand this process |
PCR makes copies of a DNA fragment from one original copy. The goal is to amplify a specific region, the target DNA or gene of interest (GoI), depending on the type or goal of research. The PCR cocktail includes the following ingredients: the DNA sample, primers (short sequences of RNA or DNA that start replication), dNTPs (free nucleotides), taq polymerase (a heat stable form of DNA polymerase derived from bacteria) and a buffer solution. There are three steps to PCR in which the temperature is cycled (in the thermo"cyler"). You need to know the steps and what happens in each! The total number of resulting DNA strands is (the number of original strands) X 2^n, where n = the number of PCR cycles. -----> In Lab 10, you will be given PCR products from our meat samples in lab and asked make some calculations. |
Agarose gel electrophoresis is a method used to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. Your PCR products are deposited in the top of the gel. Using electricity, the DNA (with a negative charge) is pushed through the gel towards the positive electrode. As your gel "runs," the DNA is separated by size. The DNA strands show up as bands under UV light and you can read the results. Your products can be compared with the ladder or marker, which has standard sized DNA fragments of KNOWN length used for comparison. In this way, you can know the exact length of your DNA samples. -----> You will MAKE & RUN an agorose gel in Lab 11 to make sure our PCR product contains the cytochrome b gene. |
Once we know we have amplified (copied) the right gene we are ready to sequence the gene. We expect the sequence (the order of As, Ts, Cs and Gs) within the cytochrome b gene to be different for different species. Samples are placed into a sequencer apparatus which can detect the order of nucleotide bases in our sample. The sequence is then cleaned and edited, |
6) BLASTing: The National Institute of Health (NIH) and National Center for Biotechnology Information (NCBI) hosts a database called GenBank, which houses all known DNA sequences. Once the sequences of our samples are ready, they are pasted into a search tool (called a BLAST) which matches them to the correct species! -----> You will be provided the sequence of successful samples in Lab 11 and asked to determine the species of origin in lab. |
1) Bushmeat Processing: Using aseptic techniques, the bushmeat samples are cut into approximately 1 cc sections. They labeled and stored in ethanol at -20 degrees Celsius. Special care is taken to ensure no cross-contamination or human contamination occurs. Samples are then carried back or shipped to the WKU biotechnology Center. -----> This has already been done. 2) Digestion & Extraction: This process is similar to the extraction we completed with the strawberry. However it involves many more steps and results in a cleaner product with far less protein. We use special "kits" as pictured to streamline the process. Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. Once extraction is complete, the DNA sample is tested to ensure an adequate amount of intact DNA was extracted from the sample. -----> This process has already been done with our meat samples. In Lab 10, you will practice extracting DNA from a strawberry to better understand this process 3) Polymerase chain reaction (PCR): PCR makes copies of a DNA fragment from one original copy. The goal is to amplify a specific region, the target DNA or gene of interest (GoI), depending on the type or goal of research. The PCR cocktail includes the following ingredients: the DNA sample, primers (short sequences of RNA or DNA that start replication), dNTPs (free nucleotides), taq polymerase (a heat stable form of DNA polymerase derived from bacteria) and a buffer solution. There are three steps to PCR in which the temperature is cycled (in the thermo"cyler"). You need to know the steps and what happens in each! The total number of resulting DNA strands is (the number of original strands) X 2^n, where n = the number of PCR cycles. -----> In Lab 10, you will be given PCR products from our meat samples in lab and asked make some calculations. 4) Gel electrophoresis: Agarose gel electrophoresis is a method used to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. Your PCR products are deposited in the top of the gel. Using electricity, the DNA (with a negative charge) is pushed through the gel towards the positive electrode. As your gel "runs," the DNA is separated by size. The DNA strands show up as bands under UV light and you can read the results. Your products can be compared with the ladder or marker, which has standard sized DNA fragments of KNOWN length used for comparison. In this way, you can know the exact length of your DNA samples. -----> You will MAKE & RUN an agorose gel in Lab 11 to make sure our PCR product contains the cytochrome b gene. 5) Sequencing: Once we know we have amplified (copied) the right gene we are ready to sequence the gene. We expect the sequence (the order of As, Ts, Cs and Gs) within the cytochrome b gene to be different for different species. Samples are placed into a sequncer apparatus which can detect the order of nucleotide bases in our sample. The sequence is then cleaned and edited, 6) BLASTing: The National Institute of Health (NIH) and National Center for Biotechnology Information (NCBI) hosts a database called GenBank, which houses all known DNA sequences. Once the sequences of our samples are ready, they are pasted into a search tool (called a BLAST) which matches them to the correct species! -----> You will be provided the sequence of successful samples in Lab 11 and asked to determine the species of origin in lab. |
Lab will precede in three parts.
1) Gel electrophoresis: You will create an agarose gel in lab. You will place a small amount of the PCR product from each bushmeat sample into the gel. If we synthesized the correct DNA, all our samples should be about 490 base pairs long. You will view these results to determine if our PCR was successful. 2) Analyze the sequence results: You will be provided with output from the DNA sequencer in the WKU Biotechnology Center for each bushmeat sample. You will then enter the sequences into a database for species identification. 3) Conclusions: Lastly, we will discuss the bushmeat crisis as a class. |
Procedure: Confirm the actual species of origin for our samples and compare it to the putative species (what is was sold as).
1. View the resulting sequences for each of our samples HERE. 2. You are assigned to determine the species corresponding to your group number in lab. View the putative species for each sample HERE.
3. Visit the National Center for Biotechnology Information (NCBI) database called GenBank. 4. Follow the steps in the slide show below to match your sequence to all those stored in GenBank. Start at THIS LINK: The Genebank BLAST Page. 5. Once you've identified the samples' species of origin, add it to your data table along with the putative species. |