Click here to enter the virtual lab. Note: The lab refers to a "DNA Size Standard" which is the same thing as the ladder discussed in the Pre-Lab.
Please review the types of questions that can be answered by PCR in the image above.
Learn the ingredients (components) required for PCR to work. You also need to know what each one is and why it is required!
PCR components. Restriction enzymes are only used in some types of analysis. We won't be using them for our purposes. Also, two types of primers are needed, a forward primer and a reverse primer. Click to enlarge.
The Steps of PCR
You need to know the three steps of PCR and should be able to describe what happens in each step. You also need to know how we determine the results of PCR...how can we know how many copies we have after the procedure?
Exercise III. Determine PCR Yield
Once we know we have enough of the cytochrome b gene, we are ready for PCR.
Procedure: How much useful DNA will we have following PCR?
1) View this 3-minute video on PCR. You need to know the ingredients for PCR and why each is used. You also need to know the three steps and what happens in each one. You do not need to know the timing of the steps nor their corresponding temperature.
1) View this 3-minute video on PCR. You need to know the ingredients for PCR and why each is used. You also need to know the three steps and what happens in each one. You do not need to know the timing of the steps nor their corresponding temperature.
2) Visit our research library to learn a bit more about PCR. You need to know the ingredients for PCR and why each is used. You also need to know the three steps and what happens in each one. You do not need to know the timing of the steps nor their corresponding temperature.
3) We use an enzyme in PCR called Taq Polymerase. It mirrors the same action of DNA polymerase in our own cells during DNA synthesis.
3) We use an enzyme in PCR called Taq Polymerase. It mirrors the same action of DNA polymerase in our own cells during DNA synthesis.
You'll review the entire process of DNA synthesis in BIOL 120. In it, DNA polymerase builds the new copies of DNA strands from the original template.
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We use Taq-Polymerase in the same way in PCR. It is an enzyme from heat resistant bacteria (Thermus aquaticus) and can therefore function at th ehigh temperatures used in th ethermocycler.
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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. |
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Next week in our last lab, Lab 11, we will start here. We will test the products of the PCR via gel electrophoresis and we will analyze the sequence of each sample to determine its mammalian source.
Lab 11: Pre-Lab
Your task in Lab 10 and Lab 11 is to identify the species of origin of a meat sample from a Kenyan butchery. You will learn about poaching, the bushmeat crisis and practice key techniques to complete DNA analysis of your sample. To prepare for Lab 11, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding pre-lab quiz in Blackboard.
What is bushmeat?
Kenya's wildlife is in decline in part due to poaching of commercially valuable species. In many areas, poaching in the form of snaring is commonplace, largely due to a lack of resources, food insecurity and poverty. Increased poaching effort has reportedly led to an increase in bushmeat in Kenya's markets and butcheries.
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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."
Commonly snared species include (from left to right), dik dik, zebra, gazelles and impala.
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Where did our meat samples originate?
Mount Kasigau as viewed from a community property bordering Tsavo West National Park.
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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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3) What will we do in lab & how will we do it?
We will be going through some of the steps required to identify the species of various meat samples in the next three labs. In order for things to go smoothly, you need to be familiar with the basic steps of DNA analysis and some of the specifics of bushmeat analysis in particular.
You will see these steps quite a few times. Start learning them now!
You will see these steps quite a few times. Start learning them now!
1) Sample PROCESSING
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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. |
2) Digestion & Extraction
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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)
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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
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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
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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
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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. |
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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. |
1) Do you know enough about gel electrophoresis?
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Following PCR, it is imperative to visualize your DNA. Last week we calculated how much we "should" have. Now we need to ensure we still have DNA and that it is the right gene. We need to ensure we have copied the cytochrome b gene. To do this, we will use a technique called gel electrophoresis.
First, review PCR from Lab 10. 
Please review the first 4m 40s of this video from Khan Academy about gel electrophoresis. Agarose gel electrophoresis is a method used in molecular biology to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. DNA-based gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several mega-bases (millions of bases).
After injecting your samples into the top of the gel, an electric field is used to push the charged DNA molecules through. The negatively charged phosphate groups of the sugar-phosphate backbone of DNA will migrate in an electric field away from the negative side (top) and toward the positive electrode (bottom).
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2) Do you know enough about DNA bar coding?
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DNA "barcoding" is like using an organism's DNA to establish its identity, similar to the bar codes on grocery items. But instead of using lines of vary widths, we use the sequence of nucleotide base pairs (As, Cs, Ts and Gs) in specific genes. Which genes to use depends on your taxa and your research question.
Review the slide presentation for a refesher on DNA structure. |
Plant barcoding studies use one or a few plastid regions and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA.
Animal barcoding studies use a region in the mitochondrial cytochrome c oxidase 1 gene (“CO1”) or the cytochrome b gene (CTYb).
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3) What will we do in lab & how will we do it?
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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. |
Click here to take your Pre-Lab Quiz.
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Lab 11: ProtocolYour task in Lab 11 is to identify the species of origin of a meat sample from a Kenyan butchery.
Exercise I. Agarose Gel Electrophoresis Exercise II. Identify the Species of Origin Exercise III. Conclusions |
Lab 10 & 11 Objectives (click to enlarge).
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Exercise II. What is the species of origin?
Once we've confirmed amplification via PCR through the gel electrophoresis, the samples are sequenced using a sequencing machine, which provides a read-out of the sequences of nucleotides within the sample. This process can be done in the WKU Biotechnology Center, or the samples can be shipped out for analysis.
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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. 
Sequence results show the order of nucleotides in our gene of interest for each sample. The order indicates species.
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. |
This is a sequencer. PCR products are placed inside and the sequence of As, Tc, Cs and Gs is determined and the output is generated on the monitor.
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Exercise III. Class Discussion
We are going to discuss our results and this project as a class. Before we start here are some questions we might discuss:
- Why is it important to understand bushmeat use in Kenya?
- Is bushmeat use for private needs different than a commercial enterprise wherein poached meat is sold to the public? Why or why not?
- Could these types of practices happen here?
- Do you think they could have an impact on conservation initiatives or public attitudes toward wildlife?
- What factrs in Kenya are contributing to this crisis in Kenya?
- How can or should we move forward?
Collaborators
ACKNOWLEDGEMENTS: CO-AUTHORS. Science is a collaborative process. Simon Kisaini (at left) has been working to conserve Kenya's wildlife since his youth and continues to do so now through Wildlife Works in Kasigau. He designed our sampling and field preparation protocols. Naomi Rowland (at right) is the Lab Coordinator for the WKU Biotechnology Center and developed and tested the molecular protocols for this research.
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Lab 11 BIOL 120 CONNECTIONS Section 1.6: Doing Biology Big Picture 1: How to Think Like a Scientist Chapter 4: Nucleic Acids Chapter 15: DNA and the Gene Chapter 20: The Molecular Revolution Chapter 54: Biodiversity and Conservation Biology *BIOL 122 |
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