Lab 12: Pre-Lab

Over the next several years BIOL 121 students will be testing meat samples from Kenya, through DNA analysis, to determine if poaching and bushmeat use is threatening conservation efforts.  We are currently testing the procedures that will be used for this research project. You will be provided the output of various steps so you can complete the process of species identification. In labs 10-12, your task will be to identify the species of origin of a meat samples from Kenyan butcheries. You will learn about poaching, the bushmeat crisis and practice key techniques to complete DNA analysis of your samples. To prepare for Lab 12, please review this pre-lab. Once you feel confident regarding the below topics, and have your Lab Notebook ready from last week, complete the corresponding LABridge in Blackboard.

• Introduction/Review
• Do you know enough?
• What will we do in lab?
• LABridge

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Review of Gel Electrophoresis

​Last week we ensured we still have DNA and that it is the right gene (the right length). We also made some calculations so we have an estimate of the amount of CYTb fragments we may have in our sample

​Please review this information on gel electrophoresis.

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Shorter DNA molecules move faster and migrate further down the gel. Longer ones migrate slower remain closer to the top of the gel. You can tell how long each fragment is by comparing them to the ladder, usually placed in the first and last lanes. The ladder contains fragments of known size so you judge how long your unknow fragments are via comparison. See the example in the side bar

• 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 PCR samples (plus a special dye) 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). 
• After allowing the sample to "run" for a specified time period, you can use various combinations of dye and light to visualize where your DNA stopped. These are called "bands."
• The DNA will form a distinct band in the gel upon stopping. By comparing the distance traveled by each sample to fragments of known size (located in the ladder or marker, injected into the first lane of the gel), it's possible to determine the size of the fragments in each sample. 

For example! In this gel, there is just one fragment in each sample. We can tell the sample in lane 2 barely amplified enough in PCR. There is just a light band at about 650bp. Sample three's segment is about 600bp, 4 is about 450 bp, and the sample in lane 5 is about 400 bp long.

​Do you know enough about sequencing & BLASTing

After amplification through PCR and visualization through gel electrophoresis, these are our last steps to get to species identification. Sequencing allows us to see the actual nucleotide sequence code in our GoI. The order of As, Ts, Cs and Gs is specie-specific in the cytochrome b gene. Once we know the sequence we will use a powerful search database to identify the actual species of origin. 

Please review the five key steps below.

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1- 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.

2- Sequencer Output: Sequence results show the order of nucleotides in our gene of interest for each sample. The order indicates species.

3- Following some clean-up we get output that looks like this...a long chain of nucleotides, within our GoI, from each unknown sample

4- We then submit our sequence through NCBI Genback, a comprehensive database that contains publicly available nucleotide sequences for more than 300,000 organisms obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects.

5- We submit to NCBI through the BLAST (Basic Local Alignment Search Tool) database. We paste our sequence into the nucleotide BLAST index. The software will search the ( > 100 million) sequences stored in the NCBI GenBank for those that most closely match our unknown sequence, and provide a list of likely species of origin

Read the article below. It was the first phase of the research we are now continuing in BIOL 121.

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Read this article!

​3) What will we do in lab & how will we do it?

In lab 12 we will complete analysis of our bushmeat samples! We will work through steps 5 and 6, making some changes based on our online only format.

• Sequencing: You will be provided with output from the DNA sequencer in the WKU Biotechnology Center for each bushmeat sample.
• Blasting: You will then enter the sequences into a database for species identification.

​Lastly, you will be asked to make some conclusions based on our results, and to think about what those conclusion may mean for the bushmeat crisis overall.

Please review the steps and sequence below, with a focus on steps 5 & 6

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1) Sample PROCESSING

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 11, 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 sequencer apparatus which can detect the order of nucleotide bases in our sample. The sequence is then cleaned and edited,

6) BLASTing

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 12 and asked to determine the species of origin in lab.

Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 12: Protocol

Your task in Lab 12 is review the role of bushmeat in global pandemics. You will identify the species of origin of our meat samples from a Kenyan butchery and discuss conservation efforts which may conflict with human needs.
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Exercise I. In our Current Context
​Exercise II. Identify the Species of Origin
Exercise III. Conclusions

Labs 10 - 12 Objectives (click to enlarge).

• Exercise I​
• Exercise II
• Exercise III
• Exam Prep

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​Exercise I. In our Current Context: ​How the pandemic has affected the bushmeat trade and how the bushmeat trade may cause the next one too...

There are significant fears that the pandemic will devastate conservation initiatives and local communities reliant on tourism; a worsening economic outlook will lead to more poaching and bushmeat utilization, thus threatening more wildlife. Increased bushmeat utilization places humans in endanger as well; COVID-19 was may have been transmitted to humans as part of the wildlife trade in Asia. A true accounting of the prevalence of the bushmeat trade is more important now than ever.

Procedure​

1. Please read the following article: Somerville, Keith. 2020. FOCUS Wildlife & Pandemics: COVID-19, bushmeat and poaching in Africa. Global Geneva. 14 September.
2. Please read the following article: ​Vidal, J. 2020. Tip of the iceberg’: is our destruction of nature responsible for Covid-19? The Guardian. Australian Edition, 18 March.

WARNIG: Includes some graphic bushmeat images. The Kenya Wildlife Service (KWS) has reported a 56% increase in bushmeat seizures since the start of the pandemic, as people struggle to find ways of generating income.​ In the Tsavo National Park in southern Kenya, wildlife rangers showed a reporter from the UK Telegraph the remains of a giraffe that had been killed and very thoroughly butchered. “They have really done a hell of a job on this one,” a ranger told him. Peter Rangi, a chief in Tsavo’s Marungu Ward, lamented that since the virus had stopped tourism “poverty has gone up." -Click the image for the full article.

“We invade tropical forests and other wild landscapes, which harbour so many species of animals and plants – and within those creatures, so many unknown viruses,” David Quammen, author of Spillover: Animal Infections and the Next Pandemic, recently wrote in the New York Times. “We cut the trees; we kill the animals or cage them and send them to markets. We disrupt ecosystems, and we shake viruses loose from their natural hosts. When that happens, they need a new host. Often, we are it.” -Click the image for the full article.

Poaching, including the hunting of elephants across Africa for ivory, then trafficked to Southeast Asia and China, can be expected to increase as the COVID-19 pandemic continues. (Photo: TRAFFIC)

Mount Kasigau as viewed from a community property bordering Tsavo West National Park.

Pangolins, armadillo like animals found in Africa and Asia, are believed to be a possible source for viruses, including COVID-19. They are poached and sold for food in ASian wet markets. (Photo: UNEP)

Our research area.

Exercise II. What is the species of origin?

After your reading in Exercise I, I hope you understand why this research is more important than ever. Let's finish up the analysis and see what we have. 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

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. Determine the species for each sample. View the putative (originally reported/labeled) species ID for each sample in the sidebar.
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. 
6. Complete the Lab Notebook Guide.

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.

Sequence results show the order of nucleotides in our gene of interest for each sample. The order indicates species.

Exercise III. What have we learned? (class discussion)

Procedure
Think back to where we started. If you need to go back to the Pre-Lab for Lab 10, or explore the bushmeat section of our research library.  Think about the questions below and complete the Lab Notebook Guide. We are going to discuss our results and this project as a class. 

1. Why is it important to understand bushmeat use in Kenya?
2. Is bushmeat use for private needs different than a commercial enterprise wherein poached meat is sold to the public? Why or why not?
3. Could these types of practices happen here?
4. Do you think they could have an impact on conservation initiatives or public attitudes toward wildlife?
5. What factors are contributing to this crisis in Kenya?
6. How can or should we move forward?

bushmeat report summary

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. 

Final Comprehensive Exam: Week Before Finals Week/ Regular Lab Day & Time

Bring YOUR DEVICE (iPad, laptop, etc.)

• Through RESPONDUS ONLY: It will be set up exactly like the syllabus quiz but will include a password.
• Charge your laptop.
• Update your laptop.
• Update Respondus if needed.
• Chromebooks WILL NOT work. Visit https://www.wku.edu/its/toppertech/ for a loaner device if needed and please review: https://www.wku.edu/ogden/tech_recs.php
• Do you know your log-in information? Can you access the WKU-Secure wifi?
• If you are late you will not be able to take the exam. Be on time guys!

The Exam

• During your regular lab day and time
• The exam will have T/F, multiple-choice, matching questions, fill-in-the blank and essay questions
• It will be in 2 parts: Practical & Standard
• The focus will be on showing what you can DO, with a smaller amount of questions on what your KNOW

Basic Outline of BIOL 121 Content

• Scientific method: Different steps, How to write hypothesis, Difference between null and alternative
• Reading data: Different types of statistic tests (i.e. T-test and Pearson correlation), What is their test statistic, When to use them, Different types of graphs, How to read them, When to use them
• Diabetes: Symptoms and basic facts (everything on intro of diabetes lab report), Osmosis, Hyper vs hypotonic, Insulins role and pathway, Glycemic index factors, Macromolecules and their tests
• Metabolism, Crayfish, What we did and why it worked, Cellular respiration, Photosynthesis, What we did and why it worked, Pathway we used
• DNA analysis, The 6 steps, PCR, How it works and list steps, Positive and negative control, Electrophoresis, How it works
• Bushmeat, What is it, How it affects us, Conservation standpoint, Relationship to covid-19, The process that includes the DNA processing (what steps were done)
• Know the name of all lab equipment you used and what it was for (e.g., burette, what does it look like, why did we use it, etc.)

Lab 12 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
Chapter 4: Nucleic Acids
​Chapter 8: Enzymes
​Chapter 12: The Cell Cycle
Chapter 15: DNA and the Gene: Synthesis & Repair
Chapter 16: How Genes Work
Chapter 20: The Molecular Revolution
Chapter 54: Biodiversity and Conservation Biology *BIOL 122