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.  Due to the new "online only" nature of this lab, you will be provided the output of various steps so you can complete the process of species identification without doing any bench work. 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 11, 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 would we have done in lab?
• LABridge

<

>

Gel Electrophoresis

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 (the right length). We need to ensure we have copied the cytochrome b gene. To do this, we will use a technique called gel electrophoresis. 

Please review the first 4m and 40s of this video from Khan Academy about gel electrophoresis. Review the bullet points below and the example image in the sidebar. 

---

• ​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. 

• 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 comparision.
• See the example in the side bar.

For example! In this gel, there is just one fragment in each sample. We can tell the sample in lan 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 controls?

We sue two types of controls in DNA analysis. We start running both from the very beginning, through PCR, onto to gel electrophoresis and through to sequencing. If either control results in an incorrect identification, we have to start the ENTIRE process over and all our results are considered invalid. 

A positive control

• Is a known DNA sample with target DNA.
• It should always work well in your PCR and therefore, should always show up as expected in your gel (at the right length).
• It should always show up as the right sequence for identification.
• In this experiment we used impala DNA as our positive control or C+.
• At the end of all of our steps of DNA analysis, we should determine the species ID of this C+ to always be impala.
• This would be a positive results for bushmeat. 
• This tests the success and validity of our protocol.
• If this C+ shows up to be ANYTHING BUT impala, all our results are invalid and we would need to start all over again with fresh extractions.

A negative control

• Is a know sample with NO DNA.
• Following PCR is should NOT show up on your gel at all because there should be no DNA for amplification during PCR.
•  It should never produce a sequence for identification.
• In this experiment (as in most) we used distilled water for our negative control or C-.
• At the end of all of our steps of DNA analysis, we should NEVER find any DNA from this sample.
• This tests for any contamination that may have occurred.
• For example, if we sequenced the C- and found human...we would have made a big mistake and would need start over again with fresh extractions.
• Or, for example, if we sequenced the C- and found cow or impala...we would have made another type of big mistake and would need start over again with fresh extractions.

Figure comparing and contrasting positive (C+) and negative (C-) controls. Click to enlarge.

Please review the explanation regarding positive and negative controls.

---

​3) What would we have done & what will we do in lab?

In lab 12 we will complete analysis of our bushmeat samples! We will work through steps 4 through 6, making some changes based on our online only format.

• Gel electrophoresis: You would have created, run and viewded a gel in lab this week. Instead you will complete a virtual lab on gel electrophoresis and analysize the reuslts we might have achieved through photos. 
• Sequnceing: 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 those conclusion may mean for the bushmeat crisis overall.

Please review the steps and sequence below, with a focus on steps 4-6

---

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

​Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. We use special "kits" as pictured to streamline the process. Extraction requires more steps to break through the cell and organelle membranes to free the DNA. 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 your own cheek cells.

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 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 11 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 to identify the species of origin of meat samples from a Kenyan butchery.


Exercise I. Agarose Gel Electrophoresis
Exercise II. Identify the Species of Origin
Exercise III. Conclusions

Lab 10 - 12 Objectives (click to enlarge).

• Exercise I
• Exercise II
• Exercise III

<

>

Exercise I. Did our PCR work? Have we copied the right DNA? 

We have no idea!!!! In fact we might not have ANY. The calculations you completed in Exercise I are the best case scenario. The truth is PCR often fails. There are many reasons! Too much enzyme, low quality DNA, poor primers, low primer concentrations, incorrect PCR cycling, and so on! We need to SEE our DNA to know it exists and to confirm it is the right length! We do that through a technique called gel elctrophoresis.

Had we met in lab this week, you all would get your PCR products back from the freezer. Just tiny little tubes with very little solution at the bottom. No way to tell if we have any DNA, much less if it is the right stuff! So, in lab, you would have made, loaded and run a gel to ensure all all samples amplified, the C+ and C- did what they were supposed to do, and you would have checked to make sure all our samples amplified the right gene (CYTb), by checking the length! 

​Procedure: Part 1

1. Download the Lab 12 Notebook Guide.
2. Navigate to the virtual PCR lab in the sidebar or through this url: https://learn.genetics.utah.edu/content/labs/gel/
3. Be sure you are using the Chrome browser.
4. Please allow pop-ups so you can be directed to download the FLASH player plug-in which is required for this lab.
5. If that isn't working for you, you can download the plug-in directly from here.
6. ​Work through the lab, reading and following the directions carefully. Please note that the lab refers to a "DNA Size Standard." This is the same as the "ladder" referred to in the pre-lab.
7. Take a selfie on the last lab page for your Notebook Guide.
8. View the photo gallery below to better familiarize yourself with the techniques and equipment we would have used in lab.

Lab Notebook Guide. Click to download.

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.

---

Basic gel creation process...it's like jello!

Gel chamber and electrodes pictured with combs to create loading wells.

The red and black electrodes are plugged into the power supply and the current pushes your samples down the gel.

Diagram of electrical field and direction of migration. Click to enlarge.

---

Procedure: Part 2

1. Review the diagram in the sidebar to see how we would have loaded our gels.
2. Review the information on our C+ and C-.
3. After electrophoresis is complete, a light band of loading dye appears across the bottom, however what you CANNOT see are any bands of DNA.
4. In order to visualize the DNA the gels must be stained with ethidium bromide (EtBr) and exposed to UV light. The compound forms fluorescent complexes with nucleic acids which can be viewed under UV light.  You have been provided with a photo of the gel and it's marker so you can read the results.
5. We are looking for segments that are approximatley 490bp long, part of the CYTb gene. Review why.
6. View the RESULTS!​​
7. Were all of our samples amplified in PCR? Did we amplify the right gene? 
8. Record these results in your original spreadsheet from Lab 10. 
9. Complete the Lab Notebook Guide

View the Gel Results

What gets loaded where? Click to enlarge.

What are our controls? Click to enlarge.

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.

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.

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

Exercise III. What have we learned?

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 librabry. Read the artcile in the sidebar. It was the first phase of the reseach we are now conitnuing in BIOL 121. Think about the questions below and complete the Lab Notebook Guide.

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

​Special Thanks to our 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. 

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