Lab 10

Lab 10: 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 10, 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

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.

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."

bushmeat report summary

Commonly snared species include (from left to right), dik dik, zebra, gazelles and impala.

Watch the brief clip below about the Kirk's Dik Dik, a commonly snared species in our research area.

The dik dik (Madoqua sp) is likely the most commonly snared wildlife species in the Tsavo area. They are amazing little antelopes. There are four recognized species. Dik dik mate for life and are always in pairs, so if one is snared and killed, the other often dies as well. This YouTube video was the best footage I could find on them, but it is NOT an endorsement of "Rob the Ranger."

Where did our meat samples originate?

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

View Mount Kasigau on Google Earth

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.

Over the next several years BIOL 121 students will be testing samples from the five villages that surround Mt. Kasigau: Mwakasinyi, Keteghe, Rukanga, Jora and Bungule. Every semester we will be adding to a bushmeat database, which can be used by conservation groups and the Kenyan Wildife Service to locate hotspots of poaching and bushmeat activity.

This semester's samples were sourced from 8 butcheries (view slideshow) from the villages of Rukanga (samples 1-6), Jora (sample 7) and Bungule (sample 8).

View the slideshow below. It pictures our test butcheries as well as provides their GPS coordinates. Make sure you know from which villages are samples are sourced for this round of research.

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

You might be wondering...why do we need DNA analysis to identify meat samples? As you saw in the introduction to this lab, meat sold in rural Kenyan butcheries does not look like meat sold in most places in the US. It is largely unregulated and unlabeled. Once meat is chopped up for sale, it is impossible to tell if it came from a cow, goat, or impala or elephant. In this way, poachers can sell their wildlife meat as domesticated meat products to the public.

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. In labs 10-12, your task will be to identify the species of origin of a meat samples from Kenyan butcheries.

This week you will:

Learn about DNA extraction by extracting DNA from fruit samples.
Be given the extraction results of 8 bushmeat samples and asked to determine the quantity of product we may have.
Review Polymerase Chain reaction and complete a short virtual lab.

DNA extraction includes the use of an extraction buffer and ethanol:

Our extraction buffer includes detergent to break apart lipids and proteins and "free" the DNA from the nucleus (to get it out of the cell).
It also includes salt with (+) charged sodium ions to neutralizes the (-) charge on the sugar-phosphate backbone of DNA, making it less soluble in water (so it stays together).
We will use ethanol to separate the DNA from the mixture. DNA is not soluble in ethanol so it is visible (so we can see it).

Read through the steps below. Pay careful attention to the details of steps 1, 2 and 3, and be sure you know the order of all six!

You will see these steps quite a few times. Start learning them now! Steps 1 -3 are the focus of lab this week! We will look again at step 3 next week and step 4. We will finish our analysis in Lab 12 with steps 5 and 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 10: Protocol

Your task in Lab 10 is to replicate a DNA extraction protocol using fruit samples, to determine how much DNA we may have successfully extracted from our samples, and to learn about the technique of PCR.

Exercise I. Practice Extracting DNA
Exercise II. Determine how much DNA we have.
Exercise III. Virtual PCR

Lab 10 - 12 Objectives (click to enlarge).

Exercise I
Exercise II
Exercise III

Exercise I. How Does DNA Extraction Work?

Our meat samples have been collected, processed in the field and brought back to WKU. In the WKU Biotechnology Center, Ms. Naomi Rowland (our collaborator) has completed the digestion of the samples and a procedure designed to extract the DNA. DNA extraction is an important step in DNA analysis, which is at the forefront of biomedicine and forensics. DNA Extraction Buffer is used for this purpose.
Extraction of DNA from meat samples is a lengthy and difficult process. It has already been completed for our samples. Today, you will mirror this process: you will be extracting DNA from fruit samples. You may need to review the structure of DNA (covered in Ch 4 of your BIOL 120 Pearson text).

DNA extraction includes the use of an extraction buffer and ethanol:

Our extraction buffer includes detergent to break apart lipids and proteins and "free" the DNA from the nucleus (to get it out of the cell).
It also includes salt with (+) charged sodium ions to neutralizes the (-) charge on the sugar-phosphate backbone of DNA, making it less soluble in water (so it stays together).
We use ethanol to separate the DNA from the mixture. DNA is not soluble in ethanol so it is visible (so we can see it). For our purposes we will use isopropyl alcohol (rubbing alcohol) as a good substitute.

Materials.

Filtration Supplies & Plastic Bag

Pipette & Tips

Digital Balance

Test Tubes

Procedure.

Open your Lab 10 Lab Notebook Guide.
Review your fruit choices and select two to from which to extract DNA.
Do some research online and design a hypothesis regarding which one will show higher levels of DNA precipitation. This is informal. THINK! What do these fruits have in common? Why these species? HINT! What is polyploidy?
Also, think about how you will "decide" which one produces "MORE" DNA. This in an objective exercise but you still need a plan. Here's what you can expect to see if extraction is successful.
Select your two fruits and obtain a section of each.
Clean the fruit and remove any leaves or skin.
You'll need a sample about the size of a strawberry. You should weigh each sample and get them as close to congruent as possible.
Place it in a zip lock baggie.
Smash up the fruit with for 2 minutes.
Using a small graduated cylinder, add 20mL of extraction buffer into the Ziplock bag.
Close the bag and smash again for 1 minute.
Go over to the filtering apparatus and add a small sheet of cheese cloth to the base of the funnel.
Pour the contents of Ziplock bag (i.e., fruit + buffer) into the filtering apparatus and let it drip directly into a clean test tube. It should be about 1/8 full.
Take a note of how much extract you have at this step. The goal is to use approximately the same amount of extract from each fruit.
Slowly add 5mL of cold ethanol (from the refrigerator freezer) into the tube, kept at a diagonal, so that the test tube is half full.
You should see “cotton-like” wisps of material forming at the interface of the liquids in the test tube. DO NOT STIR OR AGITATE THE CONTENTS OF THE TEST TUBE!
Take notes on the results and be sure the take photos to compare with your other extraction. The visibility of the DNA is time sensitive so don't let it sit for too long.
Get your instructor to check your DNA.
Repeat for your other fruit.
Complete Exercise I in your Lab Notebook Guide

Hmm? What's this about? Click to enlarge.

What is a berry?

Fruit + extraction buffer.

Filtering your product.

Your DNA will look like this.

Exercise II. What part of the DNA do we need?

Now, that understand the extraction process, let's move back to our bushmeat samples. Following extraction of DNA from the meat samples, we need to determine how much DNA we have. In other words...were we successful and to a high enough degree to allow for the next steps? Following extraction we have tons of DNA! But, that means that in fact we have a lot of genetic material that we do not need and certainly do not want to make copies of. DNA is conserved at different rates across species. Meaning we share different amounts and different genes. We need to find a gene (a section of DNA) that all mammals have, BUT that is a different enough in every species of mammal to afford us the ability to identify the species from the DNA sequence of the gene. The particular gene required is called the Gene of Interest or "GoI."

Animal barcoding studies use a region in the mitochondrial cytochrome c oxidase 1 gene (“CO1”) or the cytochrome b gene (CTYb).

Plant barcoding studies use one or a few plastid regions and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA.

Turns out, the DNA for ANY life, is very similar. For example we share 50% of our DNA with bananas. Within our species, we share 99.9%. Watch the video on SURPRISING Animals Related To Humans. It's fun reminder of how similar we all are!
For our analysis, we need a gene that will allow for something called bar-coding. Essentially, bar-coding is using a short segment of DNA, particularly to a certain gene, that differs enough to allow for species identification, similar to the bar codes on grocery items. But instead of using lines of varying 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. Watch the video on DNA Barcoding in the sidebar.
The region of DNA that codes for cytochrome complexes (b and c) is particularity useful in barcoding. Do you remember what cytochrome b and cytochrome c do? Think back to cellular respiration and the electron transport chain. Watch this short Cellular Respiration video on YouTube.

We will use the cytochrome b gene (CTYb) for species ID of our bushmeat samples. It meets our requirements. It is shared across all mammals BUT, differs enough between species for the sequence (of As, Ts, Cs and Gs) to give us a species-level ID.

Procedure: Estimate the number of DNA fragments (our gene of interest, cytochrome b) we may have following extraction.

Answer the questions associated with DNA structure and barcoding in your Lab Notebook Guide. Review the slideshow below if you need help!
Following DNA extraction, we test the amount of purified DNA in our samples, to ensure enough product exists for PCR to work.
We conduct this analysis using a spectrophotometer. The results for our samples are shown in the figure at right in ng/uL. Sample 8 yielded no genetic material.
Next, we need to know how much of our GoI we have. Using the total DNA yield, we can calculate the potential total number of cytochrome b (cytb) fragments we have extracted from each sample...
Open the "DNA YIELD CALCULATIONS" document in Excel from the sidebar. You will make your calculations here and copy paste the table into your notebook guide.
Of all the DNA we extracted first we need to determine how much is mitochondrial DNA? This is because the cytb gene is on mitochondrial DNA. Mitochondrial DNA (mtDNA) comprises ~ 3% of all DNA, is circular and contains roughly 16,000 base pairs (bp), on average, in mammals.
Next we need to determine how much of the mtDNA might be cytb. The cytb gene comprises ~ 7% of mtDNA. Review the figure mapping mitochondria at the bottom of this page.
Once you have determined the amount of cytb DNA we have (in ng/uL), the next column, the amount or number of strands we have, will auto-generate.
You are only completing the Lab 10 portion of the table at this time.
You will copy/paste your excel table into your Lab Notebook Guide.

DNA yield calculations in excel

Gene map of human mitochondrial DNA. In mammals, each double-stranded circular mtDNA molecule consists of 15,000–17,000 base pairs. In humans, approximately 7% mtDNA is of cytb, which we are using to make our estimates.

Exercise III. PCR Virtual Lab

Polymerase chain reaction (PCR) involves the amplification (or copying) of a specific segment or fragment of DNA to allow for continued analysis. PCR can be used to: identify individuals or species, for criminal proceedings, paternity determination or pathology. It is one of the most powerful tools of modern biology.

We would have created PCR cocktails for each of our bushmeat samples and placed into a thermocycler for synthesis. Instead, you will do a short virtual PCR lab. Read over the information in PCR below and complete the procedure.

Once DNA has been extracted, it is mixed into a particular PCR solution containing:

Taq polymerase: A type of heat-stable DNA polymerase derived from a species of bacteria living in hot springs. Because Taq polymerase continues to function normally at high temperatures, using it allows researchers to separate the DNA strands without destroying the polymerase.
Primers: Short, single-stranded sequence of RNA or DNA that enables the start of replication of a DNA sequence that is synthesized from the 3’ end of the primer. Two types are needed, a forward and a reverse primer.
Deoxynucleoside triphosphates (dNTPs): Free nucleotides to be used in constructing the new copies of DNA.
Mix buffer: Necessary to create optimal conditions for activity of Taq DNA polymerase and may contain restriction enzymes, which act like molecular scissors cutting the copied DNA strands at particular locations based on their genetic code.

The PCR mixture is placed inside a thermocycler (PCR machine). It is typically repeated about 35 times and the temperature changes are programmed by researchers and automated by the thermocycler. The process proceeds in three steps as outlined below.

Denaturation: the solution is first heated to nearly boiling—95ºC. The heat breaks the hydrogen bonds between the two DNA strands and allows them to separate.
Annealing: the temperature is dropped to around 60ºC. The exact temperature depends on the length and base composition of the primers. At this relatively low temperature, the primers can form hydrogen bonds with the single-stranded DNA. Two primer types are created, each one complementary in sequence to one of the two ends of the target DNA. To make the primers, the sequences at the ends of the target DNA must be known.
Extension: the temperature is increased to 72ºC. This is the optimal temperature at which Taq polymerase functions. The primers are essential in this process, because they provide free 3’ hydroxyl groups, to which the polymerase can add additional dNTPs. Each new dNTP that joins the growing strand is complementary to the nucleotide in the opposite strand.

Procedure

The virtual lab is available at right on YouTube.
You can navigate to it with this link as well: https:// www.youtube.com/watch?v=0jlWKw5qEP8
This lab will take through a typical PCR set-up, much like what we would have done in lab for each of our bushmeat sample DNA extractions.
Watch the entire video carefully and take notes.
Complete your Lab Notebook Guide.
Refer beck to virtual lab as needed

PCR components. Click to enlarge

Steps of PCR. Click to enlarge.

Lab 10 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
Chapter 4: Nucleic Acids
Chapter 9: Cellular Respiration
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

​Research Question: Is Bushmeat Sold in Kenyan Butcheries?