Lab 10: 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 10, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding pre-lab quiz in Blackboard.
  • Introduction
  • Do you know enough?
  • What will we do in lab?
  • LaBridge
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1) 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."
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Review "Lifting the Siege" and the Bushmeat Report Summary
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Commonly snared species include (from left to right), dik dik, zebra, gazelles and impala.

2) Where did our meat samples originate? 

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

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.  Due to the new "online only" nature of  some of these labs, you will be provided the output of various steps so you can complete the process of species identification without doing all of bench work. 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:
  1. Learn about DNA extraction by extracting DNA from fruit samples.
  2. Be given the extraction results of 8 bushmeat samples and asked to determine the quantity of product we may have.
  3. 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
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.
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.
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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 and determine how much DNA we have successfully extracted form our samples and how much DNA we have following PCR. 
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​Exercise I. How do you extract DNA?
Exercise II. Determine How Much DNA We Have After Extraction
​Exercise III. Determine How Much DNA We Have After PCR
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Lab 10 & 11 Objectives (click to enlarge).
  • Headline 1
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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 a difficult process. Today, we will mirror this process by extracting DNA from several fruits. 
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).
Procedure
  1. Review your fruit choices and select two to from which to extract DNA.
  2. 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?
  3. 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.
  4. You MUST get your hypothesis and analysis plan checked before you can proceed. Everyone should have it written down along with some research points. 
  5. Once "checked," here's the general protocol for your fruit:
  6. Clean the fruit and remove any leaves or skin. 
  7. You'll need a sample about the size of a strawberry.
  8. Place it in a zip lock baggie.
  9. Smash up the fruit with for 2 minutes.
  10. Using a small graduated cylinder, add 20mL of extraction buffer.
  11. Close the bag and smash again for 1 minute.
  12. Pour the extract into the filtering apparatus and let it drip directly into a clean test tube.
  13. Only fill the test tube so that it is 1/8 full.
  14. 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.
  15. 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!
  16. 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. 
  17. Get your instructor to check your DNA.
  18. Repeat for your other fruit.
  19. Make some conclusions. Everyone should write them down. You may need to do some more research.
  20. Be sure your instructor reviews your conclusions.
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Fruit + extraction buffer.
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Filtering your product.

Exercise II. How much DNA do we have?

Now, let's move on to our own samples. Following extraction of DNA from the meat samples, we need to determine how much DNA we have. In other words...we're we successful and to a high enough degree to allow for the next steps?
Proceedure: Estimate the number of DNA fragments (our gene of interest, cytochrome b) we may have by the end of PCR.
1) We start here! 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 ug/ng. ​Sample 8 yielded no genetic material.
2) How much cytochrome b (cytb) DNA might we have? Using these results calculate the potential total number of cytochrome b (cytb) fragments we have extracted from each sample. ​
Wait! Why are we looking for the cytochrome b gene? All DNA is NOT the same. Some genes are more conserved than others. The DNA for life, is very similar. For example we share 50% our DNA with bananas. Within our species, we share 99.%. The genes that can be used to distinguish between mammal species are specific. Cytochrome b is one such gene. The sequence of As, Cs, Ts and Gs can tell us from which species it originated. 
So now our task is to determine out of all the DNA we have extracted, approximately how much if the cytochrome b gene do we have?

​You need the following to make your calculations:
  • You should use THIS SPREADSHEET for your calculations. You will need to submit it for the post-lab.
  • The cytochrome b gene is in mitochondrial (not nuclear) DNA.
  • Mitochondrial DNA (mtDNA) comprises ~ 3% of all DNA, is circular and contains roughly 16,000 base pairs (bp), on average, in mammals.
  • The cytb gene has approximately 1,200bp and comprises ~ 7% of mtDNA.
  • Once you have used the above to determine the amount of cytb DNA we have (in ug/ng), use THIS CALCULATOR to estimate the number of copies we have of the gene in each sample.
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DNA yield following extraction
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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.

Lab 10 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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