1. Genetic Research Using Bioinformatics: WET LAB:
DNA Barcoding: From Samples to Sequences
PowerPoint slides to accompany
Using Bioinformatics:
Genetic Research

2. How DNA Sequence Data is Obtained for Genetic Research
Obtain Samples: Blood , Saliva,
Hair Follicles, Feathers, Scales
Genetic Data
Compare
DNA Sequences to One Another
Extract DNA from Cells
Sequence DNA
…TTCACCAACAGGCCCACA…
TTCAACAACAGGCCCAC
TTCACCAACAGGCCCAC
TTCATCAACAGGCCCAC
GOALS:
Identify the organism from which the DNA was obtained.
Compare DNA sequences to each other.
Image Source: Wikimedia Commons

3. From Samples to Sequences
Obtain samples
Purify the DNA
Copy your gene
Make sure you copied your gene
Obtain DNA sequence data
Aquarium, zoo, grocery
Lab 1: DNA Purification
Lab 2: Polymerase Chain Reaction
Lab 3: Agarose Gel Electrophoresis
Lab 4: PCR Purification and DNA Sequencing

4. DNA Purification Overview
Chop tissues and add detergents (disrupt cell membranes), proteinase K, and heat.
Small “Spin Columns” contain DNA-binding material with small holes.
Columns bind DNA, other cellular debris washes through column.
“Elute” the DNA, or remove it from the column.
1. Break open the cells. 2. Separate the DNA from the rest of the cell debris. 3. Remove DNA from the spin column and suspend the DNA in buffer for future use.

5. DNA Purification Using “Spin Columns”
1. Tissues Chopped and Broken Open with Detergents
DNA Purification Using “Spin Columns”
1. Chop up tissues and break open the cells with detergents. 2. Separate the DNA from the rest of the cell debris using spin column and centrifugation. 3. Suspend the DNA in buffer for future use. 2.
Spin Column 3.
DNA in Buffer

6. DNA Purification Overview
Chop tissues and add detergents (disrupt cell membranes), proteinase K, and heat.
Small “Spin Columns” contain DNA-binding material with small holes.
Columns bind DNA, other cellular debris washes through column.
“Elute” the DNA, or remove it from the column.
1. Break open the cells. 2. Separate the DNA from the rest of the cell debris. 3. Remove DNA from the spin column and suspend the DNA in buffer for future use.

7. Lab 2: Copying the DNA Barcoding Gene Using Polymerase Chain Reaction (PCR)
1. Obtain samples.
2. Extract the DNA.
3. Copy your gene.
4. Make sure you copied your gene.
5. Obtain DNA sequence data.

8. From Samples to Sequences
Obtain samples
Purify the DNA
Copy your gene
Make sure you copied your gene
Obtain DNA sequence data
Aquarium, zoo, grocery
Lab 1: DNA Purification
Lab 2: Polymerase Chain Reaction
Lab 3: Agarose Gel Electrophoresis
Lab 4: PCR Purification and DNA Sequencing

9. The Power of PCR

10. PCR Ingredients 1. DNA “template” Your purified DNA sample
1.7 ml Microfuge Tube
PCR Tube & Bead
1. DNA “template” Your purified DNA sample
2. Taq Polymerase Heat-stable DNA polymerase
3. Deoxynucleotides (dNTPs) Building blocks of DNA
4. Primers Small pieces of DNA bind to your gene
5. Buffer and water Maintain pH of reaction

11. Genetic Researchers Developed Primers for DNA Barcoding
Pool COI-2: mammals, fish and insects
Pool COI-3: amphibians, reptiles and mammals
Credit: Ivanova et al Universal primer cocktails for fish barcoding. Mol Ecol Notes.

12. Lab 3: Did your PCR work? Analyzing PCR Results with Agarose Gel Electrophoresis
1. Obtain samples.
2. Extract the DNA.
3. Copy your gene
4. Make sure you copied your gene.
5. Obtain DNA sequence data.

13. From Samples to Sequences
Obtain samples
Purify the DNA
Copy your gene
Make sure you copied your gene
Obtain DNA sequence data
Aquarium, zoo, grocery
Lab 1: DNA Purification
Lab 2: Polymerase Chain Reaction
Lab 3: Agarose Gel Electrophoresis
Lab 4: PCR Purification and DNA Sequencing

14. Agarose Gel Electrophoresis
Molecular Weight Standard
(DNA of Known Sizes)
Samples of DNA
Lanes:
5000 bp
2000 bp
1000 bp
750 bp

15. Agarose Gel Electrophoresis
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.

16. Agarose Gel Electrophoresis
Agarose melted in buffer to pour in mold
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.
Teeth in comb make wells
Start at o minutes, run to 2:44
Gel mold
Tape or gaskets at top and bottom of mold

17. Agarose Gel Electrophoresis
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.
10 ml of DNA
Loading Buffer
6X concentrated
Adds blue color
Usually contains glycerol (helps samples “sink” into wells”)

18. Agarose Gel Electrophoresis
Top: Positive Electrode
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.
Samples in blue dye loaded into wells to top of gel
Start at o min, run to 2:52
Bottom: Negative Electrode

19. Agarose Gel Electrophoresis
Power Supply
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.
Electrodes
Red = Positive
Black = Negative
Voltage/Amps
Gel Box & Gel

20. Agarose Gel Electrophoresis
Molecular Weight Standard
1000 bp
750 bp
500 bp
60 ng DNA
25 ng DNA
Size
Amount
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.
Separating pieces of DNA based on size.

21. Agarose Gel Electrophoresis
Ethidium Bromide & UV Light
1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel.
Fast Blast™ Stain & Visible (White) Light

22. Preparation of PCR Samples for DNA Sequencing
1. Obtain samples.
2. Extract the DNA.
3. Copy your gene.
4. Make sure you copied your gene.
5. Obtain DNA sequence data.

23. From Samples to Sequences
Obtain samples
Purify the DNA
Copy your gene
Make sure you copied your gene
Obtain DNA sequence data
Aquarium, zoo, grocery
Lab 1: DNA Purification
Lab 2: Polymerase Chain Reaction
Lab 3: Agarose Gel Electrophoresis
Lab 4: PCR Purification and DNA Sequencing

24. Sanger Method of DNA Sequencing
1. DNA “template”
Your purified PCR sample
2. Taq polymerase
Heat stable DNA polymerase
3. Deoxynucleotides (dNTPs) and Dideoxynucleotides (ddNTPs)
Building blocks of DNA. The ddNTPs stop the reaction at random points.
4. Primers
Specific for your gene of interest
5. Buffer and water
Image Source: Enzo at Polish language Wikipedia, Wikimedia Commons.

25. PCR Purification
1. Mix
1. Mix the PCR product with a DNA Binding Buffer. 2. Separate the PCR product from the rest of the PCR reaction using a spin column. 3. Elute PCR from the spin column.
2. Bind & Wash
3. Elute