WET LAB: DNA Barcoding: From Samples to Sequences

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

4. DNA Purification Overview 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. • 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.

5. DNA Purification Using “Spin Columns” 1. Tissues Chopped and Broken Open with Detergents 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 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. • 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.

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

9. The Power of PCR

10. PCR Ingredients 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. 2007. 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 • Aquarium, zoo, grocery • Lab 1: DNA Purification • Lab 2: Polymerase Chain Reaction • Lab 3: Agarose Gel Electrophoresis • Lab 4: PCR Purification and DNA Sequencing • Obtain samples • Purify the DNA • Copy your gene • Make sure you copied your gene • Obtain DNA sequence data

14. Agarose Gel Electrophoresis Molecular Weight Standard (DNA of Known Sizes) Samples of DNA Lanes: 1 2 3 4 5 6 7 8 9 10 2000 bp 5000 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 1. Make the agarose gel. 2. Prepare your sample. 3. Load your sample on the gel. 4. Run the gel. 5. Visualize the gel. Agarose melted in buffer to pour in mold Teeth in comb make wells 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 Bottom: Negative Electrode

19. 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. Power Supply Electrodes Red = Positive Black = Negative Voltage/Amps Gel Box & Gel

20. 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. Separating pieces of DNA based on size. Molecular Weight Standard 1 2 3 4 5 6 Size Amount 1000 bp 60 ng DNA 750 bp 25 ng DNA 25 ng DNA 500 bp

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

24. 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 Sanger Method of DNA Sequencing 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