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

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

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

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.

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

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.

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.

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

The Power of PCR

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

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.

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.

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

Agarose Gel Electrophoresis Molecular Weight Standard (DNA of Known Sizes) Samples of DNA Lanes: 1 2 3 4 5 6 7 8 9 10 5000 bp 2000 bp 1000 bp 750 bp

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 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

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”)

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

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

Agarose Gel Electrophoresis 1 2 3 4 5 6 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.

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

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.

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

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.

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