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WET LAB: DNA Barcoding: From Samples to Sequences
PowerPoint slides to accompany
Using Bioinformatics: Genetic Research
Chowning, J., Kovarik, D., Porter, S., Grisworld, J., Spitze, J., Farris, C., K. Petersen, and T. Caraballo. Using Bioinformatics: Genetic Research. Published Online October 2012. figshare. http://dx.doi.org/10.6084/m9.figshare.936568
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How DNA Sequence Data is Obtained for Genetic Research
Genetic Data
…TTCACCAACAGGCCCACA…
Extract DNA from Cells
Sequence DNA
CompareDNA
Sequences to One Another
Obtain Samples: Blood , Saliva, Hair Follicles, Feathers, Scales
TTCAACAACAGGCCCACTTCACCAACAGGCCCACTTCATCAACAGGCCCAC
GOALS:• Identify the organism from which the DNA was obtained.• Compare DNA sequences to each other.
Image Source: Wikimedia Commons
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• 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
From Samples to Sequences
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DNA Purification Overview1. 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.
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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.
1. Tissues Chopped and Broken Open with Detergents
2.Spin Column
3.DNA in Buffer
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DNA Purification Overview1. 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.
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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.
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• 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
From Samples to Sequences
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The Power of PCRNumber of PCR Cycles (n) Copies of DNA (2n)
0 11 22 43 84 165 326 647 1288 2569 512
10 102420 1,048,57630 1,072,741,824
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PCR Ingredients
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
PCR Tube & Bead
1.7 ml Microfuge Tube
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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.
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LAB 3:DID YOUR PCR WORK?
Analyzing PCR Results with Agarose Gel Electrophoresis
1. Obtain samples.2. Extract the DNA.3. Copy your gene4. Make sure you copied your gene.5. Obtain DNA sequence data.
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• 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
From Samples to Sequences
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Agarose Gel ElectrophoresisMolecular Weight
Standard (DNA of Known Sizes)
1 2 3 4 5 6 7 8 9 10
Samples of DNA
2000 bp
1000 bp750 bp
Lanes:
5000 bp
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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.
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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.
Teeth in comb make wells
Tape or gaskets at top and bottom of mold
Gel mold
Agarose melted in buffer to pour in mold
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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 l of DNA• Loading Buffer
– 6X concentrated– Adds blue color– Usually contains
glycerol (helps samples “sink” into wells”)
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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.
Top: Positive Electrode
Bottom: Negative Electrode
Samples in blue dye loaded into wells to top of gel
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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
Gel Box & Gel
ElectrodesRed = PositiveBlack = Negative
Voltage/Amps
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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.
1 2 3 4 5 6
Molecular Weight Standard
1000 bp
750 bp
500 bp
60 ng DNA
25 ng DNA
25 ng DNA
Size Amount
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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.
Ethidium Bromide & UV Light
Fast Blast™ Stain & Visible (White) Light
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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.
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• 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
From Samples to Sequences
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Sanger Method of DNA Sequencing1. DNA “template”Your purified PCR sample
2. Taq polymeraseHeat stable DNA polymerase
3. Deoxynucleotides (dNTPs) and Dideoxynucleotides (ddNTPs)
Building blocks of DNA. The ddNTPs stop the reaction at random points.
4. PrimersSpecific for your gene of interest
5. Buffer and water
Image Source: Enzo at Polish language Wikipedia, Wikimedia Commons.
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PCR Purification1. 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.
1. Mix
2. Bind & Wash
3. Elute