lab 6: d1s80 dna fingerprinting€¦ · lab 6: d1s80 dna fingerprinting . lab objectives •to...
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Lab 6: D1S80 DNA Fingerprinting
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Lab Objectives
• To understand how the D1S80 allele can be used to distinguish between individual human beings through the process of DNA fingerprinting
• To understand how polymerase chain reaction works (PCR)
• To learn how gel electrophoresis is used to separate DNA
Notebook
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D1S80
• VNTR (variable number tandem repeat) – non coding region
• Located on chromosome 1
• Number of repeats varies from person to person (14 to 41 copies)
• Repeat is 16 bp: GAGGACCACCAGGAAG
• Length of the D1S80 region can therefore be from 224 bp to greater than 800 bp
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Human Identity Techniques
• Fingerprints
• Biometry (facial measurements)
• Iris and retina patterns
• Voice recognition
• DNA fingerprinting
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PCR (Polymerase Chain Reaction)
• Goal of PCR – Locate, isolate and make (“amplify”) millions of copies of a gene from one sample (equivalent to DNA replication)
• Use primers to target the gene you want to amplify
• Primers are short pieces of DNA that have a sequence complementary to either end of the target gene
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dNTPS
• Deoxyribose nucleoside triphosphates
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Primers
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Taq polymerase
• Bacterial enzyme that is able to replicate DNA at high temperatures
• Human DNA polymerase would denature at the temperatures required by PCR
• Isolated from Thermus aquaticus in 1976 – found in hot springs and hydrothermal vents
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PCR
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PCR (30 cycles)
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Applications of PCR
• Cloning: make millions of copies of one gene from an entire genome
• DNA sequencing
• Mutagenesis: make a desired mutation in a gene of interest
• Mutation analysis: examine mutations in your genes
• Pathology: presence of harmful bacteria/viruses in your body
• Forensics: who done it?
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PCR of the D1S80 Region
• Heterozygous for D1S80 – two different sized D1S80 regions
• How many bands will appear on a gel?
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PCR of the D1S80 Region
• Homozygous for D1S80 – two copies of the same sized D1S80 regions
• How many bands will appear on a gel?
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Procedure Overview
• Part A: Isolation of DNA from Cheek Cells
• Part B: PCR of the D1S80 VNTR region
• Part C: Electrophoresis of PCR Product
• Part D: DNA Fingerprinting Analysis
Notebook
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PCR Tube Identifier _______
• D1S80 gene specific primers
• dNTPs (A, T, C, G)
• Salts (Tris-HCl, KCl, NH4Cl)
• pH of 8.9
• OneTaq® DNA Polymerase
Notebook
PCR Master Mix Components
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Gel Electrophoresis of PCR Product
1. Add 2 µL of PCR loading dye to your DNA sample
2. Load 20 µL of your DNA sample into one well of a 2% agarose gel (made with Gel Green)
3. Load 20 µL of the DNA ladder into lane 5 of the gel
4. Run the gel at 70 volts for 30 minutes
Notebook
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Data Table 1: DNA Marker Size
Notebook
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Data Table 2: D1S80 PCR Samples
Notebook
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D1S80 VNTR Analysis
• How many repeats of the D1S80 VNTR do you have on chromosome 1?
(If you are heterozygous, calculate the number of repeats for each allele)
Notebook