Forensic DNA Fingerprinting: Using Restriction Enzymes STAM Winnipeg Oct 2013 Inspire your students...
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Transcript of Forensic DNA Fingerprinting: Using Restriction Enzymes STAM Winnipeg Oct 2013 Inspire your students...
![Page 1: Forensic DNA Fingerprinting: Using Restriction Enzymes STAM Winnipeg Oct 2013 Inspire your students – tomorrow’s researchers.](https://reader030.fdocuments.us/reader030/viewer/2022032803/56649e365503460f94b25443/html5/thumbnails/1.jpg)
Forensic DNA Fingerprinting: Using Restriction Enzymes
STAM Winnipeg Oct 2013
Inspire your students – tomorrow’s researchers
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Forensic DNA Fingerprinting: Using Restriction Enzymes
Bio-Rad (Canada) Life Science Education – Biotechnology Explorer Consultant
Robert Malyk BSc., MEd.
Senior Biology Teacher (retired)
Ridley CollegeSt. Catharines, ON Canada
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Why Teach DNA Fingerprinting?
•Real-world connections
•Tangible results
•Link to careers and industry
•Laboratory extensions
•Standards-based
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Forensic DNA Fingerprinting
Kit Advantages• Standards Based
Aligns with AP Biology Lab 6
• Use of real restriction enzymes and electrophoresis of real DNA fragments
• Lab can be completed in two 45 minute sessions
•Sufficient materials for 8 student workstations
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• DNA structure
• DNA restriction analysis (RFLP)
• Agarose gel electrophoresis
• Molecular weight determination
• Simulation of DNA Fingerprinting
• Plasmid mapping
The Forensic DNA Fingerprinting Kit Can Help You Teach:
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DNA Fingerprinting Real WorldApplications • Crime scene
• Human relatedness
• Paternity
• Animal relatedness
• Anthropology studies
• Disease-causing organisms
• Food identification
• Human remains
• Monitoring transplants
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Workshop Time Line
• Restriction digest of DNA samples
• Introduction to DNA Fingerprinting and RFLP analysis
• Electrophoresis on Agarose gels
• Analysis and interpretation of results
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DNA FingerprintingProcedureOverview
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LaboratoryQuick Guide
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DNA Fingerprinting ProceduresDay One
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DNA Fingerprinting ProceduresDay Two
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DNA Fingerprinting ProceduresDay Three
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Chromosomes are Comprised of DNA and Proteins
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Karyotyping
Identifies Genetic Anomalies
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DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus
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Model of DNA
DNA is Comprised of Four Base Pairs
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Deoxyribonucleic Acid (DNA)
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DNA Schematic
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DNA Restriction Enzymes
• Evolved by bacteria to protect against viral DNA infection
• Endonucleases = cleave within DNA strands
• Over 3,000 known enzymes
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Enzyme Site Recognition
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences (e.g. GAATTC)
PalindromeRestriction site
Fragment 1 Fragment 2
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5 vs. 3 Prime Overhang
• Generates 5 prime overhang
Enzyme cuts
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Common Restriction Enzymes
EcoRI– Escherichia coli– 5 prime overhang
Pstl– Providencia stuartii– 3 prime overhang
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The DNA DigestionReaction
Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2+ is an enzyme co-factor
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DNA DigestionTemperature
Why incubate at 37°C?
• Body temperature is optimal for these and most other enzymes
What happens if the temperature is too hot or cool?
• Too hot = enzyme may be denatured (killed)
• Too cool = enzyme activity lowered, requiring longer digestion time
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Restriction Fragment Length PolymorphismRFLP
Allele 1
Allele 2
GAATTCGTTAAC
GAATTCGTTAAC
CTGCAGGAGCTC
CGGCAGGCGCTC
PstI EcoRI
1 2 3
3Fragment 1+2Different Base PairsNo restriction site
+
M A-1 A-2
Electrophoresis of restriction fragments
M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments
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AgaroseElectrophoresisLoading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
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AgaroseElectrophoresisRunning
• Agarose gel sieves DNA fragments according to size– Small fragments
move farther than large fragments
Power Supply
Gel running
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Analysis of Stained Gel
Determinerestriction fragment
sizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction fragments
• Determine size of DNA fragments
Identify the related
samples
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Molecular Weight Determination
Size (bp) Distance (mm)
23,000 11.0 9,400 13.0
6,500 15.0
4,400 18.0
2,300 23.0
2,000 24.0 100
1,000
10,000
100,000
0 5 10 15 20 25 30
Distance, mm
Siz
e, b
ase
pai
rsB
A
Fingerprinting Standard Curve: Semi-log
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DNA Fingerprinting Lab Extensions
• Independent studies
• Plasmid DNA isolation (mini-preps)
• Plasmid mapping using restriction enzymes
• Southern blot analysis
• Introductory labs to electrophoresis:
Kool-Aid/FastBlast
pH indicator in buffer
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Plasmid Mapand Restriction Sites
Laboratory Extensions
BamHI: EcoRI: HindIII:EcoRI+Hind III:
1 linear fragment; 7367bp2 fragments; 863bp / 6504bp3 fragments; 721bp/2027bp/3469bp
5 fragments; 721bp/863bp/947bp/1659bp/2027bp
BamHI
7367bp
EcoRI
863bp
6504bp
Hind III
721bp 2027bp
3469bp
EcoRI+ HindIII
2027bp
1659bp
947bp
863bp
721bp
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Bio-Rad’sElectrophoresisEquipment
• Electrophoresis Cells
• Power Supplies
• Precast Agarose Gels
PowerPac™ Mini PowerPac™ Basic
PowerPac™ HC PowerPac™ Universal
Mini-Sub® Cell GT Wide Mini-Sub Cell GT