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Forensic DNA Fingerprinting:
Using Restriction Enzymes
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Stan Hitomi Coordinator – Math & Science San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Sherri Andrews, Ph.D.
Curriculum and Training Specialist Bio-Rad Laboratories Essy Levy, M.Sc.
Curriculum and Training Specialist Bio-Rad Laboratories
Forensic DNA
Fingerprinting
Kit
Instructors
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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 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 World
Applications • 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
Fingerprinting
Procedure
Overview
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Laboratory
Quick Guide
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DNA
Fingerprinting
Procedures
Day One
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DNA
Fingerprinting
Procedures
Day Two
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DNA
Fingerprinting
Procedures
Day Three
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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
O CH
2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
O
Phosphate
Phosphate
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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
(eg GAATTC)
Palindrome
Restriction 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
– Eschericha coli
– 5 prime overhang
Pstl
– Providencia stuartii
– 3 prime overhang
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The DNA
Digestion
Reaction Restriction Buffer provides optimal conditions • NaCI provides the correct ionic strength • Tris-HCI provides the proper pH • Mg
2+ is an enzyme co-factor
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DNA Digestion
Temperature
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
Polymorphism
RFLP
Allele 1
Allele 2
GAATTC
GTTAAC
GAATTC
GTTAAC
CTGCAG
GAGCTC
CGGCAG
GCGCTC
PstI EcoRI
1 2 3
3 Fragment 1+2
Different
Base Pairs
No restriction site
+
M A-1 A-2
Electrophoresis of
restriction fragments
M: Marker
A-1: Allele 1 Fragments
A-2: Allele 2 Fragments
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Agarose
Electrophoresis
Loading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
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Agarose
Electrophoresis
Running
• 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
Determine
restriction 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 p
air
sB
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 Map
and
Restriction Sites
Laboratory
Extensions
BamHI:
EcoRI:
HindIII:
EcoRI+Hind III:
1 linear fragment; 7367bp
2 fragments; 863bp / 6504bp
3 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’s
Electrophoresis
Equipment
• Electrophoresis Cells • Power Supplies • Precast Agarose Gels
PowerPac™ Mini PowerPac
™ Basic
PowerPac™ HC PowerPac
™ Universal
Mini-Sub® Cell GT Wide Mini-Sub Cell GT