Forensic%20DNA%20Fingerprinting:

1
(No Transcript)
2
Forensic DNA Fingerprinting Using Restriction
Enzymes
3
Forensic DNA Fingerprinting KitInstructors

• Stan Hitomi
• Coordinator Math Science
• Principal Alamo School
• 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
• Bio-Rad Curriculum and Training Specialists
• Sherri Andrews, Ph.D.
• sherri_andrews_at_bio-rad.com
• Leigh Brown, M.A.
• leigh_brown_at_bio-rad.com

4
Why Teach DNA Fingerprinting?

• Real-world connections
• Tangible results
• Link to careers and industry
• Laboratory extensions
• Standards-based

5
(No Transcript)
6
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

7
The Forensic DNA Fingerprinting Kit Can Help You
Teach

• DNA structure
• DNA restriction analysis (RFLP)
• Agarose gel electrophoresis
• Molecular weight determination
• Simulation of DNA Fingerprinting
• Plasmid mapping

8
DNA Fingerprinting Real WorldApplications

• Crime scene
• Human relatedness
• Paternity
• Animal relatedness
• Anthropology studies
• Disease-causing organisms
• Food identification
• Human remains
• Monitoring transplants

9
Workshop Time Line

• Restriction digest of DNA samples
• Introduction to DNA Fingerprinting and RFLP
  analysis
• Electrophoresis on Agarose gels
• Analysis and interpretation of results

10
DNA FingerprintingProcedureOverview
11
LaboratoryQuick Guide
12
DNA Fingerprinting ProceduresDay One
13
DNA Fingerprinting ProceduresDay Two
14
DNA Fingerprinting ProceduresDay Three
15
DNA is Tightly Packaged into Chromosomes Which
Reside in the Nucleus
16
Model of DNADNA is Comprised of Four Base
Pairs
17
Deoxyribonucleic Acid (DNA)
18
DNA Schematic
O
Phosphate
P
O
O
Base
O
O
CH2
Sugar
O
P
O
O
Phosphate
Base
O
O
CH2
Sugar
OH
19
DNA Restriction Enzymes
Evolved by bacteria to protect against viral
DNA infection Endonucleases cleave within
DNA strands Over 3,000 known enzymes
20
Enzyme Site Recognition
Restriction site
Palindrome
Each enzyme digests (cuts) DNA at a specific
sequence restriction site Enzymes recognize
4- or 6- base pair, palindromic sequences (eg
GAATTC)
Fragment 2
Fragment 1
21
5 vs 3 Prime Overhang
Enzyme cuts
Generates 5 prime overhang
22
Common Restriction Enzymes
EcoRI Eschericha coli 5 prime overhang
Pstl Providencia stuartii 3 prime overhang
23
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

24
DNA DigestionTemperature

• Why incubate at 37C?
• 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

25
Restriction Fragment Length PolymorphismRFLP
PstI
EcoRI
GAATTC CTTAAG
CTGCAG GACGTC
Allele 1
1
2
3
GAATTC CTTAAG
CGGCAG GCCGTC
Allele 2
3
Fragment 12
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

26
AgaroseElectrophoresisLoading

• Electrical current carries negatively-charged
  DNA through gel towards positive (red) electrode

Buffer
Dyes
Agarose gel
Power Supply
27
AgaroseElectrophoresisRunning

• Agarose gel sieves DNA fragments according to
  size
• Small fragments move farther than large
  fragments

Gel running
Power Supply
28
Chemistry in action.OrAsk your friendly
chemistabout electro-phoresis
andelectrolysis.
29
Chemistry of electrophoresis and
electrolysisElectric fieldsand electric
currents
30
SDS-PAGE, DNA electrophoresis and the need for a
gel matrix (sieving medium)Without a viscous
medium, all molecules move at the same rate in
electric fieldGel structure retards larger
solute particlesDNA molecules, SDS- proteins
have equal charge/massElectrophoresis occurs
between the electrodes (field-driven).
31
Electrolysis always occurs during
electro-phoresis.Cathode produces H2 at twice
the rate that anode produces O2Current is
carried by solute ions. Electrons arent soluble
in H2O.Example TAE buffer tris
suppliescations (), acetatesupplies anions
(-).Electrolysis occursat the electrodes.
32
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

33
Analysis using Verniers Logger Pro
softwareAdaptable to many forms of Gel
Analysis Take or Import Gel Image Set
Origin Set Scale Set Standard Ladder Add
LanesLeaves more time for students to Explore
34
Standard Curve and AnalysisEasy to View Results
include Gel Image Standard Curve
Data Table One convenient page with all the
Results
35
Molecular Weight Determination
Fingerprinting Standard Curve Semi-log

• 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

36
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

37
Plasmid Mapand Restriction SitesLaboratory
Extensions
BamHI EcoRI HindIII EcoRIHind III
1 linear fragment 7367bp
2 fragments 863bp / 6504bp
3 fragments 721bp/2027bp/3469bp
5 fragments
721bp/863bp/947bp/1659bp/2027bp
38
Bio-RadsElectrophoresisEquipment
PowerPac Mini
PowerPac Basic

• Electrophoresis Cells
• Power Supplies
• Precast Agarose Gels

PowerPac HC
PowerPac Universal
Mini-Sub Cell GT
Wide Mini-Sub Cell GT
39
VerniersAnalysis Equipment

• White Light Transilluminator
• BlueView Transilluminator
• White Digital Bioimaging System
• Blue Digital Bioimaging System

White Light Transilluminator
BlueView Transilluminator
White Digital Imaging System
Blue Digital Bioimaging System
40
Webinars

• Enzyme Kinetics A Biofuels Case Study
• Real-Time PCR What You Need To Know and Why You
  Should Teach It!
• Proteins Where DNA Takes on Form and Function
• From plants to sequence a six week college
  biology lab course
• From singleplex to multiplex making the most out
  of your realtime experiments
• explorer.bio-rad.com?Support?Webinars