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Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
DNA profiling is the use of molecular genetics methods to determine the exact genotype of a
DNA sample to distinguish one human being from another
Used in forensics to investigate crime scenes, missing persons, mass disasters, paternity,
evolutionary links, …
Crime scenes often contain biological evidence (blood, semen, hair, saliva, bones, skin) from
which DNA can be extracted
Humans have ~3 billion bp and >99.5% do not vary, however a small percentage (<0.5%) does
differ
Varying regions are called “polymorphic” due to nt differences between one individual and
another and polymorphisms are in regions of our chromosome (called loci) that do not have any
known function - this DNA is satellite DNA (specifically short tandem repeats/microsatellite)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Ways to detect unique sequences within mammalian DNA
1. Restriction digestion of chromosomal DNA works for
some organisms BUT not in mammals:
EX: humans have 3 billion base pairs with 1 million
restriction fragments formed from a single restriction
enzyme digest, nt differences between people will result in
different restriction digestion products - but TOO difficult
to isolate a single band on a gel from this large number of
fragments
2. To characterize a specific gene use blot hybridization
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
RFLPs
Genome of each of us is unique (exception: identical twins)
Variation in sequences between individuals is most pronounced in DNA that does not code for proteins
Hypervariable regions called “polymorphic sites”
Polymorphic sites - variation due to small insertions, deletions, or point mutations in restriction sites
SO .. Restriction fragment with a polymorphic site may differ from one person to the next resulting in:
RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)
Pattern of bands on a blot hybridization sometimes called a “DNA fingerprint” because can use it to identify an
individual
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
History of Forensics
Early use of science for criminal investigations - use of photographs to document crime scenes
Past ~100 years - use of fingerprinting
First genetic evidence collected for investigations - blood group typing (A, B, AB, and O)
Test is very fast and straight-forward. BUT 40% of population is type O, so not useful if several
suspects are type O.
1980s - first use of DNA-based forensic test called restriction fragment length polymorphism
analysis (RFLP); discovered by Alec Jeffries, focus on VNTRs (variable number of tandem repeats)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Restriction fragment length polymorphism analysis (RFLP)
Although technique is powerful and has great discriminating potential, it is laborious, cannot be
easily automated and is time-consuming
Depends on DNA sequences being several hundred base pairs in length, so DNA must be of
reasonable quality (not degraded) and it requires a large amount of DNA (not always able to
get enough DNA from the crime scene)
SO turn to using STRs - short tandem repeat DNA (smaller repeat lengths than VNTRs)
Easier to analyze than RFLPs because:
1. due to small size they are easily replicated by PCR
2. Because they can be amplified by PCR, STR tests can be automated, with several tests being run
at the same time
3. Even degraded DNA can give OK results
4. Even a single piece of hair (or a drop of blood, cheek cell, or piece of bone) can give enough
genetic material for successful analysis
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Advances in PCR have made a huge impact
MODERN FORENSIC DNA
PROFILING MAKES IT POSSIBLE TO
DISTINGUISH ANY TWO PEOPLE ON
THE PLANET (EXCEPT IDENTICAL
TWINS), LIVING OR DEAD
Polymerase chain reaction (PCR)
Denature dsDNA
Anneal primers to now ssDNA
Polymerization with thermostable DNA pol
(DNA synthesis - get 2 copies from 1)
One cycle can be repeated over & over
After cycle 30, > 1 billion identical
molecules (230 = 1.07 x 109)
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
DNA profiling performed at many loci to improve the power of discrimination
CODIS - COmbined DNA Index System - is a federally maintained database of DNA obtained from
crime scenes and convicted violent offenders; started by FBI in 1998
CODIS examines 13 loci, or markers, that are uniformly distributed across the human genome
Also includes AMEL (determines gender)
The 13 loci reveal no medical
or health info about a person;
called “anonymous” markers
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Why 13 loci?
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
This week’s Lab
Scene: the Highway Motel, Room #13
The motel manager hears loud voices, a woman screams, and a shot rings out. The manager runs to the
window in time to see the receding lights of a car leaving in a hurry. The door to room #13 is open. The
manager runs to the open door to see a man lying face down in a pool of blood. He calls 911. The
police arrive, and begin to examine the crime scene. An apparent homicide, but with no obvious clues
as to who committed to crime. Or..? A forensic specialist is called in to examine the crime scene and
evidence is collected. In addition, four suspects samples are collected.
You will perform PCR analysis on a single locus, the BXP007 locus - start this TODAY
Run PCR products on a 3% agarose gel, visualize products, compare to the allele ladder and to the crime
scene sample, determine who committed the crime!
In addition to running your PCR products on the agarose gel you will also run your restriction digests
from the genetic engineering lab.
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Set up PCR in previous lab period:
Items needed:
Ice bath containing one yellow tube labeled MMP (blue liquid inside), 5 other tubes labeled CS (purple
tube), A (green tube), B (blue tube), C (orange tube), and D (pink tube).
5 PCR tubes (these are smaller than we usually use, 0.5 microliter size)
5 normal size microfuge tubes used as adaptors (1.5 mL size)
Foam float
Marker
P20 pipettor
PCR block
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Label PCR tubes (0.5 microliter size) “CS”, “A”, “B”, “C”, and “D”
Also put your group name on the tube
Put 0.5 microliter tubes inside 1.5 mL tubes and put 1.5 mL tubes into foam float and set on ice, keep all
samples on ice throughout the set up of these samples
Set up the tubes as follows:
PCR tube label
CS
A
B
C
D
DNA template
20 microliters crime scene DNA (purple tube)
20 microliters suspect A DNA (green tube)
20 microliters suspect B DNA (blue tube)
20 microliters suspect C DNA (orange tube)
20 microliters suspect D DNA (pink tube)
Master mix + primers*
20 microliters MMP (yellow tube)
20 microliters MMP (yellow tube)
20 microliters MMP (yellow tube)
20 microliters MMP (yellow tube)
20 microliters MMP (yellow tube)
Add all the DNA templates to their respective tubes first - change tips for each DNA!
Next add MMP to each tube, mix by flicking the tube and/or by gently pipetting the the solution up and
down - use a fresh tip each time!!
I will start the PCR cycles up in my research lab
*MASTER MIX + PRIMERS contains Taq DNA polymerase buffer, MgCl2, dNTPs, Taq DNA polymerase, DNA primers specific for the
BXP007 locus
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
PCR cycle:
STEP
Initial denaturation
Thermal cycling
Final extension
FUNCTION
Denature
Denature
Anneal
Extend
Extend
TEMP
94˚C
94˚C
52˚C
72˚C
72˚C
TIME
2 min
30 sec
30 sec
1 min
10 min
Hold
Hold
4˚C
hours
# of CYCLES
1 cycle
}
35 cycles
1 cycle
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
On Wednesday - electrophorese PCR products
Materials needed:
3% agarose gel with ethidium bromide
5 PCR samples from before break
1x TAE running buffer
Orange G loading dye “LD” (orange liquid)
Allele ladder “allele ladder” (also orange liquid!)
P20 pipettor
Gel box
Power supply
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Electrophorese PCR products
1. Add 10 microliters of Orange G loading dye “LD” to each PCR reaction tube (5 of these); mix well
2. Set up gel apparatus, rotate gel from pouring position to loading position, add 1x TAE to gel box,
carefully pull out comb
3. Load 20 microliters of each sample into wells on the gel
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Allele ladder
crime scene
suspect A
suspect B
suspect C
suspect D
20 uL
20 uL
20 uL
20 uL
20 uL
20 uL
4. Run gel at 120 volts for ~45-60 minutes (do not let orange dye front migrate off the gel)
5. Visualize results on UV light box
Crime Scene Investigation
DNA Profiling / DNA Fingerprinting
Study questions:
1. Why do you need to perform PCR on DNA evidence from a crime scene?
2. Did your samples all generate PCR products? If not, give reasons to explain why.
3. Does the crime scene DNA sample have a genotype that matches any of the suspects? If so, which
one matches?
4. If you had a pool of 13 suspects and only one suspect had a genotype that matched the BXP007 locus
found at the crime scene would you be satisfied that you had identified the perpetrator? Why or hy
not? Explain.
USEFUL WEBSITES
Folding of RNA molecules
http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi
Molecular Biology tools
http://www.molbiol.net/
http://www.molbiol.net/biolinks/alphabar/PCR%2520and%2520Primer%2520Design.shtml
http://ntdb.chem.cuhk.edu.hk/tools.htm
Oligonucleotide calculator
http://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/9/index.html
http://www-medlib.med.utah.edu/masspec/mongo.htm
http://www.schepartzlab.yale.edu/
Protein information
http://www.basic.nwu.edu/
Chemistry calculators
http://users.pandora.be/educypedia/education/calculators.htm
Good websites of “tools”
http://www.csb.yale.edu/people/steitz/Toolkit/toolkit.htm
http://szewczak.com/
Plasmid information
MacPlasmap