Forensic Characterization of
Bloodstains using Digital Imaging
• Bloodstains that are on an
item that’s dark or has a
complex pattern can be
difficult to see
• A digital imaging system
with an infrared filter
attached can make
bloodstains more visible
by filtering out the
background color
Forensic Characterization of
Bloodstains cont’d
• Micro Crystal Tests
– Takayama
– Tiechman
• Advantages
– More specific then chemical test
• Disadvantages
– Not as sensitive
– More susceptible to interference
Forensic Characterization of
Bloodstains: Individualizing
• ABO Blood Typing
• Enzyme Typing
• DNA Analysis
Ancient
– RFLP: Restriction Fragment Length
Polymorphism
– STR: Short Tandem Repeats
Forensic Characterization
of Semen: 1. VISUALIZATION
MANY BODY FLUIDS
FLUORESCE WITH
ALTERNATE LIGHTING
SOURCES
Forensic Characterization
of Semen: 2. PRESUMPTIVE TESTING
ACID PHOSPHATASE ENZYME /
FOUND IN LARGE CONCENTRATIONS
IN SEMEN
Forensic Characterization
of Semen: 3. CONFIRMATION
A. MICROSCOPIC IDENTIFICATION
B. DETECTION OF P30, A MALE
OF SPERM
PROSTATE PROTEIN. USEFULL FOR
VASECTOMIZED MALES
Intact spermatozoa on a vaginal
slide
Forensic Characterization
of Saliva
• Evidence commonly
tested for the presence
of saliva includes:
Detection of Amylase
(breaks down starch in gel)
– Cigarette butts
– Envelope flaps
– Swabs taken from the
body of sexual assault
victims
– Bottles, cans, & straws
Gel contains starch that is broken down (circles ) in the
presence of Amylase enzyme
Safety First—safeguards while
handling biological evidence
• Wear gloves
• Keep contaminated surface away from
face—protect those mucous membranes
• Properly dispose of gloves/wash hands
Goals of biological evidence
collection
• Collect as much sample as possible from a single
source—keep it concentrated
• Ensure that the sample is not inadvertently mixed
with other biological samples—change gloves if
contaminated
• Handle the sample in a manner that minimizes
deterioration—air-dry quickly (and no heat or
sunlight exposure)
Further recommendations for
collection of biological evidence
• Handle as little as possible—submit the
item with the stain still on it
• If stain is on a large porous surface (e.g., a
rug) cut out the stain area (plus unstained)
• Collect with slightly moistened (with dist.
water) cotton swab—keep it concentrated,
and take a control swab too
Taking care to avoid
contamination
• Don’t allow one evidence stain to come into
contact with other biological samples, including
transfer from tools and gloves
• Don’t talk or cough over evidence stains
• Collect and package stains separately
• Clean tools (e.g., tweezers) thoroughly, with
distilled water stream, dry with tissue, repeat
• May use disposable tools
Packaging biological evidence
• Allow stains to air-dry as much as possible before
placing in paper bag or envelope—do not use
plastic
• Use separate paper containers for each item and
package stains and controls separately
• Ensure that the paper container is large enough to
allow circulation around the evidence item
• For garments, use clean paper to prevent different
stains from contacting each other
Proper collection and procedures
are critical to avoid
Contamination
How else will you detect it and
reduce/avoid contamination?
Typically, it looks like a mixture:
A combination of DNA from
two/more persons
Contamination at autopsy—teeth
from decomposed body
Contamination at autopsy—
mixed reference
Contamination at autopsy—
resolution
Contamination in the lab
From sample
with high
level of DNA
To sample
with low
level of DNA
PCR Product Contamination—
the Thousand to One Nightmare
It only takes
a minuscule
amount of
amplified
product…
…to cause a
typing
disaster
Contamination—Prevention
• Sample items one-at-a-time
• Separate evidence samples from
reference samples
• Use protective gear
• Separate work areas with dedicated
equipment
Monitoring for Contamination—
Controls ‘R’ Us
Bloodstain (Evidence)
Substrate Control
Reagent Blank—for Evidence
Victim’s Reference Sample
Reagent Blank—for References
Negative Amplification Control
Quality Control Sample
Positive Amplification Control
Summary 1
• Screening samples for biological evidence includes both physical (e.g.
alternate light sources) and chemical (presumptive tests) detection
methods.
• 3 steps- Visualization, Presumptive testing and Confirmation
– Blood detection include observation of color, microcrystalline tests, luminol
detection, chemical presumptive tests based on hemoglobin's peroxidase-like
activity, antibody species tests, and DNA analysis.
– Semen detection includes observation of fluorescence, Acid phosphatase
detection methods, then microscopic examination, P30 testing and DNA
– Saliva detection includes microscopic examination, amylase detection and DNA
Summary 2
• Safety- In collecting biological evidence, safety is
paramount. Wear gloves- away from face- Properly
dispose of gloves/wash hands
• Collect as much sample as possible - keep it concentratednot inadvertently mixed -change gloves if contaminated
• Handle the sample in a manner that minimizes
deterioration—air-dry quickly (and no heat or sunlight
exposure)
• Prevent contamination- Sample items one-at-a-timeSeparate evidence samples from reference samples- Use
protective gear- Separate work areas with dedicated
equipment- Clean the area, tools and run QC
• Monitor for contamination with controls
I. Intro to DNA : Facts and Jargon
DNA: Deoxyribonucleic acid
Different in every *individual
The same in every **cell of an
individual's body
*except for identical twins that have the same DNA "The time honored
method of cloning humans"
** diseased individuals may be mosaics
DNA Facts and Jargon Review
Where is it? How is it stored?
DNA is found in every
*cell= basic unit of life
Inside nuclei (organization center for the cell) and mitochondria (ATP powerhouse of the
cell) & chloroplasts for plants- (making our food via photosynthesis)
Nuclei are not found in red blood cells
In white blood cells, saliva, skin, hair fingernails, urine, feces, vomitus, earwax etc.
DNA in the Cell
In nuclei, mitochondria and chloroplasts (plants) organized in
chromosomes (wound around histones)
“DNA double bagging”
DNA Spaghetti Metaphor
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
DNA function
What’s it do?
DeoxyriboNucleic Acid : blueprints of life
Replication, Information Storage and Mutation- RIM
Central Dogma
information flow--------------->
DNA------->RNA------>protein
transcription
translation
Like Phone numbers- Units the same- Difference is
in the Sequence
http://www.ncbi.nlm.nih.gov/genome/guide/
DNA Organization and Inheritance
Human Genome Contains 23 Pairs of Chromosomes
It is inherited from your mom and dad
1
2
3
4
5
6
7
8
9
10 11 12
13 14 15 16 17 18 19 20 21 22 X
Y
Sex-chromosomes
Where’s Daddy?
PCR product size (bp)
11
12
8
14
14
11 12
8
14
12
DNA Structure
What is it?
Bases (AGCT) form the stairs of the ladder, are faithfully
paired and exhibit differences.
P
S-A : T-S
P
P
S-G : C-S
P
P
S-A : T-S
P
P
S-G : C-S
P
Hydrogen bonds
5’
3’
5’
A=T
GC
A=T
GC
hybridized
strands
3’
T=A
Phosphate-sugar backbone
T=A
CG
CG
A=T
A
T
G
C
G
CG
CG
G
C
3’
A=T
T=A
A
T
denatured
strands
C
5’
3’
GC
Sugars (S) and phosphates (P) form the sides of the ladder (identical for all DNA).
Bases (AGCT- Asian Guys Can Teach) form the stairs of the ladder, are faithfully
paired by hydrogen bonds and exhibit differences. A : T and G : C (DNA is where
its AT)
DNA Structure
• Primary genetic material
is composed of two
complementary strands
• Form a double helix or
twisted ladder
• Sides are sugar phosphate
and the steps are base
pairs
• Four Bases- 2 Purines –
Adenine and Guanine and
2 Pyrimidines- Cytosine
and Thymine
• Asian Guys are Pure!
DNA Structure
Nucleotides are the building blocks themselves
composed of PBS
Nucleotides-PBS the only
channel I want my kids to
watch
Phosphate (negative charge)
Base (AGCT-Asian Guys Can Teach)
Sugar (deoxyribose-5C)
Phosphate-Sugars
Connected by phosphodiester linkages
DNA Structure
2 Complimentary,
Antiparallel Strands
held together by Base Pairs- H Bonds
A:T held with 2 H Bonds
.
G:C held with 3 H Bonds
Molecule of Heredity Limerick
Steve Lee
The molecular structure today
Is heredity’s DNA
With nucleotides
completely comprised
of a sugar and phosphate and base
Adenine and thymine can base pair
Forming two hydrogen bonds for one stair
Cytosine and guanine
pair with three in between
and are equal in size when compared
The bases you see are so keen
They include thymine and adenine
Cytosine and one more
with guanine can store
all the info with rungs in between
DNA strands are just not the same
One is coding and one is called lame (anticoding)
They are opposite
in direction and this
is called antiparallel in name
The sides of the ladder you know,
are sugar and phosphate which show
that Franklin was right
double helix is tight
ten base pairs per turn in a row
Complimentary nature of strands
lets replication proceed just as planned
with A paring to T
and G pairing to C
the fidelity is precise and quite grand
•
DNA Structure/Function Teaching Metaphors
and Acronyms
Cell Biology
– Spaghetti in Bags
• Function:
– RIM- Pacific Rim
– Information Storage- Phone Number analogy
• Structure:
– PBS- The only station I want my kids to watch
– Asian Guys Can Teach: AGCT, Asian Guys are Pure
– DNA is where its AT
– Hybridization- Battleship, DNA velcro (David Letterman)
– Repeat Sequences- Trains- number of box cars