1

Lecture 2: DNA analysis

Lecture 3: Bloodstain Pattern Analysis
Lecture 4: Fingerprints

Forensic Biology
Dr. B. L. Lim
Associate Professor
School of Biological Sciences
bllim@hku.hk
Tel: 22990826
Announcements
1. Collect hair/fiber for the lab in advance!
2. Carry hair/fiber to the lab in a plastic bag (Sept. 27 or Oct. 4,
9:30 - 12:30 pm)
1. Venue: Lab C/F/G/H, 1
st
Floor, KBSB building
2. Draw the patterns on the lab report 1 (Part I)
3. Tape the hair/fiber on report 1 (Part I)
4. Submit report 1 (Part I) immediately after the lab
5. Submit report 1 (Part II) after the lecture on 9 Oct.
II. Quiz 1
1. Date: During the lecture on 2 Oct (Wed)
2. Time: 15 mins
3. Format: 10 MCQ
4. Covering topics in Forensic Biology (lectures 2 - 4)
5. 7.5% of final grade
6. Bring calculator with Trigonometric function (No mobile phone please!!)

I. Practical 1
Outline
1. Introduction to Forensic DNA analysis
2. The development of forensic DNA testing
methods
3. Searching for biological evidence at scene
4. Forensic genetics and method
5. Result interpretation and evaluation of DNA
evidence
6. Case sharing and discussion
Recommended book
Fundamentals of Forensic DNA Typing, John M. Butler,
Elsevier Academic Press.
First use of forensic DNA testing to
convict a murder
Two young school girls were raped and murdered
in a small town in UK (1983, 1986)
Semen was identified to be type A of ABO blood
types and PGM 1+ (10% local population were of
these types). Both were from the same man.
Blood typing results cannot be used to identify a
suspect. It can only be used to exclude a suspect
Suspect Richard Buckland admitted to be the
murderer of one of the girls.


First use of forensic DNA testing to
convict a murder
Prof. Alec Jeffreys was invited to perform DNA analysis
(RFLP multi-locus probes)
Richard Buckland, a 17-year-old with learning disabilities,
was the first person in the history proved to be innocent
by DNA testing
Over 4000 blood samples of male adults, aged 17-34,
from three local villages were collected for DNA analysis.
In a pub, the owner heard from a conversation that a
baker gave his blood to police for examination, but under
the name of his colleague, Colin Pitchfork, for 200.
The murderer Colin Pitchfork was the 4583th blood donor
in 1987. He
2
Conventional blood grouping
Based on antigens on RBCs (i.e.. ABO blood groups)
and electrophoretic polymorphisms of plasma
proteins (e.g. Gc grouping) and red cell enzymes (e.g.
PGM grouping).
Can only be used to EXCLUDE a suspect
Low power of discrimination
almost 40% of local Chinese population are of type O
of ABO blood types; the rarest type is type AB (6%)
Unable to detect degraded samples
Low sensitivity. e.g. unable to detect limited amount
of samples such as epithelial cell debris from hands or
even tiny bloodstains.
Forensic DNA analysis

1. High power of discrimination
-identify/differentiate an individual
2. Able to analyze degraded samples
3. Highly sensitive
- require very limited amount of samples
4. Possibility for automation
- avoid human error *
(Murphys Law: everything that can go wrong will
go wrong)
- possible for high throughput analysis (i.e. many
samples can be analyzed at the same time)
- save human resource

Forensic Science

Identify the Victim/Remains


Identify the Criminal (Murderer/Rapist)
Evidences for Courts
Who is the victim?

What injuries are present?

When did the injuries occur?

Why and how were the injuries produced?
Forensic Pathology


Forensic Odontology

-Teeth examination
-Facial reconstruction
Source of photo: southwalespolicemuseum.org.uk
Dr. Edmund Locard (1877-1966)
The Sherlock Holmes of France
Locard Exchange Principle

With contact between two
items, there will be an
exchange
Searching for Evidence
Every contact leaves a trace (Edmund Locard)
Transfer of evidence
person to person
person to object
object to object

3
Evidence
Physical
identification
Chemical
identification
Biological
identification
Searching for Evidence
Physical evidence
Shoe print, glass fragments, fiber and etc
Biological evidence
Bloodstains, semen stains, saliva, hair and etc
Precaution of Exhibits Collection
Avoid contamination
Wear clean latex gloves for collecting evidence
items/exhibits
Exhibits must be packaged separately into paper bags,
and properly marked with case number, item number,
collection date, and initialed across the package seal
in order to maintain chain of custody which requires
proper documentation to prove the chain of
possession from the time of collection until entered in
evidence in court
Collection of Exhibits
Small exhibits
Cigarette end
Special exhibit
A bottle of drinking water without the cap
Evidence
Physical
identification
Chemical
identification
Biological
identification
Forensic Biology
What are useful biological
materials?
4
Forensic Serology


A Bloody Mess
Find a Stain at a Crime Scene

Is it Blood?

Is it from human or not?

Blood Typing
- A, B, AB, O
Is it blood?
Presumptive test
- very sensitive
- can have false positive
Confirmatory test
- not as sensitive
- specific to hemoglobin
1. Phenolphthalein color test

- based on oxidizing activity of hemoglobin
H
2
O
2
H
2
+ [O] color

- turns pink if it is blood, remain colorless
if blood is not present
Fe
3+
[heme]+
Non-Human Blood?
Presumptive test
5
2. Hemastix

strips
Non-Human Blood?
Blood?
Detection of
heme of
hemoglobin
3. Luminol Fluorescent blood detector

How does Luminol detect blood?
Reacts with heavy metals
to glow in the dark. e.g.
iron, copper, chlorine
H
2
O
2
+ AH
2
2H
2
O + A
Fe in Heme
Even at areas cleaned after a murder!
Confirmatory Tests
Takayama and Teichmann tests
Chemicals that form crystals
with hemoglobin
Highly specific
Examine under
microscope
6
If it is blood, is it
human blood?
Any test specific to human sample?
Test sample
Rabbit serum that has
been sensitized to human
blood.
Precipitin test
antigen + antibody
= precipitate

Species Identification tests (Ouchterlony Gel)
Anti- Anti-
OK! So it is human blood!

But, so ?
Blood groups?
7
The Nature of Blood
1. Plasma (55%)

2. Blood solids
Red blood cells White blood cells Platelets
In 1901 Dr. Karl Landsteiner


A - A antigen only
40-42% population


B - B antigen only
10-12% population
AB - Both A and B antigens
3-5% population

O - Neither antigen
43-45% population
Antibody and Agglutination
- Human Blood group determination
8
Microscopic view
of normal red blood
cells
Microscopic view of
agglutinated red
blood cells
Other proteins in Blood:

- Rh factor - 85% population

- Polymorphic enzymes
Phosphoglucomutase
PGM1 58%
PGM2 6%
PGM2-1 36%
Adenosine deaminase
Haptoglobin
Transferrin
.

Matching of Blood Type and Isozymes
Question:
What is the possibility of a suspects blood sample sharing the
following characteristics with the blood sample collected at the crime
scene ?

Blood Group A
Basic subtype A2
Protein AK
Enzyme PGM 2

42%
25%
15%
6%
Answer: 0.00094 (0.42 X 0.25 X 0.15 X 0.06) or
1 in 1063
DNA. What it is
Only 4 base type in DNA, but its sequence does vary
9
Look at the neighbor to your left and
to your right. You're 99.9% identical.
Why DNA?
- DNA is the building block detailing
hereditary information

- individually unique (except monozygotic
twins)

- present in virtually all body tissues
Blood
Teeth
Saliva
Tissue
Bone
Hair
Semen
Sources of DNA
How much DNA is needed for forensic analysis?
1 nanogram (ng)
1 ng = 10
-3
ug = 10
-6
mg = 10
-9
g
10
What should we compare?
DNA Polymorphism (many forms)

-Regions of DNA which differ from person
to person

- 99.9% of DNA is the same in all
humans.

- Only 0.1 % of DNA (3 million
bases) differs from one person to
another.



DNA Technologies used in Forensic Science
1. Restriction fragment length polymorphism (RFLP)

2. Polymerase Chain Reaction (PCR)

3. Short Tandam Repeats (STR)

4. PCR-STR

Basic forensic genetics
Human body is comprised of many cells.
Each cell contains DNA (except red blood cells).
The chromosomes in each cell are identical.
Chromosomes are comprised of many genes (or
alleles).
Genes are in pair; one comes from our mother and
another from our father.
Forensic DNA analysis is to measure the length of
these pair of alleles.




Child
Father
Mother
11
Restriction fragment length polymorphism (RFLP)
Rules of Inheritance
The DNA Fingerprinting Process

1. DNA extraction
2. Restriction enzyme
digestion (To cut)
3. Gel electrophoresis
(To separate)
4. Transfer of DNA to a
filter
5. Hybridization with a
specific DNA probe
(To bind)
6. Autoradiography (X-
Ray film, detect)
RFLP
Kary B. Mullis

Polymerase Chain Reaction (PCR)

1993: Nobel Prize for Chemistry
PCR Amplification
Patent sold for US$300M in 1991

DNA polymerase
dATP, dTTP, dGTP, dCTP
10 - 20 ul
DNA/ DNA
Buffer
Reagents required for PCR
Primer
Sample DNA/template DNA
12
PCR
Denaturation 95


Annealing 60


Extension 72






dsDN
A
ssDNA
dsDNA

Taq DNA polymerase
dATP, dTTP, dGTP, dCTP
Primer


dNTP
New strand
Polymerase Chain Reaction
36 cycles theoretically will produce = 2
36
= 60 billions copies
Amplify one molecule of DNA into billions
of copies in a few hours

Identifies an individual based on
microscopic amount (1 ng) of evidence




Much more sensitive than RFLP!
HK Government Lab
Short tandem repeats (STR) or
Variable number tandem repeats (VNTR)

1985 Alec Jeffreys discovers multilocus VNTR probes
Repeats of 4-6 nucleotides
13
15 CODIS Core STR Loci with
Chromosomal Positions
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
PCR-STR typing analysis
STR stands for short tandem repeat.

AAGTAAGTAAGTC AAGT one
repeat unit
TTCATTCATTCAG TTCA

PCR-STR typing is to measure the length of this pair
of repeat sequence at a particular locus.
This pair of repeat sequences is the alleles. They are
usually of different length.


1. DNA extraction

2. PCR
- Specific primer pair for each loci
- Each primer pair linked with a specific fluorescence probe
- Each primer pair produces PCR products within specific size
range

3. Separation and Detection of the PCR products by capillary
electrophoresis and laser detection, respectively.
Three steps of PCR-STR
Instrument for STR Analysis
Amplified DNA
loaded into
autosampler
DNA migrates
through capillary
DNA size analyzed
by computer
Capillary Electrophoresis + Laser Detection
Example: All possible alleles in 3 STR loci
Markers
Crime Scene


Suspect 1



Suspect 2



Suspect 3


Suspect 4
14
If all 15 core loci are
genotyped, probabilities of 1: X
quadrillions (10
15
) can be found.
How did the forensic scientist figure out the blood stain on the
van were from two individuals?
Criminal + Victim
For an individual, no more than 2 alleles should be found at each locus.
One from mother, one from father!

1. Set up in 1998 by FBI

2. Links all 50 states

3. Two PCR with 15 STRs

4. 2 DNA databases
- Convicted Offenders Index
- Forensic Index (Crime scene)

Effective Repeat Offenders
5,000,000 profiles
188,000 profiles
As of Aug, 2007
The Criminalistics and Quality Management Group
carries out various types of laboratory analysis and
conducts crime scene examinations. A total of 1871
cases were attended in 2011(628 in 2004). The number
of exhibit items submitted for forensic DNA analysis
increased sharply from 4846 in 2004 to 9059 exhibit
items. At the end of 2011,the number of DNA data
stored within the database had increased to 39,949.
The DNA Database Section regularly compares new DNA
data with those in the DNA database at two levels:
(a) convicted offenders' DNA profiles (b) between
outstanding profiles from unsolved cases. In 2011
there were respectively 150 and 17 pairs of matches,
providing important investigative leads to the
Police Force.
(HKSAR 2011 Yearbook)
HKSAR Government Lab
DNA database: since Jan 2001
Exonerate innocent persons wrongly accused of crimes
15
Mix-up man unlikely to trace biological parents
(STANDARD) 02 25 (2008)
32
() 02 24

A 31-year-old man at the center of Hong Kong's first baby mix-up case in local
public hospitals may not be able to find his biological parents, a Hospital
Authority spokesman said yesterday.
Establish paternity and other family relationships
11 Sept., 2001 26 Dec., 2004
Identify crime and catastrophe victims
The number and identity of the victims were unknown.
20,000 pieces of human remains from the World Trade Center
site.
Remains of only 1585 of the 2792 known victims were identified.
Died in prison and Rumor of Escape of King Louis XVII
Louis-Charles Louis XVI Marie Antoinette
1789 French Revolution
1795 Louis Charles died in prison
Heart stolen by a doc during autopsy
2000 DNA test of the heart
2004 Buried the heart in the royal cemetery with
his parents after 209 years
End of Plantagenet Dynasty (1154-1485)
Beginning of Tudor Dynasty (1485-1603)
Battle of Bosworth Field (22 Aug 1485)
Henry Tudor, Earl of Richmond, the first English monarch of
the Tudor dynasty, defeated Richard III, the last king of the
House of York.
In 2012, an archaeological excavation was conducted on a city council car park using
ground-penetrating radar on the site once occupied by Greyfriars, Leicester. The
University of Leicester confirmed on 4 February 2013 that a skeleton found in the
excavation was, beyond reasonable doubt, that of Richard III.
Please bring along with you an electronic calculator with Trigonometric
function and a ruler with scale in mm.
Next lecture and quiz
Dr. Henry Cheung, former Government Lab Scientific
Officer.
Acknowledgement