MCJ – 2023

BIOLOGICAL
EVIDENCE
&
FORENSIC DNA
ANALYSIS
 Forensic Serology
Definition :
IDENTIFICATION,
CHARACTERIZATION and
INTERPRETATION
of blood, semen and other body fluids
(saliva/urine), usually found in dried stain form
on items of physical evidence.
 Forensic Serology
Definition :
 In practice, examination of other
associated biological evidence such as
hairs, nail clippings, soil are included.

 In summary - all these can be classified as

Biological Evidence
 Importance of
Importance of Biological
Biological Evidence
 Indicates that a crime has been committed.
- Bloodstains on victim’s clothes, tears, cuts

 Link between suspect and victim.

- Blood, hair could be transferred to the suspect during the
act of committing the crime.

 Link between suspect and scene of crime.

- Semen left at the crime scene in rape cases.
 Importance of Biological Evidence
• Corroborate statement of victim/suspect.
- In accident cases, e,g, hit-and-run, the presence of traces of
bloodstains or tissue on the vehicle can disprove driver’s denial

• Identify/Exonerate the suspect

DNA profiles from blood or seminal stains can be used to
identify or exonerate the suspect.

• Provide leads in investigation

Blood types or presence of semen can provide further leads to law
enforcement officers
 Types of Body Fluids/Stains

 Blood/Bloodstains
- most important and most frequently encountered - all types of
crimes e.g. murder, grievous hurt, assaults, sexual assaults, etc

 Semen/Seminal stains
- sexual offences e.g. rape, sodomy, etc

 Saliva/Saliva stains
- Various crimes : evidence can range from cigarette butts to love
bites.

 Urine /Urine stains

• Most common, well-known and perhaps most
BLOOD EVIDENCE
important evidence in the world of criminal
justice today.

• Found most often in "crimes of violence such

as homicide, assault, and sexual assault.“

• May be in the form of fresh liquid, coagulated,

dried, or as a small drop or stain,

• Each form involves a different method of

preservation and collection

• It can link a suspect to the victim or crime scene

• Bloodstain patterns reveal a great deal about

the position and movement during crime
 BLOOD GROUPING
There are many grouping systems in blood
• ABO ; Rh; Duffy; I; Kell; Kidd
• Lewis; Lutheran; MNSs; P

These grouping systems give blood

its uniqueness and identity
The most common grouping system is ABO
 THE ABO SYSTEM
• The most reliable , consistent,
versatile, not easily degradable
• ABO antigens are inherently stable
• Four different classes - A, B, O, AB
• Group A could be further sub-classed into
group A1 and A2
 Forensics of Blood/Bloodstains

Five specific questions as guidelines

for determining the nature of a crime:
1. Is the sample, blood?
2. Is the sample, animal blood?
3. If the sample is animal blood, from which
species did it ~ come from?
4. If the sample is human blood, what type is it?
5. If its is human blood whose blood is it?? –
forensic DNA analysis?
 Properties of Bloodstains

 Intensely coloured, bright red when fresh

 Dry and aged stains - red to brown colour.
 Extreme conditions - brown, sometimes
green/mouldy
 Can be detected using Infrared light;
 Ultraviolet light not useful as blood doesn’t
fluoresce.
 Examination of Bloodstains
Many of the techniques and procedures used in the examination
and analysis of blood involves simple basic biochemical reactions.

The whole process is includes:

(i) Preliminary Examination
(ii) Screening Tests
(iii) Confirmatory Tests
Problems in Visual Detection of Body Fluid Stains

 Sometimes may be difficult to see even if large.

 Small stains are always difficult to find, particularly when
searching a large area.
 Not all stains of appropriate colour & texture are body fluid
stains.

Presumptive tests
 Presumptive Tests
◗ Search tests - biochemical colour tests
◗ Capable of being applied to large area
◗ Rapid tests
◗ Sensitive (to minimise sample used)
◗ Has some degree of specificity - able to exclude non-body
fluids
◗ Harmless/cheap
◗ Unambiguous result - differentiate +ve/-ve
 Presumptive Tests : Bloodstains

How do you know if the stain is a bloodstain?

1. Kastle-Meyer Test (color) KM test

2. Luminol (photoluminescence)
 Luminol
• Crime scene investigators may search the
area using a high-powered light that reveals
tiny specks of blood or they may use
luminol.

• Luminol is a reagent that reacts with the

iron found in hemoglobin to cause
luminescence (produce a bluish green light).

• It is sprayed across the crime scene and

will show up the finest traces of blood
(even if washed).
 Human or Animal Origin?
How do you know the bloodstain is human blood?
- Precipitin Test (human antisera)
• Bring together an extract of bloodstain/tissue
and a suitable precipitating antisera.
• When antisera meets the corresponding antigen Known Antisera
(Human)
found in the extract, a precipitation reaction
may occur, resulting in a white band at the White
precipitate
interface
• Visible precipitate within 10 - 20 mins. Extract of tissue
or bloodstains
• View with oblique lighting on a dark background
• Run controls at the same time
 Distribution of Blood Types of
Malaysian Population (%)
ABO SYSTEM
O A B AB
Malays 36.8 25.2 32.0 6.0
Chinese 45.9 23.3 25.5 5.3
Indians 34.9 22.0 35.8 7.3

cf. Typical distribution in USA

43 42 12 3
 BLOOD SPATTER
The location, distribution, appearance of bloodstains
and spatters may be useful in interpreting and
reconstructing the events that produced the bleeding

• position, shape
• trajectory, origin
• direction
• dropping distance
• angle of impact
 Questions Answered by
Blood Spatter Interpretation

1. The distance between the target surface and the

origin of blood at the time of blood shed

2. The point(s) of origin of the blood

3. Movement and direction of a person or an object

4. The number of blows, shots, etc. causing the

bloodshed and/or the dispersal of blood

5. Direction of travel and impact angles

6. The object used to cause the bloodshed

 Questions Answered by
Blood Spatter Interpretation

7. Sequencing of multiple bloodshed events

8. Interpretation of contact or transfer patterns

9. Type and direction of impact that produced

the bloodshed

10. The position of the victim and/or object during

bloodshed

11. Movement of the victim and/or object

after bloodshed
 Items of Evidence involving
Blood/Bloodstains

 Clothing and other articles of victim

 Clothing and other articles of suspect
 Blood swabs from scene of crime of murder/assault/shooting
 Weapons used in the commission of the crime eg. parang,
wood, etc.
 Reference blood specimens of victim & suspect
EXAMINATION AND
IDENTIFICATION
OF SEMINAL STAINS
 Why is seminal stain identification important?

• Evidence in sexual assault cases

• Positive identification that ejaculation occurred
• Can help prove a crime was committed
• Can identify the perpetrator
• Two steps:
1. Stains must be located
2. Stains are tested to determine identity
 Identification of Semen
What is semen?

 Semi-fluid mixture of:

• Cells
• Enzymes
• Other organic and inorganic materials

 Sperm cells are most important component

as these are specific to semen.
 SEMEN – Seminal Plasma

Seminal plasma - contains a complex range of organic and inorganic constitu

Acid phosphatase - an enzyme that secreted in seminal fluid, (also in other bo

Concentration of Acid Phosphatase is up to 400 times greater in semen than i

Acid phosphatase activity is 50-1000 times greater in human semen than in an

 Spermatozoa
 Consists of head, tail and middle piece
 1/500 in. long
 Head is oval, flattened
 Middle piece connects head and tail
 Sperm from different species vary in size and shape.
 Bull and human sperm, - paddle-shaped heads
 Dog - similar but smaller (1/3 size);
 Rat sperm - hook-shaped heads
 Chicken sperm - spindle-shaped, almost difficult
to distinguish from the mid-piece.
 SEMEN - Spermatozoa
• Oligospermia – males who have low sperm count (less
than 2.0 x 107 cells per ml)

• Azoospermia - males who have no spermatozoa e.g.

vasectomy

• Vasectomized, oligospermic, and aspermic males can still

produce normal amounts of seminal fluid
 Spermatozoa (contd.)

 Sperm count of < 50 million - indicates infertility

 Contains half the normal chromosome complement
 Sole function – reach and fertilise the female egg cell
(indicated by long tail and high motility)
 Bacteria attacks tail first during degradation
 Seminal Stains

 Spermatozoa visible under microscope.

 Important forensic value
 Seminal stain – can be detected by UV lighting
(not conclusive)
 Presumptive Test - The AP test
 Confirmatory Test – Microscopic detection
of spermatozoa
 Identification by DNA analysis
 Properties of Seminal Stains
 Freshly dried - crusty and white in colour.
 Wear and handling - crust will
disappear, leaving pale discolouration.
 Fluoresces under UV light but optical
brighteners incorporated in fabrics
and washing powders also fluoresce.
Visual & Tactile Examination
 Presumptive Tests: Seminal Stains

Acid Phosphatase (AP)Test

 Test for enzyme acid phosphatase
present in high concentrations in seminal
fluid.
 In the presence of acid phosphatase the
colour change will be from yellow to
purple.
 Reaction time – 30 secs.
 Longer reaction time –of more than 30 sec
means it is not seminal stains
 Microscopic Examination - Detection of Sp

The most reliable confirmation for the presence of semen is the

positive visual identification of sperm cells. Several staining
techniques to stain sperm cells

 Nuclear Fast Red/Picroindigocarmine (Christmas Tree stain)

Two Dyes used:
Green – Stains tails
Red – Stains heads
 Detection of Seminal Stains
Another confirmatory test for semen is the detection of
prostate specific antigen (PSA) or p30

 p30 – a glycoprotein produced by the prostate gland

 p30 - found exclusively in seminal fluid
 Concentrations of 200,000 to 5.5 million ng per mL
 Useful in the absence of spermatozoa
 Sensitivity of the p30 test is 4 ng/mL (which means semen can be
detected in samples that have been rinsed or washed.
 Aids in the identification of semen in aged evidence samples
 May be considered definitive
 Detection of Seminal Stains
p30 (or PSA – Prostate Specfic Antigen)

Most laboratories use a

simple PSA cartridge
 p30 (or PSA – Prostate Specfic Antigen)

The advantages of a PSA determination are: ·

• PSA - An accepted marker for detecting semen in criminal cases.
• PSA test - not presumptive like the AP test.
• Detection of PSA - possible in cases where no spermatozoa.
• PSA can be recovered at detectable concentrations in 30 year
old semen stains.
• Semen samples can show positive PSA results even at a
dilution factor of 1:200,000
• PSA is detectable in post-ejaculate urine and male urine from
adult men and can be detected in urine of eleven year old boys.
 Persistence of Seminal Constituents in the Human Vagina
• Living, motile sperm survive 4-6 hrs in
vaginal cavity of a living female

• Non-motile sperm may be found 3-6 days

after intercourse

• Intact sperm (with tails) not normally found 16

hrs after sex, but have been found 72 hrs after in
rare instances

• Finding seminal acid phos. decreases with time

after sex, with little chance of finding it after 48
hrs

• PSA, or p30, will not be detected beyond 24

hrs after sex
 Items of Evidence involving Seminal Stains

• Clothing/Articles of victim
• Clothing/Articles of suspect
• Vaginal swabs (low and high)
• Anal and/or oral swabs
• Bedsheet, mattress cover, blanket,
towels, tissue paper
• Mattress, cushions
• Condoms
• [Reference blood specimens of victim &
suspect]
 Saliva

 99% water, pH 6.8 – 7.0

 Contains the digestive enzyme – amylase (also known
as alpha (ά amylase).
 Cast off cheek cells are normally present in saliva
 Adults make about 1.0 -1.5 litres of saliva per day
 SALIVA
 Found as evidence on objects that come into
contact with a person’s mouth
 Cigarette butts, toothpicks, bottles/glasses,
handkerchiefs
 Pieces of cloth used as gags
 Spittle at crime scene can be useful evidence
 Cases - Murder, rapes ……burglary.
 Identifying Saliva Stains

 Presumptive: Fluoresce/ Alternate light

 Amylase Screening- enzyme found at high level in
saliva. Doesn’t confirm the presence of salivary
amylase. Two tests: (i) Starch-iodine test (ii)
Phadebas Reagent
 Confirmatory: ◦ DNA testing
 Forensic Characterization of Saliva
• A simple test for saliva involves mixing starch,
iodine, and a sample of the presumed saliva together.
• Starch and iodine are a deep blue color when mixed
together. The amylase breaks down starch, however,
and the color fades
(takes about 15 mins @ 37 oC).
 Presumptive Tests : Saliva
Press Test –

i. Phadebas reagent (tablets dissolved in water) sprayed on

filter paper, then allowed to dry

ii. Phadebas paper placed on area to be tested

iii. Paper is sprayed with water and pressed against area
iv. Paper is left on area and watched for blue color change
v. Can be watched for up to 40 minutes for color change
Hair Examination
 Introduction

• Hair is encountered as physical evidence in a wide variety of crimes.

• Hair is considered class evidence. Although hair analysis evidence

is not accepted as reliable for the purpose of positively identifying a
specific person, it still has value as physical evidence.

• When properly collected and submitted to the laboratory

accompanied by an adequate number of standard/ reference samples,
hair can provide strong corroborative evidence for placing an
individual at a crime scene.

• Approximately 100 head hairs are shed by an individual each day.

These hairs are shed on clothing and on items in the
environment.
 Human Hair Characteristics

A. Macroscopic
Color (in reflected light) Structure : Shaft form
• White • Straight
• Blonde • Arced
• Red • Wavy
• Brown • Curly
• Black • Twisted
• Tightly coiled
• Crimped
Overall shaft
thickness
Shaft length range in
• Fine
centimetres or inches
• Medium
• Coarse
 Human Hair Characteristics
B. Microscopic
Color (in transmitted Natural pigmentation
light)
• Colourless (White ) Pigment size
• Blonde • Coarse
• Red • Medium
• Brown • Fine
• Black
Pigment aggregation
• Streaked
Pigment aggregate size
• Clumped
• Large
• Patchy
• Medium
• Small Pigment density
• Absent
 HAIR EVIDENCE
• Because of its tough outer coating, hair does not easily
decompose.
• Hair found at crime scenes or secondary locations can be
analyzed.
• The physical characteristics of hair can offer clues to the
broad racial background of an individual.
• Chemical tests can provide a history of the use of drugs
and other toxins, indicate the presence of heavy metals, and
provide an assessment of nutritional deficiencies.
• When the follicle of a hair is present, DNA evidence may
be obtained and can lead to individual identification
 Hair Evidence
• Forensic analysis of hair centers on color and structure,
determined through microscopic magnification.
• If the hair has been pulled out, it should include the
follicle, and that helps to see the hair's full length.
• The way the pigment is distributed helps to identify
hairs from particular individuals.
• The center of the shaft is the medulla, which is also
valuable for species differentiation.
 HAIR EVIDENCE
• As a general rule, forensic hair comparisons involve either head
hair or pubic hair.

• The collection of 24-50 full-length hairs from all areas of the

scalp will normally ensure a representative sampling of head
hair.

• A minimum collection of two dozen full-length pubic hairs

should cover the range of characteristics present in pubic
hair.

• Hair samples are also collected from the victim of suspicious

deaths during an autopsy.
 Significance and Use
A hair examination is usually used to determine if
the item is
•1 A hair.
• From a human or another animal.
• From certain body areas.
• Characteristic of a certain racial group.
• Characteristic of a particular growth phase.
• Damaged.
• Diseased.
Shed hair
Pulled out hair
 HAIR COLLECTION AND PRESERVATION
• “Reference” hair must always be collected from the victim of a crime as
well as all suspects

• About 24-50 hairs from all over the head, 24 from pubic area

• Entire hair length collected – pulled from root

• The hair collected must be from the same body parts, since a pubic
hair found on a rape victim cannot be compared to the head hair of a
suspect.

• Rape victims must have their pubic area combed for perpetrator’s hair.
Comb is packaged separately

• Dead victims – hair collected during autopsy

Methods of Hair Recovery
Some of the methods used to
collect hairs from clothing and
bedding items are :
• Hand picking
• Tape lifting
• Vacuum Sweeping
• Combing
 HAIR EXAMINATION
POSSIBLE CONCLUSIONS
1. That the hairs are similar to the reference hair samples in
terms of microscopic characteristics and that they could
have come from the same source.

2. That the hairs are not similar to the reference hair samples
and that they did not originate from the same source

3. That no conclusion could be reached

 Conclusion
 Forensic Serology is a comparison science

 From comparison of serology results, the forensic scientist

can at best conclude the suspect was a possible source of the
deposited semen, bloodstain or hair or maybe able to
absolutely exclude the suspect as a source.

 The implementation of DNA testing in forensic samples has

dramatically enhanced the value of evidence, providing near-
absolute identification.
Forensic DNA Profiling
– Role in Crime
Investigation
 What is DNA ?
DEOXYRIBONUCLEIC ACID
• DNA is genetic material found in cells.

• DNA is inherited by an individual from

its biological parents.

• DNA is material that governs inheritance of eye

color, hair color, stature, bone density and
many other human traits.
 At fertilization in humans,
the ovum and the sperm
+
combine

to produce a child.
 Cell
Tissue

Nucleus

Human body consists of

tissues made of cells generally
having nucleus at the center.
Nucleus
contains
chromosomes

Chromosomes are
made up of
DNA and proteins
 The Cell Nucleus Contains Paired Chromosomes

 Human Cells contain 46 chromosomes(called the Human

Genome) within their nuclei.

 The nucleus of every cell in every human contain 22 pairs

of chromosomes,

 In addition,
a) the cells of every female contain an additional pair of ‘X’
chromosome (XX)
b) the cells of every male contain just one ‘X’ and another unique
one called the ‘Y’ chromosome (XY)
 Human Genome
• 23 pairs of chromosomes = 46 chromosomes
• 3,200,000,000 pieces of information on 23 chromosomes
DNA is made up of two strands
of double helix structure

A long but narrow string-like object

 There are four types of basic units
called NUCLEOTIDES.

A = Adenine T = Thymine

G = Guanine C = Cytosine
 The chemical structure of everyone's DNA
is the same.

 The only difference between people (or

any animal) is the order of the base pairs.

 There are so many millions of base pairs

in each person's DNA that every person
has a different sequence.
 “Your DNA sequence is unique amongst all DNA sequence
of any human that has ever lived and will live for quite some
time to come.
Unless you have an identical twin, in which case you do
have someone who has the same DNA sequence.
But apart from that your DNA sequence is yours and yours
alone.”
….. Dr. Eric S. Lander, Prof. & Director of the
Massachusetts Institute of Technology (MIT) Centre for
Genome Research.
 CEL

NUCLEUS

CHROMOSOME

DNA

GENES
GENE - genetic material on a
chromosome that code for a visible trait
or functional protein. E.g eye colour,
hair colour, bone density
LOCUS - Specific location(s) of DNA. May
or may not represent a gene

ALLELE - One of the possible forms of a

given gene, contained on either
chromosome and can be the same or
different lengths (homozygous or
heterozygous
POLYMORPHISM:
 The existence of a gene in several forms.
The more polymorphic the gene, the more
variations of the trait it encodes.
 The polymorphisms tested in forensics are
length polymorphisms. ◦
 Reported as a number
 The number indicates the length of the DNA
repeat sequence.
 DNA is the same

Saliva Teeth
Blood

Hair Bone
Urine Tissue Sperm

from any part of the body

 Factors Leading to DNA Degradation
Degradation refers to any decrease in quality of the DNA, including
breaks between individual nucleotides in the DNA, damaged
bases, and fractures in the phosphate backbone

 Time
 Temperature
 Humidity
 Ultraviolet Light
 Exposure to chemicals
 Some facts:
 Each human cell has 6 billion bases

 The total length of DNA in each human cell is 2 metres

 An average person has 75 trillion cells.

 The total length of DNA in a person is 150 trillion metres.

 Therefore each person has DNA long enough to

go to the sun and back 500 times.

 DNA does not change with age – What you are

born with, you die with
 D N A

Profiling
A new technology with
multiple applications
 DNA PROFILING
• A great breakthrough in the development
of forensic science

• The technique of DNA- Profiling was

originally developed in 1985 at Leicester
University, UK by Professor Alec
Jeffreys

• Discovered Forensic “DNA Fingerprinting”

based on the hypervariable regions of
human DNA
 WHAT IS DNA PROFILING?
 The name given to a scientific testing process that positively
identifies an individual from his/her genetic material.
 A forensic technique in criminal investigations, comparing
criminal suspects' profiles to DNA evidence so as to assess
the likelihood of their involvement in the crime.
 It is also used in paternity testing, to establish immigration
eligibility, and in genealogical and medical research.
 A major tool in forensic investigation of crime, paternity, body
identification, etc
 DNA Profiling/Typing:
 Is
a term used in Forensic DNA analysis
where several genetic loci is analyzed.
 DNA results from a number of genetic loci
analyzed constitute a DNA profile of a person.
 Increase in the number of genetic loci analyzed
will increase the uniqueness/rarity of the DNA
profile.
 FORENSIC DNA ANALYSIS IS A POWERFUL TOOL BECAUSE :
• Each person's DNA is unique (with the exception of identical twins).
Therefore, DNA evidence collected from a crime scene can implicate
or eliminate a suspect, similar to the use of fingerprints.

• It can analyze unidentified remains through comparisons with DNA

from relatives.

• When evidence from one case is compared with evidence from another,
those cases can be linked to the same perpetrator locally and
nationally.

• Biological evidence from crime scenes when collected and stored

properly, forensically valuable DNA can be found on evidence that
may be decades old.
 DNA TECHNOLOGIES USED IN FORENSIC INVESTIGATIONS
1. Restriction Fragment Length Polymorphism (RFLP)
RFLP was one of the first applications of DNA analysis to forensic investigation.

2. Short Tandem Repeat (STR) Analysis.

Short tandem repeat (STR) technology is used to evaluate specific regions (loci)
within nuclear DNA.

3. Mitochondrial DNA Analysis

mtDNA analysis uses DNA extracted from the mitochondria.

4. Y-Chromosome Analysis
The Y chromosome is passed directly from father to son,

5. X-Chromosome Analysis
In forensic investigations such as complex kinship analysis
 POLYMERASE CHAIN REACTION (PCR)
• PCR is a an extraordinarily powerful technique used to
amplify a specific region of DNA (genetic locus),by
repeated cycles of denaturing and replication to an amount
large enough to be tested or analysed.

• In criminal forensics, PCR is used to amplify DNA evidence

from small samples that may have been left at a crime
scene.

• The PCR technique was invented by Dr. Kary Mullis in

1983. Was awarded the Nobel Prize in Chemistry in 1993.
POLYMERASE CHAIN REACTION

 FASTER RESULTS

 LESS LABOUR INTENSIVE

 LOW AMOUNTS OF DNA

 DEGRADED DNA
FORENSIC DNA IDENTIFICATION KIT - GlobalFiler - 24-locus STR kit

• 24-locus multiplex (21

autosomal + 3 sex
chromosome – amelogenin,
1 Y-STR, Y indel)
• High discrimination power
• High sensitivity
• Tolerance to inhibitors.
PROCEDURES

IN FORENSIC

DNA ANALYSIS
 Steps in DNA Sample Processing
Sample Obtained from Crime
Scene or Paternity BIOLOGY
Investigation
DNA DNA
Extraction Quantitation

PCR Amplification
of Multiple STR markers

TECHNOLOGY
Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination

Comparison of Sample Genotype to Other Sample Results Generation of Case Report with Proba
GENETICS
 DNA-Profiling
involves following
steps

1. Isolation and purification of DNA.

2. Quantitation of the DNA.

3. Amplifying the DNA.

4. Separation by capillary electrophoresis.

5. Detection of specific loci by laser.

 1. EXTRACTION OF DNA

BLOOD
HAIR WITH ROOT

BONE
SPERMATOZOA URINE
TISSUE
DENTAL
DENTAL
PULP
PULP

SALIVA
 DNA PROFILING BY PCR-STR TECHNIQUE

Blood / Bloodstains

Extraction

STEPS
DNA Extract
IN
Quantitation of DNA Extract
DNA
PCR Amplification on DNA Extract
ANALYSIS
Analysis of PCR Products

RESULT : DNA PROFILE OF BLOOD / BLOODSTAIN

DNA PROFILING BY PCR-STRTECH NIQUE

SE MINAL STAIN

STEPS
EXTRACTION

DNA EXTRACT FROM SPERM CELLS DNA EXTRACT FROM NON-SPERM CELLS
IN
DNA
QUANTIT ATIO N OF DNA EXTRACTS
ANALYSIS
PCR AMPLIFICAT ION ON DNA EXTRACTS
(SEMINAL
ANALYSIS OF PCR PRODUCTS
STAINS)
DNA PROFILE OF SPERM CELL
DNA
S PROFILE OF NON- SPERM CELLS
2. Amplification

Solution

DNA

Thermalcycler
 Multiplex PCR
 Over 24 Markers Can Be Copied at Once

 Sensitivities to levels less than 1 ng of DNA

 Ability to Handle Mixtures and Degraded Samples

 Different Fluorescent Dyes Used to Distinguish STR

Alleles with Overlapping Size Ranges
PCR amplification - the number of copies of the target is doubled every cycle.
 3. Electrophoresis
• The amplified DNA in a sample is
separated by electrophoresis in a
genetic analyzer
• The analyzer has a gel-filled capillary
tube through which the DNA travels.
• DNA fragments move through the gel
tube by size, smallest first
• A laser reads the fluorescent marked
DNA loci
 A sample print out
for one person
showing the loci
tested.
Different colours help
with the interpretation
 Sampel Male “A” Male “B Male “C”
Comparison of
D8S1179 10, 13 10, 14 11, 15
D21S11 30, 30 30, 33 27, 29 Alleles
D7S820 8, 12 12, 12 11, 12
CSF1PO 10, 12 10, 12 10, 12 Alleles obtained
D3S1358 14, 17 16, 16 15, 15 from three
THO1 8, 10 9, 9 6, 9 unrelated different
D13S317 9, 14 8, 8 8, 11
individuals
D16S539 11, 14 9, 11 12, 12
D2S1338 18, 21 19, 19 19, 19

D19S433 14.2, 15.2 15, 15.2 14.2, 15

vWA 14, 19 16, 17 17, 18

TPOX 8, 9 9, 11 8, 11
D18S51 16, 16 12, 15 15, 16 They are
Amelogenin XY XY XY
D5S818 10, 12 12, 12 10, 13 drastically different
FGA 23, 26 23, 23 22, 23

individual specific
Paternity
&
Kinship
 TABLE OF DNA PROFILES
STR
Amelogenin THO1 TPOX CSF1PO D3S1358 vWA FGA D5S818 D13S317 D7S820
Locus

“A”
Blood Sp. XY 6, 9 9, 11 11, 13 15, 17 14, 16 22, 24 8, 10 10, 12 9, 11
of AF

“B”
Blood Sp. XX 7, 8 8, 8 10, 12 14, 15 15, 18 23, 25 8, 10 9, 12 10, 13
of mother

“C”
Blood Sp. XY 7, 9 8, 11 11, 12 15, 15 14, 18 23, 24 10, 10 10, 12 10, 11
of child
 SUMMARY OF DNA-STR RESULTS OF A PATERNITY CASE

STR
Amelogenin THO1 TPOX CSF1PO D3S1358 vWA FGA D5S818 D13S317 D7S820
Locus

“A”
Blood Sp. XY 6, 9 9, 11 11, 13 15, 17 14, 16 22, 24 8, 10 10, 12 9, 11
of AF

“B”
Blood Sp. XX 7, 8 8, 8 10, 12 14, 15 15, 18 23, 25 8, 10 9, 12 10, 13
of mother

After eliminating the alleles from the mother

“C”
Blood Sp. Y 9 11 11 15 14 24 10 10 11
of child
 SUMMARY OF DNA-STR RESULTS OF A PATERNITY CASE

STR
Amelogenin THO1 TPOX CSF1PO D3S1358 vWA FGA D5S818 D13S317 D7S820
Locus

“C”
Blood Y 9 11 11 15 14 24 10 10 11
Sp. of
child

Compare the remaining alleles with that of the alleged father

“A”
Blood XY 6, 9 9, 11 11, 13 15, 17 14, 16 22, 24 8, 10 10, 12 9, 11
Sp. of AF

Match
 Rape Case.
Evidence:
Seminal Stains
 TABLE OF DNA PROFILES
STR
Amelogenin THO1 TPOX CSF1PO D3S1358 vWA FGA D5S818 D13S317 D7S820
Locus

“S”
Blood Sp. XY 6, 9 9, 11 11, 13 15, 17 14, 16 22, 24 8, 10 10, 12 9, 11
of suspect

“T”
Sperma- XY 6, 9 9, 11 11, 13 15, 17 14, 16 22, 24 8, 10 10, 12 9, 11
tozoa
from swab

“R”
Seminal 8, 9, 10, 11, 14, 15, 14,15 22, 23 9, 10 9, 10
stains XY 6, 8, 9 8, 10
from 11 12, 13 17 16,18 24, 25 12 11, 13
panties

“A”
Blood Sp. XX 7, 8 8, 8 10, 12 14, 15 15, 18 23, 25 8, 10 9, 12 10, 13
of victim
 Interpretation of DNA Results
COMPARING DNA PROFILES :
Typically there are three possible laboratory outcomes:

 If the DNA profiles from the evidentiary and known samples are
consistent at each locus, laboratory analysts can interpret this
finding as a "match," "inclusion," or "failure to exclude.“

 If the two profiles are not consistent at each locus, the finding can
be interpreted as a "nonmatch" or "exclusion.“

 If there are insufficient data to support a conclusion, the finding

is often referred to as "inconclusive."
 Interpretation of DNA Results
COMPARING DNA PROFILES :

1. DNA PROFILE FROM SAMPLE “A” DOES NOT MATCH DNA

PROFILE FROM SAMPLE “B”

CONCLUSION :
SAMPLE “A” AND SAMPLE “B” COME FROM
DIFFERENT SOURCES
 Interpretation of DNA Results
COMPARING DNA PROFILES :

2. DNA PROFILE FROM SAMPLE “A” MATCHES

DNA PROFILE FROM SAMPLE “B”

CONCLUSION :
SAMPLE “A” AND SAMPLE “B” COULD HAVE
COME FROM THE SAME SOURCE
 Interpretation of DNA Results
 Once a DNA match is observed, forensic scientists estimate
the chance of finding that DNA profile in particular human
populations i. determine the significance of such a match.

 This calculation is necessary to inform the courts of the

rarity of the profile

 This will depend on how common the observed profile is in

the general population.

 Random match probabilities are most often used to interpret

evidence from single source samples.
 POPULATION DATABASES

• Population databases of major

racial and ethnic groups are used to
determine estimates of the rarity of
DNA profiles.

• These databases sometimes consist of

as few as 100 profiles from unrelated
persons

• Allow a reliable estimate of the

chance of observing a given DNA
profile in a larger population.
 POPULATION DATABASES

• Each matching allele is assumed to

provide statistically independent evidence

• The probability of a random match is

calculated by applying the multiplication
rule to the probability of the allele found at
each locus.

• The scientific validity of the multiplication

rule depends on whether the matches at
each allele are actually statistically
independent (Hardy Weinberg Principle).
MALAYSIAN POPULATION DATABASE

• Malay
• Chinese
• Indian
• Kadazandusun
• Bajau
• Murut
• Iban
• Bidayuh
• Melanau
 Power of Discrimination ( P.D ) – Red Group

Malay (%) Chinese (%) Indian (%)

D5S818 91.3 92.3 89.2
FGA 96.9 96.5 95.9

Combined Power of Discrimination

- More than 99.99999999% ( 1 in 1 quadrillion )

Combined Power of Exclusion (Paternity)

- More than 99.999% ( 1 in 100,000)
 Applications of DNA Profiling
DNA fingerprinting can be used for various purposes.

• Crime Investigation

• Family Matters

• Medical Diagnosis

• Pedigree Analysis

• Seed-stock Identification

• Defence Records
 Human Identity Testing

 Forensic cases -- matching suspect with evidence

 Paternity testing -- identifying father
 Historical investigations
 Missing persons investigations
 Mass disasters -- putting pieces back together
 Military DNA “dog tag”
 Convicted felon DNA databases
DNA – not 1st choice
 Quality Assurance
 Designated work area for specific work process
 Processing of crime and reference samples
at different time.
 Reagent Blank Control.
 Positive Control.
 Negative Control.
 Reanalysis of samples.
 Proficiency Testing.
 FORENSIC CASE OF INTEREST

DNA PROFILING – Serial rapist identified.

Murder Case: Body Identification
BLOODSTAINS ON JEANS
The infamous blue dress
Y-STRs and mtDNA
 Human Genome
23 Pairs of Chromosomes + mtDNA

Located in cell nucleus

Autosomes
2 copies
Located in
per cell mitochondria
(multiple copies in
cell cytoplasm)

mtDNA
1 2 34 5 6 7 8 9101112 16,569 bp

Y
Mitochondrial
131415 16171819202122X DNA
Sex-
Nuclear DNAchromosomes
3.2 billion bp 100s of copies
per cell
Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 2.3, ©Elsevier Science/Academic Press
 Role of Y-STRs and mtDNA
Compared to Autosomal STRs
• Autosomal STRs provide a higher power of discrimination and are the
preferred method whenever possible

• Due to capabilities for male-specific amplification, Y-chromosome STRs

(Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential
extraction is not possible such as fingernail scrapings)

• Due to high copy number, mitochondrial DNA (mtDNA) may be the only
source of surviving DNA in highly degraded specimens or low quantity samples
such as hair shafts
 Different Inheritance Patterns
Lineage Markers

CODIS STR Loci

Autosomal Y-Chromosome Mitochondrial

(passed on in part, (passed on complete, but only by (passed on complete, but
from all ancestors) sons) only by daughters)
Maternal Inheritance of mtDNA

1st
generation

2nd
generation

Multi -generation
Paternal Inheritance of Y-STRs

1st
generation

2nd
generation

Multi -generation
 Lineage Markers:
Y-STRs and mtDNA

Advantages Disadvantages

• Extend possible reference samples • Lower power of discrimination

beyond a single generation (benefits due to no genetic shuffling with
missing persons cases and genetic recombination
genealogy)
• Family members have
• Family members have indistinguishable haplotypes
indistinguishable haplotypes unless unless mutations have occurred
mutations have occurred
 Y-STRs permit extension of possible
reference samples in missing persons
cases

uncle 3rd cousin

? (paternal)
 Why the Y Chromosome?
• Applications
– forensic investigations (98% of violent crime by men)
– genealogical purposes
– evolutionary studies

• Advantages to Human Identity Testing

– male component isolated without differential extraction
– paternal lineages

• Needs
– population studies to evaluate diversity of haplotypes
– robust assay for accurate characterization of Y markers
 Forensic Advantages of Y-STRs
• Male-specific amplification extends range of cases accessible to
obtaining probative DNA results (e.g., fingernail scrapings, sexual assault
without sperm)

• Technical simplicity due to single allele profile; can potentially recover

results with lower levels of male perpetrator DNA because there is not a
concern about heterozygote allele loss via stochastic PCR amplification;
number of male contributors can be determined

• Courts have already widely accepted STR typing, instrumentation,

and software for analysis (Y-STR markers just have different PCR
primers)

• Acceptance of statistical reports using the counting method due to previous

experience with mtDNA
 Scenarios Where Y-STRs
Can Aid Forensic Casework
• Sexual assaults by vasectomized or azoospermic males (no sperm left
behind for differential extraction)

• Extending length of time after assault for recovery of perpetrator’s

DNA profile (greater than 48 hours)

• Fingernail scrapings from sexual assault victims

• Male-male mixtures

• Other bodily fluid mixtures (blood-blood, skin-saliva)

• Gang rape situation to include or exclude potential contributors

Mitochondrial DNA (mtDNA)
 Comparison of Nuclear and Mitochondrial DNA

Advantages of mtDNA testing:

• Higher copy number per cell

• Results with highly degraded DNA
• Results with limited sample

Disadvantages of mtDNA testing:

•Low power of discrimination

•Labor intensive
•Expensive

http://www.fbi.gov/hq/lab/fsc/backissu/july1999/dnaf1.htm
Utility of mtDNA Analysis

Cortical window: left tibia

Mongolian Model shot and blown to bits

Bone fragments of model found on hill

 The Tsunami of 2004

Over 150,000 people lost their lives

 Trace DNA Analysis

Trace DNA is meant to describe the minute quantities of DNA

transferred through skin contact, which can be successfully analyzed and
follow the general principle of trace evidence

To obtain DNA profiles from cells and debris transferred through skin
contact (referred to hereinafter as trace DNA analysis) are such that a
vast number of cases now have the potential to be aided or even resolved
by this technology.
Trace DNA analysis is a valuable tool and has a the potential to
provide investigators with additional information.
Contamination prevention is of utmost importance.
Adequate communication between investigators and scientists.
Trace DNA has the potential to be an effective tool in reducing the level
of volume crime.
 DNA Database
• One tool for investigating and identifying
perpetrators of crime
• A database is an organized file or files
of data that can be searched to retrieve
information
• DNA databases compare crime scene
evidence to a database of DNA
profiles obtained from convicted
offenders for investigative leads.
Forensic DNA Database of Malaysia

• 116,534 DNA profiles were obtained

and stored in the Forensic DNA
Databank of Malaysia (FDDM) by
2019,

• Since 2013, police have solved 60 cases

with the use of the Forensic DNA Databank
Malaysia (FDDM).
The Significance or Success of Forensic DNA Pro

 its high potential to EXCLUDE wrongly

accused suspects in violent crimes
such as murders, homicides and rapes.

 its high CERTAINTY for individual

identification compared to
conventional blood group or enzymes
typings.
 Conclusion
DNA analysis of body fluid stains is an
extremely powerful tool for the
identification of an offender but if the
evidence is not properly recognized,
documented, collected and preserved, then
it is of no value in a criminal investigation
and the forensic laboratory may not be able
to use this tool in the interests of justice.