IDABEL BERNABE-PAGULAYAN. DMS; PhD..Crim.

Forensic Chemist Consultant, Registered Criminologist

 What is DNA?
- Deoxyribonucleic Acid (de–ak–si–ri–bo–n(y)u–kle–ik)
- is a chemical substance found in all cells of living
organism whose composition have been passed on
from parents to offsprings. It is called as the
genetic or hereditary material. A person’s DNA
is the SAME in every cell. It is contained in
blood, semen, skin cells, tissue, organs, muscle,
brain cells, bone, teeth, hair, saliva etc.

- chemically, it is an acid and is composed of three

sub-units, namely:
1. the phosphate group
2. a deoxyribose sugar, and
3. one of the four bases – Adenine (A), Thymine (T),
Cytosine (C) and Guanine (G)
FROM THE WHOLE TO THE (MICROSCOPIC) PARTS

The There Each One The Genes

are
human is a nucleus chromosome chromosomes
segments
body nucleus contains 46 of every are filled of
DNA
contains inside chromosomes pair with that contain
100 each arranged is from tightly instructions
trillion human in 23 each coiled to
make
cells cell pairs parent strands proteins
of the
building
BIOLOGICAL EVIDENCE AMENABLE
FOR DNA ANALYSIS;
• blood and bloodstains
• semen and seminal stains
• hair with follicle/root
• saliva and buccal cells
• tissues and skin cells
• organs
• bone marrow and bones
• teeth
ADVANTAGES OF DNA ANALYSIS VS
CONVENTIONAL SEROLOGICAL METHOD
• DNA is stable – it can be isolated from
material that is months or even years old
• DNA can be recovered from a wide variety
of biological resources like blood, semen,
hair, saliva and bone
• DNA can be replicated in the laboratory from
a small amount of initial material through the
process of PCR (Polymerase Chain Reaction)
• DNA shows greater variability from one individual
to the next
LINE-UP of CASES where DNA ANALYSIS
can be of help:

• Sexual assault cases like RAPE

• Murder
• Homicide
• Robbery
• Hit and run
• Extortion
• Paternity cases
• Identification of remains in mass disaster
 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”
■ Criminal DNA databases
■ Population genetic databases
 IDENTIFYING DNA EVIDENCE
Possible location of
EVIDENCE DNA on the evidence
Source of DNA

baseball bat or similar handle, end sweat, skin, blood, tissue

weapon
hat, bandanna, or mask inside sweat, hair, dandruff
eyeglasses nose or ear pieces, lens sweat, skin
facial tissue, cotton swab surface area mucus, blood, sweat, semen, earwax
dirty laundry surface area blood, sweat, semen
toothpick tips saliva
used cigarette cigarette butt saliva
stamp or envelope licked area saliva
tape or ligature inside/outside surface skin, sweat
bottle, can, or glass sides, mouthpiece saliva, sweat
used condom inside/outside surface semen, vaginal or rectal cells
blanket, pillow, sheet surface area sweat, hair, semen, urine, saliva
“through and through bullet outside surface blood,tissue
 IDENTIFYING DNA EVIDENCE

To avoid contamination of evidence that may

contain DNA, always take the following precautions:

• Wear gloves. Change them often.

• Use disposable instruments or clean them thoroughly before
and after handling each sample.
• Avoid touching the area where you believe DNA may exist.
• Avoid talking, sneezing, and coughing over evidence.
• Avoid touching your face, nose, and mouth when collecting and
packaging evidence.
• Air-dry evidence thoroughly before packaging.
• Put evidence into new paper bags or envelopes, not into plastic bags.
Do not use staples.
 GUIDELINES FOR COLLECTING AND
SUBMITTING DNA EVIDENCE
DOCUMENTING, COLLECTING, PACKAGING and PRESERVING DNA
EVIDENCE from CRIME SCENE

If DNA evidence is not properly documented, collected, packaged and

preserved, it will not meet the legal and scientific requirements for admissibility
in a court of law.

• If DNA evidence is not properly documented, its origin can be questioned.

• If it is not properly collected, biological activity can be lost.
• If it is not properly packaged, contamination can occur.
• If it is not properly preserved, decomposition and deterioration can occur.
DOCUMENTING DNA EVIDENCE
• Photograph the evidence before it is touched, moved or collected.
• Note the location and condition of the evidence.
• Note and sketch relationships of the evidence relative to the crime scene and other
objects present.
• Note and sketch the condition of the biological evidence.
COLLECTING DNA EVIDENCE
COLLECTING KNOWN SAMPLES from VICTIM/SUSPECT
1) Blood
• Only qualified medical personnel should collect blood samples from a person.
• Collect at least two 5-ml tubes of blood in purple-top tubes with EDTA as an
anticoagulant for DNA analysis.
• Identify each tube with the date, time, subject’s name, location, collector’s name,
case number, evidence number.
• Refrigerate, DO NOT FREEZE blood samples. Use cold packs, not dry ice during
shipping.
• Pack liquid blood tube individually in Styrofoam or cylindrical tube containers with
absorbent material surrounding the tube
2) Bloodstain
• Prick the left ring finger of the subject with a blood lancet and stain a drop of blood in
a clean sterile white cotton cloth or gauze.
• Air dry the stain for at least two hours out of direct sunlight without any heat source
such as dryer.
• Label the stain with date, time, subject’s name, location, collector’s name, case
number and evidence number.
• Pack in a clean white envelope and seal it.
• Submit to the laboratory as soon as possible.
3) Saliva
• Use clean cotton swabs to collect saliva samples. Rub the inside surfaces of the
cheeks and gums thoroughly. Air dry the swabs and place in a clean paper envelope
with sealed corners. DO NOT use plastic containers.
• Identify each sample with the date, time, subject’s name, location, collector’s name,
case number and evidence number.
• Saliva samples do not need to be refrigerated if properly dried.
• Submit to the laboratory as soon as possible.
4) Hair
• Using a tweezer carefully pluck at least 10 strands of hair (head hair, armpit hair, beard
or moustache), ensure the presence of root with sheath material. Wrap in a clean paper
and place in an envelope with sealed corners. Note: DO NOT CUT the hair!!!
• Identify each sample with the date, time, subject’s name, location, collector’s name,
case number and evidence number.
• Hair samples do not need to be refrigerated.
• Submit to the laboratory as soon as possible.
COLLECTING BLOOD/BLOODSTAIN
1) Liquid/Dried Blood
• Absorb suspected liquid blood onto a clean cotton or swab. Air dry the cloth
or swab and pack in clean paper or an envelope with sealed corners. Do not
use plastic containers.
• Absorb suspected dried blood onto a clean cotton cloth or swab moistened
with distilled water. Air dry the cloth or swab and pack in clean paper or an
envelope with sealed corners. Do not use plastic containers.
2) Blood on Surfaces or Water
• Absorb suspected liquid blood or blood clots onto a clean cotton cloth or swab. Air
dry the cloth or swab and pack in clean paper or an envelope with sealed corners.
Do not use plastic containers.
• Collect suspected blood in water immediately to avoid further dilution. Place in a
clean airtight container. Freeze the evidence and submit as soon as possible to the
laboratory.
3) Bloodstains
• Air dry suspected wet bloodstained garments. Wrap suspected dried bloodstained
garments in clean paper. DO NOT place wet or dried garments in plastic or airtight
containers. Place all debris or residue from the garments in clean paper or an
envelope with sealed corners.
• Air dry small suspected wet bloodstained objects and submit the objects to the
laboratory. Preserve bloodstain patterns. Avoid creating additional stain patterns
during drying and packaging. Pack to prevent stain removal by abrasive action or
packaging materials during shipping. Pack in clean paper. Do not use plastic
containers.
• When possible cut a large sample of suspected bloodstains from
immovable objects with a clean sharp instrument. Collect
an unstained control sample. Pack to prevent stain removal by abrasive action. Pack in
clean paper. Do not use plastic containers.
• Absorb suspected dried bloodstains on immovable objects onto a clean cotton cloth or
swab moistened with distilled water. Air dry the cloth or swab and pack in clean paper or
an envelope with sealed corners. Do not use plastic containers.
COLLECTING SEMEN and SEMINAL STAINS
• Absorb suspected liquid semen onto a clean cotton cloth or swab. Air dry the cloth or
swab and pack in clean paper or an envelope with sealed corners. Do not use plastic
containers.
• Submit small suspected dry semen-stained objects to the laboratory. Pack to prevent
stain removal. Pack in clean paper. Do not use plastic containers.
• When possible, cut a large sample of suspected semen stains from immovable objects
with a clean sharp instrument. Collect an unstained control sample. Pack to prevent stain
removal. Pack in clean paper. Do not use plastic containers.
• Absorb suspected dried semen stains on immovable objects onto a clean cotton
cloth or swab moistened with distilled water. Leave a portion of the cloth or
swab unstained as a control. Air dry the swab or cloth and place in clean paper or
an envelope with sealed corners. Do not use plastic containers.

COLLECTING SEMINAL EVIDENCE from

SEXUAL ASSAULT VICTIM(S)
• Sexual assault victim(s) should be medically examined by a medico-legal officer
or city/municipal health officer using a sexual assault evidence kit to collect
vaginal, oral and anal evidence. In the absence of a kit, collect vaginal and anal
swabs (two each) by using sterile cotton swabs in long wooden stick as well as
additional evidence like underwear and shorts worn by the victim at the time of
the crime if available.
• DO NOT forget to collect reference samples from victims:
- if living, collect saliva or buccal swabs
- if deceased, collect blood, hair with root or muscle tissues with no preservatives
like formalin or alcohol
• Refrigerate and submit the evidence as soon as possible to the laboratory.
COLLECTING SALIVA
• Absorb suspected liquid saliva onto a clean cotton cloth or swab. Leave a portion of the
cloth unstained as a control. Air dry the cloth or swab and pack in a clean paper or an
envelope with sealed corners. Do not use plastic containers.
• Submit suspected small, dry saliva-stained objects to the laboratory. Pack to prevent
stain removal by abrasive action. Pack in clean paper or an envelope with sealed corners.
Do not use plastic containers.
• When possible, cut a large sample of suspected saliva stains from immovable objects
with a clean sharp instrument. Pack in a clean paper. Do not use plastic containers.
• Pick up cigarette butts with gloved hands or clean forceps. Do not submit ashes. Air dry
and place the cigarette butts from the same location (ashtray) in clean paper or an envelope
with sealed corners. Do not submit the ashtray unless latent print examinations is
requested. Package the ashtray separately. Do not use plastic containers.
• Pick up chewing gum with gloved hands or clean forceps. Air dry and place in clean
paper or an envelope. Do not use plastic containers.
• Pick up envelopes and stamps with gloved hands or clean forceps and place in a clean
envelope. Do not use plastic containers.
COLLECTING HAIR
• Pick up hair carefully with clean forceps to prevent damaging the root tissue.
• Air dry hair mixed with suspected body fluids.
• Package each group of hair separately in clean paper or an envelope with sealed
corners. Do not use plastic containers.
• Refrigerate and submit as soon as possible to the laboratory.

COLLECTING TISSUES
• Pick up suspected tissues with gloved hands or clean forceps.
• Collect 1-2 cubic inches of red skeletal muscle.
• Place tissue samples in a clean, airtight plastic container without formalin
formaldehyde.
• Freeze the evidence, place in Styrofoam containers, and ship overnight on dry ice.
SUBMITTING DNA EVIDENCE
REQUESTING EVIDENCE EXAMINATIONS
All requests for evidence examinations should be in writing addressed to the
Deputy Director for Technical Services, Attention: Forensic Chemistry
Division, NBI, Taft Avenue, Manila and contain the following information:
• The requesting and submitting contact person’s name, agency, address
and telephone number.
• Description of the nature and the basic facts concerning the case
(brief history of the case).
• The name(s) of and descriptive data about the individual(s) involved
(subject, suspect, victim) and the agency-assigned case identification
number.
• A list of the evidence being submitted herewith(enclosed) or under
separate cover.
• State what types of examinations are requested.
• State where the laboratory report should be sent.
• Chain of Custody of Evidence
PACKAGING AND TRANSPORTING EVIDENCE
• Take precautions to preserve the evidence.
• Place porous evidence in individual protective covering such as paper envelopes. Stabilize
the evidence to avoid movement or friction during transport.
• Wrap and seal each item of evidence separately to avoid contamination.
• Place the evidence in a clean, dry and previously unused inner container.
• Seal the inner container with tamper-evident tape.
• Affix EVIDENCE and appropriate BIOHAZARD labels to the inner container.
• Affix the evidence examination request and all case information between the inner and
outer container.
• Place sealed inner contained in a clean, dry and previously unused outer container with
clean packaging material.
• Completely seal the outer container so that opening of the container would be evident.
• Address the outer container as follows:

FORENSIC CHEMISTRY DIVISION

Attention: DNA Laboratory
National Bureau of Investigation
Taft Avenue, Manila 1000
 COLLECTION OF DNA EVIDENCE
EVIDENCE CONDITION LOCATION COLLECTION MODE
BLOOD Liquid Person Collect in EDTA tubes.
Liquid Scene a. Use syringe, collect into EDTA tube.
b. Transfer onto cotton cloth. Air dry.

BLOOD Clot Scene a. Collect clot in test tube.

b. Transfer onto cotton cloth. Air dry.

BLOOD Wet Clothing Air dry at room temperature. Package in

paper bag.
Wet Object Air dry at room temperature. Package in
paper bag.
Collect sample with syringe. Place sample
Wet Water
in plastic container. Freeze sample.

DRIED Crust Person, Scene, Scrape crust into paper packet.

BLOOD Object Collect control blank.
Stain Weapon
Collect item directly.
EVIDENCE CONDITION LOCATION COLLECTION MODE
DRIED Stain Small object Collect entire item.
BLOOD Stain Vehicle,Uphol- Cut out stained area. Package separately.
stery,Carpet, Collect control.
Wallpaper,Wood
Unmovable surface, a. Scrape into paper packet. Collect control.
Stain
concrete wall b. Transfer onto moistened cotton thread.
Air dry thread.
Unmovable surface Tape lifting. Place in container.
Spatters

SEMEN Liquid Victim a. Collect sample with rape kit.

b. Collect sample with swabs.
Keep in refrigerator.
Liquid Object, Scene
a. Collect liquid semen into tube.

Wet Clothing b. Transfer onto cotton cloth.

Dried stain Clothing, Carpet Air dry.
Upholstery Air dry. Package separately.
Unmovable surface Collect as is. Package
separately.
Cut out section with stain.
Collect control.
Scrape sample into paper
 EVIDENCE CONDITION LOCATION COLLECTION MODE
URINE Liquid Person Direct deposit in container. Keep
refrigerated.

SALIVA Liquid Scene Use syringe transfer into test

tube. Keep refrigerated.

TISSUE, ORGAN Fresh Scene Place in container. Keep

BONE refrigerated.
Dried Scene Place in container.

HAIR With tissue Scene Collect hair with tissue

in container. Keep
refrigerated.
With blood Scene Separate hair from
blood. Air dry. Collect
Intact hair Scene in paper packet.
Pick up sample with
clean forceps. Place in
Fragments Scene paper packet.
Tape lift. Package in
Control Person
container.
Pulled (at least 20).
HOW IS DNA TYPING DONE?
DNA typing or profiling is a procedure wherein DNA extracted from the
evidentiary sample as well as from the reference biological samples obtained
from the victim and suspect are analyzed and processed to generate a particular
pattern or profile for each samples. This profile is unique for each person except
from identical twins. The patterns are compared either with that of a known
individual to determine a match, or a set of possible relatives to determine
consanguinity.
In individual identification, the pattern obtained from the evidentiary sample is
compared with that of a suspect. If the patterns are different, definitely it has not
originated from the suspect. If it is SIMILAR, the probability that the
evidentiary sample arose from the suspect and not from a random individual in
the population is calculated using a formula based on well-accepted concepts of
statistical probabilities and population genetics.
In cases of determining consanguinity, DNA from the subject and his/her
relatives are analyzed and compared. DNA fragments of an individual are
contributed by his/her father and mother. Identification of mass disaster
victims are done in the same way, the DNA of unidentified victim and
relatives are analyzed and compared. In paternity cases, the DNA fragment
contributed by the father should be observed in the alleged father. Then,
the probability that the alleged father is the father of the child is calculated
as a ratio between that of the alleged father and any random male in the
population.
There are several types of DNA tests that can be performed. The NBI
currently employs the PCR-based testing using the Short Tandem Repeats
(STR) systems like PE-ABI Profiler and Promega’s PowerPlex 16 and
PowerPlex Y Systems of analysis.
 STAGES OF ANALYSIS
1. EXTRACTION – to obtain the DNA material from the specimen. Two
commonly used methods are Chelex, DNA IQ (rapid methods) and
organic extraction.

2. QUANTITATION - to determine the amount of DNA material extracted

from the sample. NBI make use of the QUANTIBLOT kit which is
human specific and highly sensitive up to picogram level.

3. DNA AMPLIFICATION by PCR (Polymerase Chain Reaction)

– to make many copies of specific DNA fragment. PCR is a synthesis
reaction that is repeated for a number of cycles and results in
exponential accumulation of the specific DNA fragment. The thermal
cycler is the machine that does this PCR and is compared to a Xerox
machine.
 PCR PROCESS
5’ 3’
Starting DNA
Template
3’ 5’

Separate
Forward primer
strands
5’ 3’ (denature) 5’ 3’

Add primers
3’ 5’ (anneal) 3’ 5’
Reverse primer

Make copies
(extend primers)

Repeat Cycle,
Copying DNA
Exponentially
 Thermal Cycling Parameters

94 0C 94 0C 94 0C
94 0C
T
E
M
720C 72 0C
P
E
72 0C
R
A
T
U
60 0C 60 0C 60 0C
R
E
Single Cycle

Time
Typically 25-35 cycles
performed during PCR
The denaturation time in the first cycle is
Lengthened to ~10 minutes when using
AmpliTaq Gold to perform a “hot-start” PCR
Three (3) steps in PCR:
a) DENATURATION – template DNA is made single-stranded by heat
denaturation
b) ANNEALING – temperature is lowered, annealing of primers to template
will occur
c) EXTENSION – temperature is raised again which favors specific
annealing of primers and extension by DNA polymerase.

4. DNA TYPING/PROFILING – to determine the genotype of the

donor/source of the DNA material. The NBI make use of the fully
automated hi-tech equipment called ABI-PRISM 310 GENETIC
ANALYZER. which allows simultaneous analysis of fifteen (15) Short
Tandem Repeat loci plus Amelogenin for gender determination.
 Tips for Avoiding Contamination
• Pre- and post-PCR sample processing areas should be physically separated
• Equipment, such as pipettors, and reagents for setting up PCR should be
kept separate from other lab supplies, especially those used for analysis of
PCR products
• Disposable gloves should be worn and changed frequently
• Reactions may also be set up in a laminar flow hood, if available
• Aerosol-resistant pipet tips should be used and changed on every new
sample to prevent cross-contamination during liquid transfers
• Reagents should be carefully prepared to avoid the presence of any
contaminating DNA or nucleases
• Ultraviolet irradiation of laboratory PCR set-up space when the area is not in
use and cleaning workspaces and instruments with isopropanol and/or 10%
bleach solutions help to ensure that extraneous DNA molecules are
destroyed prior to DNA extraction or PCR set-up
Steps in DNA Sample Processing
Sample obtained from
Crime scene or Paternity
Investigation BIOLOGY
DNA DNA PCR Amplification
Extraction Quantitation of Multiple STR Markers

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

GENETICS
Comparison of Sample Generation of Case
Genotype to other Report with Probability
Sample Results of Random Match

If match occurs, comparison

of DNA profile to population
databases
 SHORT TANDEM REPEATS (STRs)
Fluorescent
Dye label
AATG AATG AATG

7 repeats

8 repeats

The repeat region is variable between samples while the flanking

regions where PCR primers bind are constant
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
 Commercially Available STR Kits
Power of
NAME SOURCE STR loci included Discrimination

THO1, TPOX, CSF1PO

monoplexes Promega THO1, TPOX, CSF1PO 1 : 410
Applied Biosystems
AmpFlSTR Blue D3S1358, VWA, FGA 1 : 5000
Applied Biosystems
AmpFlSTR Amelogenin, THO1, TPOX, 1 : 410
Green I CSF1PO
CTTv Promega CSF1PO, TPOX, THO1, VWA 1 : 6600
FFFL Promega F13A1, FES/FPS, F13B, LPL 1 : 1500
Gamma STR Promega D16S539, D13S317, D7S820, 1 : 1.8X104
D5S818
CSF1PO, TPOX, THO1, VWA, D16S539, D13S317, D7S820,
PowerPlex (version 1.1
and 1.2 later)
Promega D5S818 1 : 1.2X108
AmpFlSTR Profiler Applied Biosystems D3S1358, VWA, FGA, Amelogenin, THO1, TPOX, CSF1PO,
D5S818, D13S317, D7S820 1 : 3.6x109
Applied Biosystems D3S1358, vWA, FGA, Amelogenin, D8S1179, D21S11, D18S51,
AmpFlSTR Profiler Plus
D5S818, D13S317, D7S820 1 : 9.6x1010
Applied Biosystems D3S1358, D16S539, Amelogenin, THO1, TPOX, CSF1PO,
AmpFlSTR COfiler D7S820 1 : 8.4X105
Applied Biosystems D3S1358, VWA, D16S539, D2S1338, Amelogenin, D8S1179,
AmpFlSTR SGM Plus
D21S11, D18S51, D19S433, THO1, FGA 1 : 3.3X1012
D3S1358, THO1, D21S11, D18S51, VWA, D8S1179, TPOX, FGA,
PowerPlex 2.1 Promega Penta E 1 : 8.5x1010
CSF1PO, FGA, TPOX, THO1, VWA, D3S1358, D5S818, D7S820,
PowerPlex 16 Promega D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta 1 : 1.8x1017
 PATERNITY TESTING
A question could be raised as to who is the most likely father of a child
whose mother had the misfortune of having sexual encounters with two
men prior to the conception of the child. (Some real life situations include
a married woman who had sex with her husband and then raped by another
man prior to conception, a woman with simultaneous sexual relations with
two men, etc.). The traditional tests include blood typing selected protein
analysis, however, these tests are not definitive. They can only indicate
whether the alleged father could possibly be the father but since many other
persons in the population can have the same blood and protein type, these
tests are only indicative.
Biological (blood, hair or buccal swabs) samples from the child (C), the
mother (M) and the alleged father/s (AF) are obtained. DNA is then
extracted and the DNA patters compared as in the following example:
 CASE 1 CASE 2
FRAGMENT M C AF M C AF
a __ __ __
b __ __
c __ __ __
d __ __

e __ __
In Case 1, the Child (C) has fragments b and e. It shares its b
fragment with its mother (M) hence, fragment b is referred to
as its maternal allele and its e fragment the paternal allele has
been inherited from its father. Since fragment e is not present
in the alleged father, then the alleged father (AF) could not
possibly be the father of the child.
In Case 2, the child again has two different parental alleles c
and a. Fragment c is inherited from the mother and fragment a
from its father. Hence, the alleged father is likely the father of
the child because the fragments which is not contributed by the
mother and is contributed by the father is also present in the
alleged father.
 TYPES OF RESULTS
1. INCLUSION - means that the test may indicate that DNA found at the crime
scene is that of the suspect.
- in paternity testing, means that the alleged father could be the
biological father of the child
2. EXCLUSION – when the results obtained from the suspect’s sample or
known individual are not present in the results from the unknown crime scene
sample. The test results show that the sample found at the scene does not
belong to the suspect.
- in paternity testing, means that the alleged father
CANNOT be the biological father of the child.
3. INCONCLUSIVE RESULTS – include situation in which NO RESULTS were
obtained from the sample because there was not enough DNA to test or in
which results are obtained from an unknown sample from the crime scene but
there are NO samples from known individuals or suspect to test for
comparison.
INCLUSION
INCLUSION
EXCLUSION