1. Forensic DNA Typing
RANA MUHAMMAD ASIF
Ph D Scholar
Forensic Science and Molecular Biology
Forensic Scientist (DNA & Serology),
PUNJAB FORENSIC SCIENCE AGENCY
HOME DEPARTMENT
GOVERNMENT OF THE PUNJAB, PAKISTAN

2. The application of natural sciences to matters of criminal and civil laws
that are enforced by police agencies in the criminal justice system.
Scientific knowledge and technology are used to serve as witnesses in both
criminal and civil (intelligence) matters
Forensic science now has a prominent role in almost all criminal
investigations
Forensic science has shaped the world of justice, fuelling crime
investigations and signifying the progress of modern technology.
It provides accurate, thorough info to decision makers in our criminal
justice system.
Speedy DNA analysis enable police and LEAs to identify and arrest a
suspect in a very short period of time.
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Forensic Science

3. Forensics
Derived from Latin word “forēnsis”
meaning : -
1. Pertaining to the law or justice system
2. Forum,
3. Place of assembly for legal trials/
judicial proceedings
4. Public discussion/ debate/
argumentation
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4. 1. During the time of the Romans,
a criminal charge meant
presenting the case before the
public.
2. Both the person accused of the
crime & the accuser would give
speeches based on their side of
the story.
3. The individual with the best
argumentation would determine
the outcome of the case.
Forensics during the time of the Romans
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5. December 25, 2012
Locard’s Exchange Principle
Dr Edmond Locard (1877-1966)
“Father of the Crime Lab”
Lyon, France
“Every contact leaves a trace”
5
Foundations of Forensic Science
The basic concept in Forensic Science
With contact between two items, there will
always be an exchange.
This is the basis of evidence collection at a
crime scene.

6. Wherever he steps, whatever he touches, whatever he leaves, even unconsciously,
will serve as a silent witness against him. Not only his fingerprints or his
footprints, but his hair, the fibers from his clothes, the glass he breaks, the tool
mark he leaves, the paint he scratches, the blood or semen he deposits or collects.
All of these and more, bear mute witness against him. This is evidence that does
not forget. It is not confused by the excitement of the moment. It is not absent
because human witnesses are. It is factual evidence. Physical evidence cannot be
wrong, it cannot perjure itself, it cannot be wholly absent. Only human failure to
find it, study and understand it, can diminish its value.
Professor Edmond Locard
Basis of Locard’s Exchange Principle
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7. When a criminal comes in contact with an object or person, a cross-transfer of
evidence occurs.
1. The suspect’s DNA deposited on the victim’s body or clothing;
2. The suspect’s DNA deposited on an object;
3. The suspect’s DNA deposited at a location;
4. The victim’s DNA deposited on suspect’s body or clothing;
5. The victim’s DNA deposited on an object;
6. The victim’s DNA deposited at a location;
7. The witness’ DNA deposited on victim or suspect; or
8. The witness’ DNA deposited on object or at location.
Dr. Henry Lee
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How Evidences get Transfer

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9. December 25, 2012 9

10. What is DNA?
1. Deoxyribonucleic acid
2. Nucleic acid that contains
the genetic instructions used in the
development and functioning of all
known living organisms.
3. Blueprint of life
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11.  DNA is a long molecule which looks like a ladder that is curled around
continuously and features about 3.2 billion rungs that attached to the ladder
and forms a double stranded helix. It is made from repeating units
called nucleotides held together with HB and consist of three types of
chemical components:
 Simple sugar – deoxyribose Form Rails
 Phosphate group (Run in opposite directions)
 Four nitrogenous bases -Adenine (A)
- Guanine (G) Form Rungs
- Cytosine (C)
- Thymine (T)
 The order in which these nucleotides are arranged on a strand of DNA is
unique to every individual person, making DNA a very efficient tool for the
identification of a person.
Structure of DNA
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12. December 25, 2012 12

13. December 25, 2012 13

14. December 25, 2012 14
• 100 trillion cells
• 23 pairs of chromosomes
• 99.9% identical between
humans
• 0.1 % or 3 million bases
accounts for variation
• Est. 20,000 – 25,000
genes
Human Basic Genetics

15. Calculation of the quantity of DNA in a cell
1. Molecular Weight of a DNA Basepair = 618g/mol
A =: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol
G = 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol
2. Molecular weight of DNA = 1.85 x1012 g/mol
There are 3 billion base pairs in a haploid cell ~3 x 109 bp
(~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol
3. Quantity of DNA in a haploid cell = 3 picograms
1 mole = 6.02 x 1023 molecules
(1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules)
= 3.08 x 10-12 g = 3.08 picograms (pg)
A diploid human cell contains ~6 pg genomic DNA
4. One ng of DNA contains the DNA from 167 diploid cells
1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per
167 diploid genomes)
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16. DNAAnalogy
Genome = Book
Chromosome =Chapter
Gene = Paragraph
Exon = Sentence
Intron =Blank space
Nucleotides = Letters
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17. Astonishing Comparisons That Elucidate
the Giant Data Capacity in DNA
1. If the information in the human genome could be
written down using the alphabet, it would fill
1,000 books of 1,000 pages each, each page
containing 3,000 letters. The DNA strand is
therefore approximately equal to 462 volumes of
the Encyclopedia Britannica
2. Someone working at a typewriter at a rate of 300
letters per minute for 8 hours a day, 220 days a
year, would take 95 years to complete the task.
3. According to Professor Leonard Adleman of Los
Angeles South California University, just 1 gram
(0.0022 pounds) of DNA can contain the
equivalent amount of data to 1 trillion CDs.
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18. The earth is 150 billion m or 93 million miles
from the sun.
Astonishing Comparisons That
Elucidate the Giant molecule of DNA
1. Each cell has about 1.8 m of
DNA.
2. The average human has 100
trillion cells.
3. The average human has enough
DNA to go from the earth to the
sun more than 600 times.
4. DNA has a diameter of only
0.000000002 m.
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19. December 25, 2012 19
Forensic DNA Typing
Forensic DNA Typing is a technology that identifies particular individuals by their
respective DNA profiles.
It is a scientific venture and powerful investigation tool to strengthen our war against the
vicious cycle of crimes and menace of terrorism using national DNA databases i.e.,
CODIS (USA), NDNAD (UK), INTERPOL DNA Database.
DNA fingerprinting has proven to be the most scientifically valid procedure for human
identification in forensic science.
It is the most powerful crime-fighting tool we have at our disposal.
DNA fingerprinting has been convicting criminals, and freeing the innocent, since 1985.
Forensic DNA typing is:
1. Solving the crimes,
2. Protecting the innocents
3. Identifying missing persons
4. Relationship testing
a) Paternity, Maternity
b) Grand Parantage Test
c) Siblingship test, Twin Zygosity Test
d) Ancestry
e) Genetic Reconstruction

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DNA is preferably used as concrete evidence due to following
basic scientific facts: -
1. DNA does not tell lies
2. DNA can remain stable and typable for years
3. It is present in every cell (except RBCs)
4. The "genome" of any given individual (except for identical
twins and cloned organisms) is unique
5. The analysis of cells left at a scene allows for the DNA
present to be profiled
6. DNA containing cells can be found in many different sources
of biological evidences
Why DNA as evidence?

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1. DNA profiling is a highly valuable and relatively economical
forensic identification tool
2. Since 1986, it has successfully exonerated the innocents and
convicted the offenders.
3. Allow a large number of samples to compare with a high degree of
reliability.
4. Developing DNA profiling techniques fit more closely with
standard specifications
5. DNA profiling techniques are more applicable to the routine
analysis of crime scene samples, often obtained from high-volume
crime.
6. The growing DNA Databases allow to accurately and efficiently
match and manage the data.
Why forensic DNA profiling is important for LEAs?

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Advantages of DNA Typing
UNSURPASSED DISCRIMINATORY POTENTIAL
1. Complete blood group testing allows discrimination of one person in
several thousand and HLA typing one in several million.
2. DNA typing can routinely provide exclusion probabilities on the
order of one in billions
EXQUISITE SENSITIVITY
1. DNA can be amplified from very low amount of DNA. Even
Compromised DNA sample can be very successfully typed.
2. Smaller sample sizes are adequate
3. Allows rather small samples to be split and submitted for testing to
more than one laboratory

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Application to any body tissue
DNA testing can be conducted with any sample having
nucleated cells. For example hairs, semen, urine and saliva
DNA is stable in comparison to proteins
Resistant to degradation by common environmental insults
Advantages of DNA Typing

24. •Mixtures must be resolved if present
•DNA is often degraded
•Inhibitors to PCR and sample contamination
are often present
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Challenges of DNA use in Forensic Cases

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•Rapes,
•homicides,
•child molestation,
•Sodomy,
•Aggravated assaults,
•hit & run,
•burglaries,
•Robberies,
•Kidnappings
Cases in which DNA evidences can be used
•Matching suspect with evidence
•Paternity Testing
•Maternity Testing
•Post conviction DNA testing
•Missing persons Identification
•Mass disasters Investigation
•Historical investigations
•Military personnel's DNA Testing

26. •Blood Stains
•Semen Stains
•Chewing Gum
•Stamps & Envelopes
•Penile Swabs
•Sweaty Clothing
•Bone
•Teeth
•Hair
•Vaginal Secretions
Where DNA may be found?
• Hospital samples
• Clothing
• Bedding
• Weapons
• Cigarette butts
• Drinking cups
•Fingernail Scrapings
•Saliva
•Body tissues
•Condom etc
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27. Collection & Preservation
1. Biological evidence should be
allowed to air dry before
packaging.
2. Biological evidence should be
packaged in paper bags.
3. Biological evidence should be
stored under laboratory
conditions as available resources
permit - or in a
cool, dry climate, free of moisture.
4. Place liquid items in collection
tubes and refrigerate
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28. Preliminary Documentation and Evaluation of the Scene
When conducting a crime scene assessment:
•Talk to the first responding officer regarding hid/her observations and activities
•Determine the entry and exit points to the crime scene
•Document every person who is entering and exiting the crime scene and evaluate any
biohazardous safety issues that should be considered
•Identify possible locations and sources of DNA evidence
•Thoroughly document the scene with diagrams and photographs (with scale in view when
appropriate)
•Document if evidence is wet or dry when discovered
•Document any bloodstain patterns, with reference points, before collection
These factors can be very important to the future investigation of the case.
NOTE: An alternative light source may be used to locate stains, such as semen and saliva,
on evidence before the processing begins.
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29. Personal Protection & Contamination
Biological material can contain pathogens such as:
•Hepatitis
•Syphilis
•Tuberculosis
•Gonorrhea
•Measles
•HIV
Assume that all stains, wet or dry, are infectious.
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30. Personal Protection
To minimize contamination while collecting evidence at a crime scene,
use appropriate personal protective gear such as:
•Latex gloves
•Lab Coat
•Masks
•Goggles
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31. Guidelines to Protect Yourself and the Evidence
To protect yourself from biohazards and to protect the evidence from becoming
contaminated:
• Use new gloves for each piece of evidence
•Use clean or new implements to manipulate the sample
•Minimize contact with the sample (use a swab or forceps, etc.).
•If possible allow evidence to dry before packaging
•Collect and package evidence separately
•Do not fold together a bloodstained garment
•AVOID DIRECT CONTACT with the evidence sample
•Avoid coughing, sneezing and talking over evidence
•Avoid touching your face, nose and mouth during processing of evidence
•Establish a secure location for the disposal of biohazardous material like used
gloves and disposable instruments
•Establish a secure temporary storage area for biological evidence on the
crime scene
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32. General procedures for collecting DNA evidence
•Submit the entire item if possible
•Use a clean or disposable razor blade or scalpel to cut out or scrape the stain
•Air-dry the stain before packaging
•Use a new or clean razor blade or scalpel to cut out a control sample
•If cutting is not appropriate to collect the evidence, new swabs can be used.
•Collect and package evidence separately
•Appropriately label each package
•DO NOT fold a stained garment on itself (even if stains are dry). "Sandwich" the
garment between two pieces of clean paper, then fold the garment so that the
paper is protecting the stained garment
•Avoid direct contact with the evidence sample
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33. Collection Methods for DNA Evidence
1. Collection of stained item itself
2. Scraping of dried stain
3. Tape lifting of dried stain
4. Swabbing of stain
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34. Packaging and Transport of DNA Evidence
•Packaging in the breathing paper bags/ boxes, plastic bags only for
temporary storage and transport of very wet items
•Proper documentation and labeling of item (Location, association, Item
description, date, time, Collector’s identity, manner of collection and
storage info)
•Maintenance of chain of custody (chronological list of all the persons
who handled the evidence and mention of storage places) is even more
important for biological evidence
•Appropriate storage of evidence items
•Timely dispatch of evidence to crime lab
•Submission of investigation notes with DNA evidence
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35. Packaging & Chain of Custody
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36. The evolution of DNA technology
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37. DQ-alpha
TEST STRIP
Allele = BLUE DOT
RFLP
AUTORAD
Allele = BAND
Automated STR
ELECTROPHEROGRAM
Allele = PEAK
Three generations of DNA testing
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38. Mitochondrial DNA
mtDNA sequence
Sensitive but not
discriminating
Y-STRs
Useful with mixtures
Paternally inherited
Two relatively new DNA tests
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39. December 25, 2012 39
Short Tendam Repeats (STR)
1. Short tandem repeat (STR) also known as Simple sequence
repeat (SSR) are repetitive sequences of 2-7 base pairs
which continuously repeat end-to-end.
2. The number of times the STRs repeat varies noticeably in
each individual person and, therefore, allows for
identification.
3. In STR typing, size based DNA separation is performed to
resolve different alleles from one another.
4. The repetition of STR usually only needs to be counted up
to thirteen and it is at this point that we are able to make a
match in identity.

40. • Dinucleotide (CA) (CA) (CA)
• Trinucleotide (GCC) (GCC) (GCC)
• Tetranucleotide (AATG) (AATG) (AATG)
• Pentanucleotide (AGAAA) (AGAAA) (AGAAA)
• Hexanucleotide (AGTACA) (AGTACA) (AGTACA)
Tetranucleotides STRs are preferred in human identification
Types of STR Repeat Units
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41. December 25, 2012 41
Examples of STRs
Target region
(short tandem repeat)
7 repeats
8 repeats
9 repeats
10 repeats
11 repeats
12 repeats
13 repeats
DNA Profile of this locus = 8,10

42. The repeat region is variable between samples while the flanking regions
where PCR primers bind are constant
AATG
7 repeats
8 repeats
AATG AATG
Homozygote = both alleles are the same length.
Heterozygote = alleles differ and can be resolved from one another.
Primer positions define PCR product size
Fluorescent
dye label
Short Tandem Repeats (STRs)
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43. Why STRs are Preferred Genetic Markers
1. Rapid processing.
2. Abundant throughout the genome.
3. Highly variable within populations.
4. Small size range allows multiplex development.
5. Discrete alleles allow digital record of data.
6. Allelic ladders simplify interpretation.
7. PCR based which allows use of small amounts of DNA.
8. Small product size compatible with degraded DNA.
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44. December 25, 2012 45
1. Sample Obtained from Crime Scene or Paternity Investigation
2. Screening for biological evidence
3. Extraction of DNA from biological samples
4. Quantification of the obtained DNA
5. Multiplex Amplification of the CODIS-required STR loci by the
polymerase chain reaction (PCR)
6. Separation and Detection of PCR products (STR Alleles)
7. Sample Genotype detection
8. Comparison of sample genotype to other sample results
9. If match occurs, comparison of DNA profile to population
databases
10. Generation of case report with probability of Random match
Steps in DNA Typing

45. Forensic serology deals with the scientific study of blood and other bodily fluids
that are found at crime scenes. This is primarily performed for the detection and
identification of biological material (i.e., blood, semen, saliva, and urine) on
physical evidence in order to:
1. Link suspect(s) and victim(s) to each other and/or to the scene(s)
2. Include or exclude potential suspect(s) or victim(s)
3. Establish crime scene(s)
4. Identify weapon(s)
5. Corroborate case circumstances
6. Narrow down the samples for further analysis
Forensic Serology
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46. Serological Analyses
Analysts document the physical evidence, screen the
evidence thoroughly for the presence of biological
materials, and collect and preserve biological samples for
further analysis while upholding the integrity of the
evidence at all times. Based on the case information
provided and established casework management protocols,
scientists select an appropriate evidence processing scheme
which may involve chemical, enzymatic, immunological,
and/or microscopic techniques.
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47. 48December 25, 2012
Screening

48. The first step in a serological examination is the documentation and visual
examination of evidence. Biological stains may, or may not, be visible to the
unaided eye. The alternate light source (ALS) allows the scientist to visualize
biological stains invisible to the naked eye. Performed in a darkened room
while wearing colored goggles, stains will fluoresce when viewed at different
wavelengths of visible light. Questioned stains are then subjected to the
appropriate presumptive and confirmatory tests, as described below.
Visual examination of evidence
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49.  A complex mixture of cells, enzymes, proteins &
inorganic substances
 Fluid portion of blood is called the plasma (55% of blood
content)
 primarily water
 red cells (erythrocytes)
 white cells (leukocytes)
 platelets
Human Blood
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50.  Is it blood?
 What species is it
from?
 Individualization?
In criminal cases blood is the 1st most common fluid that is
found at the scenes of crime.
Blood Analysis
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51.  Screening Test:
1. Prostatic acid phosphatase
 Confirmatory Test:
1) Microscopy (Staining)
2) ABAcard® P-30 Test Strips
In criminal cases semen is the 2nd most common fluid that is found at
the scene. Semen identification is very important part of: -
1. Rape,
2. Sodomy,
3. Bestiality,
4. False accusation by a women,
5. Incest and
6. Sexual murder cases.
Semen Analysis
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52. Presumptive test for semen
The Acid Phosphatase (AP) Test is a presumptive test for
semen. Acid phosphatase is an enzyme that is present in
high concentrations in seminal material. If a purple color
change occurs within a minute, the test is considered
positive for the possible presence of semen. This is not a
conclusive test as AP is also found in other substances
(e.g., vaginal secretions, douches, and contraceptive
creams), although at lower concentrations.
Confirmatory tests for semen
1. Using compound microscopes, scientists search for
spermatozoa, or sperm cells, on slides prepared from
swabs, clothing, etc. The slides are stained using the
Kernechtrot-Picroindigocarmine Stain, or “Christmas
Tree Stain”, in which the heads of the spermatozoa are
colored red and the tails are colored green.
2. If spermatozoa are not detected, an extract of the stain
is tested for p30, a protein synthesized in the prostate
gland using two immunoassay methods: cross-over
electrophoresis and a rapid, membrane-based card test.
Semen Analysis
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53. Semen
1. Mixture of sperm and the secretions of the seminal vesicles, prostate gland,
and the bulbourethral glands.
2. Sperm 10%, Seminal fluid 90% and Epithelial Cell < 1%.
3. Contains small amounts of more than thirty elements, including fructose,
ascorbic acid, cholesterol, creatine, citric acid, lactic acid, nitrogen, vitamin
B12, potassium Choline Phosphate, Proteases, free Amino Acids,
Ergothioniene, Zinc, Calcium, Spermine, Lipids, Enzymes like
Fibrinogenase, Diastase, Acid & Alkaline Phosphatase, Glysidases, a & ß
Mannosidases a & ß Glucosidases, ß Givcouridases and various salts.
4. pH of 7.2 to 8.0, sperm concentration of 20×106 spermatozoa/ml or more,
sperm count of 40×106 spermatozoa per ejaculate
5. The average speed of semen at the moment of ejaculation is 31 mph (Faster
than a West African Jaguar.)
6. Semen acts as an antidepressant for women.December 25, 2012 54

54. 1. About 0.05 mm long
2. Develop only within the testicles from a spermatogonium
3. Spermatogonium spermatocytes spermatids
mature spermatozoan
4. Develop with in 70 -74 days
5. A man usually produces between 10 and 50 million sperms/day
6. Around 200 million to 500 million spermatozoa are released per ejaculation
and make up only about 2–5% of the volume of semen.
7. sperm determines the sex of the offspring, chromosomal pair "XX” results in a
female while "XY" results a male child.
8. Sperm carry 18,000 male genes to the female’s egg.
9. Antoni van Leeuwenhoek first observed sperm cells in 1679.
10. Sperm swim at a rate of about 1 to 4 mm (0.12 inches) per minute and wave
their tales more than 1000 times just to swim 1.25 cm or a half an inch
11. Over the course of a man’s life, man produce more than 12 trillion sperm.
12. The no of days the sperm can live inside a woman = 5
Sperm
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55. December 25, 2012 56
Human Sperm or spermatozoa

56. Steps Involved in DNA Casework Processing
Organic and Differential DNA
extraction
Separation of time and space for
evidence and reference samples
Real-Time PCR
Multiplex PCR with fluorescent labels
Capillary Electrophoresis
Crime scene DNA profile comparison
with victims, suspects or Database
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57. Blood
Hair Roots
Saliva
Sweat
Tissue
Chemical
DNA
Isolation of DNA
December 25, 2012 58

58. Semen stain
Chemical
Remove
Epithelial
DNA
Different
Chemical
Sperm
DNA
Semen stain
Epithelial DNA
Sperm DNA
Differential Isolation of DNA
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59. The Problem:
How do we identify and detect a specific sequence in
a genome?
• TWO BIG ISSUES:
– There are a LOT of other sequences in a genome that
we’re not interested in detecting. (SPECIFICITY)
– The amount of DNA in samples we’re interested in is
VERY small. (AMPLIFICATION)
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60. The Problem: Specificity
How do we identify and detect a specific sequence in a
genome?
• Pine: 68 billion bp
• Corn: 5.0 billion bp
• Soybean: 1.1 billion bp
• Human: 3.4 billion bp
• Housefly: 900 million bp
• Rice: 400 million bp
• E. coli: 4.6 million bp
• HIV: 9.7 thousand bp
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61.  Molecular Xeroxing of
targeted areas of DNA
determined to contain
information
 Used in:
 research
 diagnostics
 forensics
Polymerase Chain Reaction
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62. What Is PCR?
 In vitro technique
 Amplification of specific DNA
sequence between two regions of
known sequence
 Invented 1985 by Kary Mullis- Nobel
Prize in chemistry in 1993.
 PCR is used to target and replicate any
segment of DNA.
 Copy high numbers (1 trillion) of
target in 2-3 hours.
 A technique that involves repeated
cycles of 3 steps:
 Denature
 Anneal
 Extension
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63. Overall Principle of PCR
DNA – 1 copy
Known sequenceSequence of interestKnown sequence
PCR
Lots of copies
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64. Why PCR?
 To ‘pull the needle out of the haystack’
 Rapid & easy
 Sensitive
 Robust
 Widespread applications
December 25, 2012 65

65. The Cycling Reactions
 Denature: ‘separate D.S DNA’
 Anneal: ‘stick primers to S.S DNA’
 Extend: ‘make new DNA from template’
94 oC
60 oC
72 oC
Time
Temperature
Single Cycle
Typically 25-40 cycles
performed during PCR
94 oC 94 oC 94 oC
60 oC60 oC
72 oC72 oC
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66. Components of Reaction
 Template DNA
Taq
dTTP
dCTP dGTP
dATP
 Primers
 DNA Polymerase
 dNTPs
 MgCl2- required to activate
Taq Polymerase.
 Buffer- salt and pH balanced.
 Water
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67. Denaturation
 Temperature: 92-94C
 Double stranded DNA melts single
stranded DNA
92C
3’5’
3’ 5’
+
5’3’
5’ 3’
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68. Annealing
 Temperature: ~50-70C (dependant on the melting
temperature of the expected duplex)
 Primers bind to their complementary sequences
5’3’
5’ 3’
Forward primer Reverse primer
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69. Extension
 Temperature: ~72C
 Time: 0.5-3min
 DNA polymerase binds to the annealed primers and
extends DNA at the 3’ end of the chain
5’ 3’
Taq
5’
5’
3’
Taq5’
December 25, 2012 70

70. Extension (2)
5’ 3’
5’3’
Taq
Taq
5’
5’
December 25, 2012 71

71. Products of Extension
3’5’
3’ 5’
3’5’
3’ 5’
Taq
Taq
December 25, 2012 72

72. Cycle Begins Again………
3’5’
3’ 5’
3’5’
3’ 5’
December 25, 2012 73

73. Products after 2 cycles
3’5’
3’ 5’
3’5’
3’ 5’
3’5’
3’ 5’
3’5’
3’ 5’
December 25, 2012 74

74. Products after 3 cycles…..
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75. December 25, 2012 76
Multiple PCR amplification
PCR Cycling conditions

76. 2 Cycles
3 Cycles
4 Cycles
5 Cycles
28 Cycles
DNA
December 25, 2012 77
PCR amplification
1 Cycle

77. Amplified DNA
Analysis of amplified DNA
December 25, 2012 78
DNA
Profile
ABI PRISM 310 Genetic Analyzer

78. Sample
Tube
DNA-
-
Electrokinetic Injection Process
Electrode
Capillary
-
The amount of salt in the sample
affects how much DNA migrates
into the capillary.
Amount of DNA is inversely
Proportional to the ionic strength.
December 25, 2012 79

79. DNA Separation Mechanism
+-
DNA-
DNA-
DNA-DNA-
DNA-
• Size based separation due to interaction of DNA molecules with entangled
polymer strands
• “Gel” is not attached to the capillary wall
• Pumpable -- can be replaced after each run
• Polymer length and concentration determine the separation characteristics
December 25, 2012 80

80. Inlet
(cathode)
Outlet
(anode)
Capillary Electrophoresis (CE)
Argon Ion
Laser
Fill with Polymer
Solution
50 m x 47 cm
15 kV
- +Burn capillary
window
Data Acquisition
DNA Separation occurs in
~30 minutes...
December 25, 2012 81

81. Mixture of dye-labeled
PCR products from
multiplex PCR
reaction
Sample
Separation
Sample Detection
CCD Panel (with virtual filters)
Argon ion
LASER
(488 nm)
Color
SeparationFluorescence
ABI Prism
spectrograph
Capillary
Sample
Injection
Size
Separation
Processing with GeneScan/Genotyper software
Sample Interpretation
Figure 13.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
December 25, 2012 82

82. Internal Size Standard
500400300100 200 600
December 25, 2012 83
A sizing standard is used in all samples and allelic ladders. The known
standard is used to determine the size of the allelic ladders and the unknown
samples
Internal size standards serve two purposes:
1. Sizes of all fragments are established for a sample (relative fragment
units). This is accomplished by using the ISS to establish a correlation
coefficient, which is used to determine the size of sample fragments.
2. The ISS serves as an effective control and provides information about
instrument run conditions. For instance, if all of the peaks for the sizing
standard are not present, it suggests a temperature, run time, or injection
problem.

83. December 25, 2012 84
Allele Calls
Comparison of the unknown fragments to the migration pattern of the allelic
ladder allows determination of the genotype. Software programs use macros to
automatically make the comparison.

84. December 25, 2012 85
DNA Profile

85. DNA Testing Requires References
DNA results from one sample are always measured relative to other samples
(called references) examined at the same sites along the DNA molecule.
1. Forensic casework: Evidence compared to suspect(s)
2. Paternity testing: Child compared to alleged father(s)
3. Airplane crash: Victim remains compared to living relative(s)
4. Genetic genealogy: Y-chromosome results compared between two or
more relatives
Without reference samples and a full understanding of the genetic past, no valid
comparisons can be made
No reference samples means no association or match and no reliable
conclusion!
December 25, 2012 86

86. December 25, 2012 87
STR Data Sheet

87. Three Possible Outcomes
 Match – Peaks between the compared STR profiles have the same
genotypes and no unexplainable differences exist between the samples.
Statistical evaluation of the significance of the match is usually
reported with the match report
 Exclusion (Non-match) – The genotype comparison shows profile
differences that can only be explained by the two samples originating
from different sources.
 Inconclusive – The data does not support a conclusion as to whether
the profiles match. This finding might be reported if two analysts
remain in disagreement after review and discussion of the data and it is
felt that insufficient information exists to support any conclusion.
December 25, 2012 88

88. How Statistical Calculations are Made
 Generate data with set(s) of samples from desired
population group(s)
– Generally only 100-150 samples are needed to obtain reliable allele
frequency estimates
 Determine allele frequencies at each locus
 Count number of each allele seen
 Allele frequency information is used to estimate the rarity
of a particular DNA profile
 Homozygotes (p2), Heterozygotes (2pq)
 Product rule used (multiply locus frequency estimates)
For more information, see Chapters 20 and 21 in Forensic DNA Typing, 2nd Edition
December 25, 2012 89

89. December 25, 2012 90
DNA report

90. December 25, 2012 91

91. December 25, 2012
Source: National Institute of Justice
92
Time required for DNA testing
1. Turnaround time in US: 30 days at best, > 1year in
many states.
2. Turnaround time in UK: 23-30 days (goal: 7-14
days).

92. Thank you
for your attention
December 25, 2012 93