Forensics Criminal Investigation

1. The Indispensable
Forensic Science
Tool
DNA:

2. APPLICATIONS
FORENSICS - TO LINK A SUSPECT TO A CRIME
SCENE AND FOR CRIMMINAL EXONERATIONS
Colin Pitch Fork, or the OJ Simpson trial
TO IDENTIFY A MISSING OR DEAD PERSON
ID Saddam Hussein and his sons,
or when we find Osama Bin Ladin
TO LINK A SUSPECT TO AN EVENT
Clinton – Lewinsky Scandal

3. Why is DNA an Indispensable
Tool Forensic Science?
Study into the structure of the human
genome has led to the discovery that
portions of the DNA structure of
certain genes are as unique to each
individual as fingerprints

4. What is DNA?
Each of our 60 trillion cells
contains nuclear genetic material
called chromosomes
Arranged on these chromosomes are
genes
The gene is the fundamental unit of
heredity
Instructs the body cells to make
proteins that determine everything
from hair color to our susceptibility to
diseases.
Each gene is composed of DNA coded
with a specific sequence designed to
carry out a specific function.

5. What is DNA?
Polymer – repeating
monomers of
nucleotides.
DNA nucleotides are
composed of a
deoxyribose sugar
molecule, a
phosphorous-
containing group, and
a nitrogen-containing
molecule called a
base.

6. James Watson & Francis Crick
1953 discovered the structure of
Deoxyribonucleic Acid – DNA
Double Helix
Composed of 4 different type bases
complementary base pairing of Adenine
always with Thymine and Guanine with
Cytosine.
No restriction on how many bases are to
be sequenced on a strand of DNA.

7. Nitrogenous bases
are always paired
A-T and C-G
Held together by
weak hydrogen
bonds

8. The backbone of the
DNA chain is
supported by the
phosphorous and
sugar groups –
forming the rungs of
the “ladder”

9. A T A G C C A A C C A A
T A T C G G T T G G T T
Replication of a single strand of DNA

10. DNA at Work
DNA controls the production of Proteins
Proteins are polymers of amino acids
There are 20 known amino acids yet thousands of
different proteins can be produced performing
different functions.
The amino acids act like the letters in the alphabet
(26 letters in different combinations = thousands
of words) so 20 different amino acids in different
sequences equal about 100,000 different proteins
each with its own unique shape which determines
its function.

11. How can the DNA sequence
dictate the sequence for a
particular protein?
The template DNA is transcribed in the
nucleus by RNA (Ribonucleic Acid)
Thymine on the DNA nucleotide is
replaced by Uracil on the RNA
nucleotide so A is paired with U.
Each amino acid is attached to a triplet
complementary codon of a RNA
transcribed DNA sequence.

12. U A U C G G U U G G U U
A T A G C C A A C C A A
So, a DNA Sequence of:
Would have an mRNA sequence of:
Use the following table to determine which of the
20 amino acids would make up this sequence.
Each group of three nucleotide bases, an
RNA triplet codon, is read in order to
determine which amino acid is placed in the
sequence. The sequence determines the type
of protein that is produced.
U A U C G G U U G G U U
DNA template Strand
RNA Strand of Transcribed DNA

13. U A U
C G G
U U G
G U U

14. U A U C G G U U G G U U
mRNA Sequence:
Tyrosine Arginine Leucine Valine
Would result in this protein amino acid sequence

15. U A U C G G U U C G U U
a one base change in the mRNA Sequence:
Tyrosine Arginine Phenylalaine Valine
Would result in this protein amino acid sequence
U A U C G G U U G G U U

16. Does all my DNA code for
proteins?
99.9% of all human DNA is the same
Exons – makes up about 5% of human DNA
Codes for Proteins
Introns – makes up about 95% of human DNA.
Filler or “junk” portion of the DNA sequence.
Variaitions within intron DNA accounts for the .1%
difference between each of us and is used in DNA
fingerprinting.

17. EARLY DNA FINGERPRINTING
TECHNIQUE
Restriction Fragment
Length Polymorphisms
– RFLP “RiFLiPs”
1980 – Ray White
Describes first
Polymorphic RFLP
Genetic Marker.

18. Restriction Fragment Length
Polymorphism
DNA from several individual was
digested (cut) with a restriction
enzyme.
Detected by Southern Blot analysis.
Different banding patterns were seen
for different individuals. 1984
Sir Alec Jeffreys developed and used
multi-locus RFLPs to catch Colin
Pitchfork.

19. RFLP/ Southern
blot analysis

20. Size of the fragments cut as a result of the restriction enzyme will
influence the distance the fragments will travel within the gel during
electrophoresis. Smaller fragments will travel further than large
fragments.
small
fragments
Large
fragments

21. RFLPS/SOUTHERN GEL
DISADVANTAGES
It is expensive, time, and labor intensive with
a low throughput.
One locus is analyzed at a time.
Less sensitive – Requires a large sample size.
It is limited in obtaining DNA from a crime
scene since you need enough blood, hair, or
semen.
It requires whole genome analysis.
Needs relatively recently collected samples as the
DNA tends to degrade.

22. More than 30% of the human genome contains
repetitive DNA nucleotide sequences.
These repeat sequences or tandem repeats
vary in number between individuals and can be
inherited from both parents
Variable Number Tandem Repeats - VNTRs
15 to 35 nucleotide bases per repeat
Up to 1000 repeats
Results in DNA segments of thousands of bases in length
Short Tandem Repeats - STRs
1-7 nucleotide bases per repeat
Results in DNA segments of about 400 bases in length
Shorter segments make it less susceptible to degredation

23. VNTRs with RFLPs
SOUTHERN GEL ANALYSIS
By digesting genomic DNA with restriction enzymes
that cuts outside of these repeats it is possible to
obtain different multiples of repeats that can be
detected by hybridizing a DNA probe that detects
(hybridizes to) the single repeat.
Number of
repeats
influences the
length of the cut
fragments after
exposure to the
restriction
enzymes

24. DNA FINGERPRINT EXAMPLE
VNTR WITH SINGLE LOCUS
REPEAT
NUMBER
8
6
3
2
POSSIBLE CHILDREN
ADOPTED
CHILD
F M
5
1 2 3 4
DID CHILD 4
DEVELOP A NEW
ALLELE FOR 7
REPEATS?
IS LANE 4
FROM AN
UNRELATED
PERSON WITH
A SIMILAR
ALLELE TO
THE MOTHER?
ANAYLZE MORE
LOCI
SIMULTANEOUSLY

25. VNTR/RFLP MULTILOCUS
DNA FINGERPRINTING
Some VNTR sequence segments
are found at only a single locus
in the human genome. Probes
made of these sequences are
single locus probes and yield
this pattern when used to probe
RFLP blots of DNAs
Other VNTR sequence
segments occur at many loci in
the human genome. These loci
are dispersed among the
chromosomes. Multi-locus
VNTR probes yield patterns
like bar codes
YOU STILL NEED THE
WHOLE GENOME FOR
RFLP ANALYSIS

26. VNTRs/ RFLPS
SOUTHERN GEL ANALYSIS
One of the drawbacks of
VNTRs with Southern gel
analysis was that they
required relatively large
amounts of DNA and was
very time consuming
PCR changed everything due
to the ability to amplify a
locus in a short period of
time

27. Short Tandem Repeats (STRs)
PCR Analysis
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
7 repeats
8 repeats
AATG
THE FORENSIC
COMMUNITY
SETTLED ON
TETRANUCLEOTIDE
REPEAT

29. What is PCR?
Polymerase Chain Reaction
A technique used to amplify (copy or multiply) DNA
Can be done with minute amounts of DNA
Uses DNA’s natural replication by DNA polymerase
DNA is heated (denatured) to separate the strands of DNA
primers (short strands of DNA target specific regions of
DNA for replication) hybridize (anneal) with the separated
strands of DNA
DNA polymerase directs the rebuilding of the double-
stranded DNA molecule using a mixture of free nucleotides
PCR Tutorial
PCR animation

33. RFLPs have provided valuable information in
many areas of biology, including:
screening human DNA for the presence of
potentially deleterious genes
Uses of STR and RFLP

34. providing
evidence to
establish the
innocence of,
or a probability
of the guilt of,
a crime suspect
by DNA
"fingerprinting“

35. Paternity
DNA fingerprinting can be used to
identify a child’s parents. Each
child inherits one set of
chromosomes from each parent.
Some RFLPs are inherited from the
mother and some from the father.

36. Interpret the results of the
following DNA fingerprints
Both daughter 1 and
son 1 share RFLPs
with both the mom
and dad, while
daughter 2 has
RFLPs of the mom
but not the dad, and
son 2 does not have
RFLPs from either
parent.

37. The police use the same
analysis to determine
the identity of a person
at a crime scene. After
collecting DNA samples
from the scene and any
suspects, the police
amplify and digest the
DNA with a restriction
enzyme. The samples
are run on an agarose
gel, and the bands found
at the crime scene are
aligned with those of
the suspects’.

38. The DNA
fingerprint from
suspect 1
matches up with
the fingerprint
of the sperm
DNA from the
crime scene. You
can also see that
the female cells
from the scene
match the
victim’s DNA.

39. Capillary Electrophoresis
The preferred technique of characterization of STRs
Preformed in a thin glass column (coated with a gel
polymer) immersed in a reservoir of buffer liquid with
platinum coated high-voltage electrodes
DNA sample solution is injected into one end of the
column.
DNA will migrate under the influence of an electrical
potential at a speed that is related to the STR
fragment lengths.
The opposite side of the column is connected to a
laser beam detector that tracks the separated STRs
as they emerge from the column.
This is sensitive enough to detect fragments that are
different by only 1-4 bases in length.

43. D3S1358 VWA FGA
AMEL
D8S1179
D21S11
D18S51
D5S818 D13S317 D7S820
Profiler Plus Allelic Ladders

44. D3S1358 D16S539
AMEL
TH01
TPOX CSF1PO
D7S820
COfiler Allelic Ladders

45. What are Y-STRs and how are they
useful to the forensic scientist?
Short tandem repeats located on the human Y
chromosome
Originates only from a male donor of DNA
20 different Y-STR markers
Running a Y-STR profile is useful when
multiple males are involved in a sexual assault.
Yields only one band or peak for each STR type
Simplifies the appearance and interpretation of
the DNA profile

47. What is mitochondrial DNA and how is
it used in forensic analysis?
Mitochondria are the site of cellular
respiration in all our cells (energy producers).
Many mitochondria are found in the cytoplasm
of each cell.
Can self replicate – has its own looped DNA
mtDNA
Inherited only from the mother
mtDNA analysis is more sensitive then nuclear
DNA but is more rigorous, time consuming,
and costly.

48. What is mitochondrial DNA and how is
it used in forensic analysis?
Two regions of mtDNA have been found to be highly
variable in the human population – HV1 and HV2.
Analysis requires:
PCR
Sequencing the hyper-variable regions
FBI maintains a database containing the base
sequences from HV1 and HV2 – Currently contains
about 5,000 sequences which can be used to
determine how common or rare a specific sequence is
compared to the current database.
Not as discriminated as nuclear DNA analysis
Best used when there is no nuclear DNA available for
testing

49. What is CODIS ?
CODIS is an acronym for
Combined DNA Index
System
Essentially administered
by the FBI – provides
CODIS training and
software

50. CODIS Alleles
13 CODIS Alleles
STRs of
tetrameric
repeats
Amelogenin Gene
– determines
gender

51. DNA Profile Frequency Calculations
Genotype Probability at any STR Locus
Forensic DNA analysis requires population databases
for the STR loci evaluated.
Probability calculations are based on knowing allele
frequencies for each STR locus for a representative
human population.
Allele frequency – the number of copies of the allele
in a population divided by the sum of all alleles in a
population.

52. For a heterozygous individual, if the two alleles have frequencies
of p and q in a population, the probability (P) of an individual of
having both alleles at a single locus is
P = 2pq
If an individual is homozygous for an allele with a frequency of p,
the probability (P) of the genotype is
P = p2.
Individual A has the genotype 15, 18 at the locus D3S1358. In a
reference database of 200 U.S. Caucasians, the frequency of the
alleles 15 and 18 was 0.2825 and 0.1450, respectively. The
frequency of the 15, 18 genotype is therefore
P = 2 (0.2825) (0.1450) = .0819, or 8.2%.
DNA Profile Frequency Calculations
Genotype Probability at any STR Locus

53. MULTIPEX STR
SYSTEM
The simultaneous
extraction and
amplification
(copying) of a
combination of
different STRs

54. Since the CODIS Loci are predominantly
on separate chromosomes (only 2 are
found on chromosome 5 at opposite
ends), it insures that the probability of any
two bands being inherited together is the
product of individual occurrences i.e.
independent assortment.
This aspect strengthens the significance
of probability of occurrence for court
cases.

55. Probability for a DNA profile of Multiple
Loci
If databases of allele frequency for different loci can be shown
to be independently inherited by appropriate statistical tests,
the probability for the combined genotype can be determined by
the multiplication (product rule).
The probability (P) for a DNA profile is the product of the
probability (P1, P2, ... Pn) for each individual locus, i.e.
Profile Probability = (P1) (P2) ... (Pn)
When all 13 CODIS STR markers are included in the DNA
profile, the probability can result in extremely low numbers.
1.3 X (1016), or no more frequent than 1 in 7.7 quadrillion
individuals (7.7 million billion), which is more than a million
times the population of the planet.

56. Sample Obtained
from Crime Scene
or Paternity
Investigation
Steps in DNA Sample Processing
Biology
DNA
Extraction
DNA
Quantitation
PCR Amplification
of Multiple STR markers
Technology
Separation and Detection of
PCR Products
(STR Alleles)
Sample Genotype
Determination
Genetics
Comparison of Sample
Genotype to Other
Sample Results
If match occurs, comparison
of DNA profile to population
databases
Generation of Case
Report with Probability
of Random Match

57. Allele Frequencies
STRBase