Applications in forensics PCR and DNA fingerprinting.pptx

1. 30/11/23
Dr. U. Venkateswara Prasad MSc.,
PhD.
Assistant Professor
School of Life Science
B.S.Abdur Rahman University,

2. 2

3. What is Forensic Science?
• Forensic science (or forensics) is the
application of scientific knowledge to
answer questions of interest within
the legal system.
• Evidence from accidents and crime
scenes is collected and analyzed by
forensic scientists.
• Forensic scientists act as expert
witnesses.

4. Forensic Scientists Use Techniques
from Many Scientific Disciplines
Southern Blotting
Polymerase Chain
Reaction (PCR)
Blood Typing
• Anthropology – Analyze remains in advanced
stages of decomposition
• Genetics/Molecular Biology – Uses biological
material to determine serological and DNA
profiles
• Toxicology – Identifies poisons, drugs and/or
alcohol in tissues and body fluids.
• Chemistry – Analysis of trace physical evidence
• Pathology – Determine cause of death post-
mortem

5. Forensic Science Lessons Engage Critical
Thinking and Literacy Skills
• Read witness statements
and police report
• Analyze evidence using
forensic science techniques
• Write a persuasive essay
using the evidence from the
written documents and the
biological and physical tests.

6. DNA Sequences are Unique
• The chances of two individuals having exactly the same
DNA profile is ~30,000 million to one
• Single Nucleotide Polymorphisms (SNPs)
• Restriction Fragment Length Polymorphisms (RFLPs)
• DNA profiling is a forensic technique used to identify
individuals by analyzing differences within DNA.
CODIS –
Combined
DNA Index
System

7. A technique used by scientists to distinguish between individuals of the same
species using only samples of their DNA.
DNA fragments show unique patterns from one person to the next.
Used in paternity disputes and as forensic evidence.
Historic Examples:
1800 Photographs
Early 1900s: fingerprints, Palm prints retinal scan
Foot wear and tire imprssions, Ear, lips etc.
1985: United KingdomThe process of DNA fingerprinting was
invented by-Sir Alec Jeffreys , Dr. peter Gill Dr. Dave Werrett
at the University of Leicester in 1985.

8. DNA is Isolated from
Crime Scene Samples
• Cell membranes are disrupted to
release its intracellular
components.
• The resulting “lysate” consists of
cytoplasm, metabolites, DNA, RNA,
proteins and organelles.
• Want to learn more about forensic
blood testing?

9. • Chemical Structure of DNA is always the same.
• Important: The order of the base pairs is different!
• 3 Billion Nucleotides form our 46 chromosomes.
• In fact, no two people on Earth have exactly the same genetic
sequence, except identical twins.
• Important: Every cell contains a copy of our DNA!

10. • VNTR(variable number of tandem repeats)- recognition that variable
numbers of repeated nucleotides can be found in DNA and can be
used for identification of individuals.
• VNTR’s provide a scientific marker for identity.
How does it Work?
• DNA Fingerprinting using VNTR's
• Cut out the segment of the chromosome containing this variable number
of tandem repeats (VNTR's ).
• Identify the VNTR's for the DNA sequence of the repeat.

11. Isolation of DNA
• DNA must be recovered from the cells or tissues of the
body.
• Blood
• Hair
• Saliva
• Semen
• DNA samples have been obtained from vaginal cells
transferred to the outside of a condom during sexual
intercourse.
• Only a small amount of tissue is needed. For example,
the amount of DNA found in one drop of blood or in one
hair root is enough.

12. Stages of DNA Fingerprinting
• Step 1:
• Cells are broken down to release DNA.
• If only a small amount of DNA is
available it can be amplified using the
polymerase chain reaction (PCR).

13. RFLP – Restriction Fragment Length Polymorphism
• More accurate than PCR
• Requires a large sample of fresh DNA
• Takes longer time & costly than PCR
• When a match if found, there is no question as to whether the
suspect was at the scene.
PCR – Polymerase Chain Reaction
• Takes less time, less costly
• Requires only a small sample
• That sample is amplified using polymerases to force the small amounts
of DNA to copy themselves many times.
• Can be performed on older samples
• If no match is found, the donor was not at the scenes

14. PCR Amplifies Specific
DNA Sequences
• Denaturation (94°C) Double-
stranded DNA is “unzipped” into
single strands.
• Annealing (40-65°C) Primers base
pair with the target DNA sequence.
• Extension (72°C) Taq
polymerase extends the primer and
synthesizes a new strand of DNA.

15. RFLPs Are Used as Landmarks for
DNA Fingerprinting
• Restriction Fragment Length Polymorphisms (or RFLPs) are
heritable differences in the nucleotide sequence.
• Some RFLPS add a restriction enzyme cut site to the DNA.

16. Electrophoresis
DNA fragments are injected into
wells and an electric current is
applied along the gel.
DNA is negatively charged so it is
attracted to the positive end of the gel.
The shorter DNA fragments move faster
than the longer fragments.
DNA is separated on basis of size.

17. Step 2: Cutting, Sizing, and Sorting
Special restriction enzymes are used to cut the DNA
at specific places.
The DNA pieces are sorted according to size by a
technique called electrophoresis. The DNA pieces are
passed through a gel made from seaweed agarose.

18. RFLPs Are Used as Landmarks for
DNA Fingerprinting
• Variable Number of
Tandem Repeats (or
VNTRs) comprise short,
repetitive DNA sequences
present in multiple copies
between two restriction
sites
• 15-35 base pair sequences
• VNTRs appear between five
and 100 times.
Cat. # 334

19. DNA Fingerprinting is
Used to Solve Crimes
• Forensic scientists
identified a biological
sample at the scene of a
crime.
• DNA was extracted from
the sample, amplified by
PCR and digested with
restriction endonucleases
to create a DNA
Fingerprint.
• A match suggests that the
suspect was at the crime
scene.
Cat. # 109
Cat. #130

20. 1. Paternity Test
• By comparing the
DNA profile of a
mother and her child it
is possible to identify
DNA fragments in the
child which are absent
from the mother and
must therefore have
been inherited from the
biological father.
Application’s

21. 2. DNA Profiling can solve crimes
• The pattern of the DNA profile is
then compared with those of the
victim and the suspect.
• If the profile matches the suspect it
provides strong evidence that the
suspect was present at the crime
scene.
• If the profile doesn’t match the
suspect then that suspect may be
eliminated from the enquiry.

22. PCR
• Reaction requirements
• Template DNA – total genomic DNA isolated from an
organism that contains a target region to be amplified
• DNA primers - Short pieces of single-stranded DNA that
flank the target
• Taq DNA polymerase - Attaches nucleotides on the
growing strand of DNA
• Nucleotides (GATC) – Polymerase adds complementary
nucleotides to the template

23. PCR
• Reactions are placed in a machine called a thermal cycler.
• The machine cycles through three temperatures.

24. PCR
• Heat samples to 94°C for a minute or so to denature the
double stranded template DNA.

25. PCR
• The thermal cycler repeats the denaturing, annealing,
and elongating temperatures approximately 30 times.

26. PCR
• PCR amplification is logarithmic, meaning the number of
copies of the target is doubled every cycle.

27. Famous Cases
• Colin Pitchfork was the first criminal caught based on DNA
fingerprinting evidence.
• He was arrested in 1986 for the rape and murder of two girls.
• O.J. Simpson was cleared of a double murder charge in 1994
which relied heavily on DNA evidence.
• This case highlighted lab difficulties.
• In 2002 Elizabeth Hurley used DNA profiling to prove that Steve
Bing was the father of her child Damien.

28. 3. Personal Identification
• The notion of using DNA fingerprints as a sort of
genetic bar code to identify individuals has been
discussed.
• Diagnose inherited disorders in both prenatal and new
born babies.
• By studying the DNA fingerprints of relatives who
have a history of some particular disorder.
• Identify DNA patterns associated with the disease.
• These disorders may include hemophilia, Alzheimer's,
sickle cell anemia, thalassemia, and many others.
4.Diagnosis of Inherited Disorders

29. Drawbacks
•Human Error
– Sneezes, improper storage, failure to label samples
•Chain of Custody
–Collection of evidence must be systematically
recorded and access to evidence must be controlled.
•Collecting samples
–High temperatures can degrade DNA
–Bacteria
–Moisture

30. References
 Russel, P.J. 1998 Genetics (5th edition).
 Chawla, H.S.2005 Introduction to Plant Biotechnology(2nd edition)
 Singh, B.D. 2005 Biotechnology Expanding Horizons
 http://www.biologycorner.com/bio4/notes/DNA_fingerprint.
 http://www.scq.ubc.ca/a-brief-tour-of-dna-fngerprinting/
 http://evolution.berkeley.edu/evolibrary/news/060301_crime

31. Thank you
Mailid:bioprasad.uppu666@gmail.com