DNA fingerprinting is a technique that analyzes variations in DNA sequences at specific locations in the genome to identify individuals. There are two main methods: RFLP (restriction fragment length polymorphism) and PCR (polymerase chain reaction). RFLP involves digesting DNA with restriction enzymes, separating fragments by size, and detecting with probes. PCR amplifies specific DNA regions defined by primer sequences. Short tandem repeats (STRs) are now commonly analyzed by PCR. DNA fingerprinting is used in criminal investigations to identify suspects or victims, and in resolving medical issues like paternity disputes. DNA databases help law enforcement match crime scene evidence to suspects.

1. Dr. Saikat Saha
1st year PGT
Dept. of Forensic Medicine

2. DEFINITION
 Is a technique
 Chemically dividing DNA into fragments which form a unique
pattern
 Matching that identity profile with the obtained from similarly
testing a suspect’s blood samples

3. INTRODUCTION
The Cell-
 Smallest unit of life
 Compose all living thing
 The nucleus(one of the
many organelles) contains
genetic information that the
cell needs to survive and
reproduce.

4. THE CHROMOSOME
 Bodies genetic material
contained within it.
 23 pairs of chromosome in
each somatic cell.
 23 are from biological
mother and 23 from
biological father.

5. THE DNA
 Each chromosome are made
up of DNA or
Deoxyribonucliec Acid.
 DNA is present as double
helix structure within arm of
chromosome, looks like
spiral staircase.
 DNA is polymer of repeating
unit called nucleotide.

6. NUCLEOTIDE
 Each nucleotide is composed
of phosphate, deoxyribose
sugar, and organic nitrogen
base.
PHOSPHATE SUGAR
ADENINE GUANINE CYTOSINE THYMINE

7.  A single DNA molecule contain 50
to 500 million base pairs
 The bases are adenine(A),
guanine(G), cytosine(C), and
thymine(T).
 Bases of one strand connected to
the bases of other strand by
hydrogen bond s, and nucleotides
are linked with each other by
covalent bonds.

8.  Adenine combines only with
thymine by 2 hydrogen
bonds.
 Guanine combines with
cytosine by 3 hydrogen
bonds.
 The base sequence of one
strand is always
complementary to sequence
of the other.

9. GENE
 Each segment of DNA in a
chromosome which codes
for a particular protein is
called as a gene.
 There are 10000 genes in
human genome.
 5% of entire cellular DNA is
responsible for it, those are
called active base pairs.

10. JUNK DNA
 Rest 95% of inactive base
pairs are considered as junk
DNA..

11. VARIABLE NUMBER OF TANDEM REPEATS
 In junk DNA short sequences of
base repeat themselves over
again and again.
 The regions containing
repetitive base pairs are
demonstrating hypervariability
from person to person.
 These variants are called
variable number of tandem
repeats(VNTR) or minisatellite.
 9-100 bp in length.

12. METHOD OF DNA FINGERPRINTING
 At first DNA is need to be extracted from samples. For
extraction-
 Disruption of cells and fragmentation of cellular organelles
 Dissociation of DNA from protein by salt solution
 Addition of an extractant to phase separate the DNA.
 Differential precipitation to remove RNA and polysaccharides.
 Isolated DNA is quantitated in ultraviolate spectrometry.

13.  There are two methods of fingerprinting –
 RFLP OR Restriction fragment length polymorphism
 PCR or Polymerase chain reaction method

14. RFLP METHOD
 The process of RFLP
fingerprinting was invented
by Alec Jeffreys at the
University of Leicester in
1985.
 He was knighted in 1994.

15.  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 and was sentenced in
1988.

16. RFLP METHOD
 The isolated DNA is
completely digested with
restriction endonucleases
 This enzymes recognize
specific sequence in DNA
and cut it at this site into
various fragments.
 Called as restriction
fragment length
polymorphism.
Restriction site
FRAGMENT1 FRAGMENT2

17. RESTRICTION ENZYMES

18.  Digested DNA run on
agarose gel electrophoresis
 The different restriction
fragments are separated
varying in length between .5
to 25 kb.

19.  From the agarose gel DNA is
transferred to the nylon
membrane using Southern
blot technique.
 Addition of a probe to the
nylon membrane, tagged
with a radioactive marker
such as P32.

20.  The membrane is then
wrapped with saran wrap
and placed in a x-ray
cassette holder along with x-
ray film, and kept at 80
degree C.
 X-ray film is then washed
and fixed in reagent, then
washed and dried.
 The series of band seen on
the film.

21. TYPES OF RFLP
 Multi-locus RFLP
 Single locus RFLP
 VNTR sequences.

22. POLYMERASE CHAIN REACTION
 Dr. Karry Mullis invented this
technique in 1983.
 He got noble prize for this
famous invention.

23. • Technique devised to amplify small amounts of DNA
•
• Can be performed on DNA from a single cell
 cigarette butt, a licked stamp, root of a single hair, 1/50,000 a
drop of blood (0.1 microliters)

24.  Steps Involved:
 Isolate repeating loci from
person’s DNA using
restriction enzymes
 Design primers – short
segments of synthetic DNA
that are complementary to
DNA on either side of the
VNTR regions

25.  Add vast excess of the
primers and heat mixture
to 75 oC. This causes DNA
strands to separate by
breaking hydrogen bonds
between bases

26.  Cool to 15 oC. Primers hydrogen bond (anneal) to
complementary strands
 Add DNA polymerase and all four types of nucleotides. The
polymerase (enzyme used in DNA replication) will fill in the rest
of the two strands
 Now have two identical copies of the DNA you started with.

27. DIFFERENCE BETWEEN PCR AND RFLP
PCR RFLP
 Requires small amount of DNA
sample.
 Chances of degradation is less.
 Tests are faster and extremely
sensitive.
 Useful in decomposed
samples.
 More sensitive to
contamination.
 Requires large amount of DNA
sample.
 Sample degradation is
accelerated in warm moist
condition.
 Test are less sensitive.
 Not useful in decomposed
samples.
 Less sensitive to
contamination.

28. SHORT TANDEM REPEAT (STR) ANALYSIS
 Short repeated DNA
sequence.
 3 TO 5 base pairs in length.
 STR loci are targeted with
sequence-specific primers
and are amplified using PCR.

29. ADVANTAGE OF STR ANALYSIS
Advantages –
 this type of approach allows for somewhat 'mistake-proof'
information to be gathered even in poor conditions
 Another benefit of this type of analysis is that it can be
successfully used to monitor patients following transplant
therapy.

30. MITOCHONDRIAL DNA ANALYSIS
 Mitochondrial DNA (mtDNA
or mDNA) is the DNA
located in organelles called
mitochondria.
 All mothers have the same
mtDNA as their children,
because mitochondria of
each embryo comes from
mothers egg cell.

31. ADVANTAGES OF mtDNA ANALYSIS
 Older biological samples that lack nucleated cellular DNA such
as hair, bones and teeth can analyzed by this method.
 There are many copies of mitochondrial DNA in cell, while only
1 to 2 copies of nuclear DNA.
 It is useful in highly degraded samples.

32. DISADVANTAGES OF mtDNA ANALYSIS
• All people of same maternal line will be indistinguishable (less
discriminatory)
• More work, more time consuming, more costly

33. Y CHROMOSOME ANALYSIS
 The y chromosome passes
directly from father to
son.
 So the analysis of y
chromosome is useful in
proving relationship
among males.

34. ADVANAGES OF Y CHROMOSOME ANALYSIS
 More rapid and can be done in 2 to 3 days.
 It can be performed in small amount of DNA

35. COLLECTION OF SAMPLES
1. Liquid blood-
 In cases of paternity,
maternity disputes,
biological relations.
 2 to 5 ml of drawn blood
collected in leak proof
screw capped tubes
contained heparine, EDTA.
 Sealed and labeled.
 Blood soaked dried gauze
may be used.

36. 2.semen, vaginal swab-
 Sterile cotton ear buds are
used to take swabs.
 Air dried and placed in a
sterile tube.
 Sealed and labeled with
necessary information.

37. 3.saliva-
 Saliva in liquid state or stained area as much as possible should
be sent in dried condition.
4.Stains from scene of crime
 Swabbed with sterile cotton swab, air dried and placed in a
clean bottle and sent to the lab at room temperature.

38. 5.Urine –
 Urine about 10 ml should be freezed
 Stain as available should be sent in a dried condition.
6.Hair –
 10 to 20 in number.
 Picked up with forceps without damaging the root.

39. 7.Visceral samples-
 In case of mutilated bodies.
 Sample of 100 gm of muscles in normal saline or in 20% DMSO
saturated with NaCl.
 In case of foetus, placenta should be removed.
 Sent in normal saline or DMSO.
 Jar should be placed in thermocole box with ice.

40. 8.Bones and teeth-
 Femur and humerus are preferred.
 Molar teeth from upper and lower jaw, if molar teeth not
available other teeth may be sent.
9.Fingernail scrapings

41. SAMPLE PRESERVATION
 Freezing is the simplest procedure.
 For long storage -7o degree upto 5 weeks.
 Storage in ice upto 5 days.
 Tissue samples wrapped in a aluminum foil, placed in a plastic
bags and frozen.

42. APPLICATION
DNA profiling is used to
solve
 crimes and
 medical problems

43. CRIMINAL CASES
 Murder-
 Blood on a weapon can be
matched with blood of the
victim
 Victims blood in clothing of
accused
 Sexual crime-
 The seminal DNA obtained
from the vaginal aspirants or
swab.

44. DNA DATABASE
 Combined DNA Index
System (CODIS) is the
generic term used to
describe the FBI’s program
of support for criminal
justice DNA databases as
well as the software used to
run these databases.
 United Kingdom National
DNA Database is NDNAD

45. INDIAN PICTURE
 In 2007 the Draft Human DNA Profiling Bill was piloted by
the Centre for DNA Fingerprinting and Diagnostics.
 In February 2012 another draft of the Bill was leaked by
the Department of Biotechnology.
 Another working draft of the Bill was created in April
2012.

46.  In a first, the Indian Air Force
has initiated a project for
DNA profiling of its
personnel, selecting a high-
risk group of aircrew that
undertakes dangerous
missions for the first round.
A database of the DNA
records will be established in
Pune.
Uttarakhand floods: On rescue
mission,IAF’s Mi-17 helicopter
crashes,20 dead

47. MEDICAL CASES
 Paternity disputes-
 In paternity disputes
mother, child and alleged
father DNA is printed.
 Disputed maternity.
 Mutilated bodies in mass
disaster.
 Exchange of newborn in
hospitals.

48. EXAMPLES
 In 2002 Elizabeth Hurley
used DNA profiling to prove
that Steve Bing was the
father
of her child Damien

50. THANK YOU