By : Marvi Altaf & Group Biology Department Preston University Islamabad

1. Presented by :-
Marvi Altaf Amna Liaqat
Afshan Gohar Mehwish Khalid
Shumaila Raza Namal Khan

2. Introduction
History
Steps involved
Types
Advantages and disadvantages
Conclusion
References
Structure of DNA
DNA fingerprinting 2

3. DNA fingerprinting 3

4.  A technique used especially for identification by extracting
and identifying the base pair pattern of an individual's DNA.
 Also known as
DNA typing ,
Genetic fingerprinting ,
DNA profiling.
DNA fingerprinting 4

5. What is DNA Profiling?
A technique used by scientists to distinguish between
individuals of the same species using only samples of their
DNA. The only difference between people (or any animal) is
the order of the base pairs. The information contained in
DNA is determined primarily by the sequence of letters along
the zipper. The different sequences is the same as the word
"POST" has a different meaning from "STOP" or "POTS,"
even though they use the same letters

6. 1984
Alec
jeffreys
Leicester
university
DNA fingerprinting 6

7.  It was first used in the law courts of England in 1987 to convict
a man in a rape case.
 It has now been used successfully in many crime and paternity
cases in worldwide.
 Although 99.9% of human DNA sequences are the same in
every person, enough of the DNA is different to distinguish one
individual from another, unless they are monozygotic twins.
 DNA profiling uses repetitive sequences that are highly
variable, called variable number tandem repeats (VNTRs),
particularly short tandem repeats (STRs). VNTR loci are very
similar between closely related humans.
 The analysis of variable number of tandem repeats (VNTRs),
to detect the degree of relatedness to another sequence of
oligonucleotides , making them ideal for DNA fingerprinting.

8. DNA fingerprinting 8

9. ( DNA PROFILING )
•Cells broken down to
release DNA
•DNA strands cut into
fragments
•Fragments separated
•Pattern of fragments
analysed

10. Overall Process
http://www.youtube.com/watch?v=drC7rR7CIBg
Video of DNA
Fingerprinting Process

11. DNA fingerprinting 11

12. Isolation of the DNA!
In order for a DNA fingerprint to be made
you must first gather DNA from the person
you are fingerprinting.
There are many things you could use to
gather DNA…
1. Blood
2. Hair
3. Skin
4. Saliva
5. Or any other cell
with DNA in it.
Human Cheek Cells

13. Restriction Enzymes :
1) Used to cut DNA into pieces that are specific to
each individual persons DNA.
Specific Enzymes cut the DNA
into pieces at specific places as
well.
For Example…
• EcoR1 cuts only when the
sequence GAATTC occurs.

14.  The sections of DNA that are cut out are called
restriction fragments.
 This yields thousands of restriction fragments of all
different sizes because the base sequences being cut
may be far apart (long fragment) or close together
(short fragment).

15. 2) Transfer of DNA to nylon:
The distribution of DNA pieces is transferred to a
nylon sheet by placing the sheet on the gel and soaking
them overnight.
3) Probing :
Adding radioactive or colored probes to the nylon
sheet produces a pattern called the DNA fingerprint.
Each probe typically sticks in only one or two specific
places on the nylon sheet.
4) DNA fingerprint :
The final DNA fingerprint is built by using several
probes (5-10 or more) simultaneously. It resembles the
bar codes used by grocery store scanners

17. Types of DNA fingerprinting
methods are…
 Electrophoresis.
 Polymerase chain reaction (PCR).
 Restriction fragment length polymorphism (RFLP).
 Random Amplified Polymorphic DNA(RAPD).
 Amplified fragment length polymorphism (AFLP).

18.  Method to separate DNA or RNA molecules by size.
 Carried out by applying electric field which attracts
negatively charged nucleic acid molecules till the end.
 Shorter ones move faster.
DNA fingerprinting 18

19. Electrophoresis…
Electrophoresis is
a separations technique that
is based on the the mobility
of ions in an electric field.
Positively charged ions
migrate towards a negative
electrode and negatively-
charged ions migrate
toward a positive electrode.
Ions have different
migration rates depending
on their total charge, size,
and shape, and can
therefore be separated.

20. Its purpose is to amplify a lot of double stranded DNA
with same size and sequence by enzymatic method and
cyclic condition.
DNA fingerprinting 20

21.  The polymerase chain reaction (PCR) was developed by Karry
Mullis of the Cetus Corporation in 1983.
 In this process, the DNA sample is denatured into the separate
individual strands.
 Specific DNA primers are used to hybridize to two
corresponding nearby sites on opposite DNA strands in such a
fashion that the normal enzymatic extension of the active
terminal of each primer (that is, the 3’ end) leads toward the
other primer.
 In this fashion, two new copies of the sequence of interest are
generated.

22.  Repeated denaturation,
hybridization, and
extension in this fashion
produce an exponentially
growing number of
copies of the DNA of
interest.
 The PCR analysis
amplified isolated
regions on the strands of
the DNA under
examination.

23.  A procedure allowing DNA in electrophoresis gel
to be transferred to nitrocellulose membrane.
DNA fingerprinting 23

24. Restriction
Fragment
Length
Polymorphis
m
Amplified
Fragment
Length
Polymorphism
Random
Amplification
Polymorphic
DNA
Single
Sequence
Repeats
Inter Simple
Sequence
Repeats
DNA fingerprinting 24

25.  Analyze difference in homologous sequence that can be
detected by the presence of fragments of different length
after digestion of DNA samples.
 Simple language- study of morphology of DNA from
different sources which are digested using restriction
enzymes into fragments.
DNA fingerprinting 25

26.  RFLP analyzes the length of the strands of the DNA
molecules with repeating base pair patterns.
 The basic technique for detecting RFLPs involves
fragmenting a sample of DNA by a restriction enzyme,
which can recognize and cut DNA wherever
a specific short sequence occurs, in a process known as
a restriction digest.
 The resulting DNA fragments are then separated by
length through a process known as agarose gel
electrophoresis, and transferred to a membrane via the
Southern blot procedure.

27. DNA…
 It is a type of PCR reaction, but the segments of DNA that
are amplified at random.
 RAPD creates several arbitrary, short primers (8–12
nucleotides), then proceeds with the PCR using a large
template of genomic DNA, hoping that fragments will
amplify.
 By resolving the resulting patterns, a semi-unique profile
can be gleaned from a RAPD reaction.
 RAPD does not require any specific knowledge of the
DNA sequence of the target organism: the identical 10-mer
primers will or will not amplify a segment of DNA,
depending on positions that are complementary to the
primers' sequence.

28. Amplified fragment length polymorphism…
 This technique was also faster than RFLP analysis
and used PCR to amplify DNA samples. It relied
on variable number tandem repeat (VNTR)
polymorphisms to distinguish various alleles, which
were separated on a polyacrylamide gel.
 By using the PCR analysis to amplify the minisatellite
loci of the human cell, this method proved quicker in
recovery than the RFLP.
 However, due to the use of gel in its analysis phase,
there are issues of bunching of the VTRN's, causing
misidentifications in the process.

29.  Analysis of uniqueness present in the genome of all
eukaryotes.
 A nucleotide repeat sequence such as (dC-dA)n , (dG-
dT)n as many as 50,000 times with 'n' varying from 10-60.
DNA fingerprinting 29

30. SSR working
NNNNNNNN CACACA NNNNNNNNN NNNNNNNN
NNNNNNN GTGTGTGTGTGTG NNNNN CACACA NNNNNNNN
SSR
A
B
Gel
electrophoresis
DNA fingerprinting 30
GTGTGTGT

31.  Analysis of inter simple sequence repeats is carried
out i.e those nucleotides which occurs in between
SSR.
 No. of nucleotides varies from individual to
individual.
DNA fingerprinting 31

32. ISSR working
NNNNNNNN CACACA NNNNNNNNN GTGTGTGT NNNNNNNN
NNNNNNNN GTGTGTGTGTGTG NNNNN CACACA NNNNNNNN
ISSR
A
B
Gel
electrophoresis
DNA fingerprinting 32

33. Advantages and Disadvantages
Advantages Disadvantages
RFLP Paternity test, Criminal test,
Genetic diseases.
Slow and more tedious
process, Required more DNA.
AFLP Positional cloning of gene of
interest, High reproducibility,
Many loci can be analyzed
simultaneously.
Complex procedure, Non-
identical comigrating bands
can cause noise.
RAPD Genetic relation, Genetic
diversity, Analyze mixed
genome samples, Applicable
when limited DNA available,
low expense.
Markers are dominant i.e. they
can’t distinguish whether
DNA sequence amplified is
heterozygous or homozygous.
SSR and ISSR Genotyping, Gene profiling,
DNA fingerprinting
Point mutation on the primer
annealing site can lead to
occurrence of null alleles.
DNA fingerprinting 33

34. Applications…
1) Diagnosis and Developing cures for inherited
disorders :
 DNA fingerprinting is used to diagnose inherited
disorders in both prenatal and newborn babies in
hospitals around the world.
 These disorders may include cystic fibrosis, hemophilia,
Huntington's disease, familial Alzheimer's, sickle cell
anemia, thalassemia, and many others.
 Early detection of such disorders enables the medical
staff to prepare themselves and the parents for proper
treatment of the child.
 In some programs, genetic counselors use DNA
fingerprint information to help prospective parents
understand the risk of having an affected child.

35.  In other programs, prospective parents use DNA
fingerprint information in their decisions concerning
affected pregnancies.
2)Biological Evidence to Identify Criminals:
 Where fingerprints are not available but biological
specimens are available like blood or semen stains, hair,
or items of clothing at the scene of the crime then these
items may prove to be valuable sources of DNA of the
criminal.
 Since the year 1987, innumerable cases have been
solved with the help of DNA fingerprint evidence.

36. ( DNA PROFILING )
Uses Of DNA Fingerprinting:
Medical researchers, forensic biologists,
genealogists and anthropologists all use DNA
fingerprinting to identify individuals or to
determine the relation between individuals or
groups of individuals. Information gained from
DNA fingerprinting can answer questions
concerning the structure and migration of
human populations, establish paternity and
place a suspect at the scene of a crime.

37. ( DNA PROFILING )
Benefits and limits of DNA Fingerprinting:
Benefits of DNA Fingerprinting:
The most important benefit of DNA fingerprinting is that there
is strong similarities shown between genetic fingerprints of
parents and children. This is a benefit because a child's
genetic fingerprint is made up of half the father's genetic
information and half of the mother's information. This means
that the bands of a child's genetic fingerprint will match the
bands on both of their parents, making it possible to establish
paternity and maternity tests.

38. Limitations of DNA Fingerprinting:
An uncontaminated DNA blood
sample taken from a crime scene.
One of the main problems with the process of DNA
fingerprinting is that the sample can be easily
ruined. The tiniest pieces of genetic junk can
contaminate DNA samples, causing them to be
useless. Although DNA fingerprinting requires a
good sample to work with, this problem can be solved
by using the newer technique called PCR. PCR can
use extremely small samples of DNA and produce a
much faster result. But this also means the DNA
samples that PCR uses are even more likely to be
contaminated because of their size, as it is harder to
find a small sample with hardly any
contamination. Another limitation of fingerprinting is
that the procedure is so complex and hard to read the
DNA patterns, that sometimes the juror finds the
evidence almost invisible.

39. ( DNA PROFILING )
DNA Fingerprinting In Plants:
The basic methodology of DNA profiling in plants involve first the extraction
of DNA from plant cells, quantification and quality assessment of extract. The
further steps are of two types,
1) PCR based. - RAPD, ISSR, SSR
2) Non PCR based. – RFLP.
The in the PCR based techniques diluted DNA is mixed with a master mix
comprising the PCR buffer, DNTPS, primer, water and the Taq polymerase
enzyme in a PCR eppendorf tube .The mixture is loaded into the PCR. The
PCR is pre-programmed for appropriate number of cycles and temperature
variations depending on the technique. After required cycles, the samples are
subjected to electrophoresis, either AGE or PAGE, depending on the
technique. The staining is done for revealing the banding pattern.

40. Conclusion:
With a beginning as a mere forensic tool, the world of DNA
fingerprinting has a gone a long way in revealing the genetic
identity of living beings. With massive evolution in concepts
and techniques it has given a lot to various fields cutting across
the spectrum. In plants it has not only helped in identifying
species but also in defining a new realm in plant genomics,
plant breeding and in conserving the biodiversity. With world
paving way for developments in biotechnology, DNA
fingerprinting promises a very powerful tool in our future
endeavors.

41. DNA analysis remains the key to linking suspects to
biological evidence and to identifying individuals in
crimes and disasters.
The establishment of paternity in custody and child
support litigation.
DNA profiling is used to diagnose inherited disorders
and human diseases.
DNA fingerprinting 41

42. [1] DNA Fingerprinting in Plants and Fungi by Kurt Weising, Hilde Nybom,
Markus Pfenninger, Kirsten Wolff, Wieland Meyer
[2] Jeffreys AJ, Wilson V and Thein SL Hypervariable ‘ minisatellite ' regions in
human DNA. Nature 1985 314: 67-73.
[3] SAMPLING TECHNIQUES FOR GENETIC ANALYSIS, Tapir specialist group.
[4] DNA Amplification & PCR, New England Biolabs.inc
[5] Restriction Fragment Length Polymorphism (RFLP), probe, NCBI.
[6] Ovidiu Paun and Peter Schönswetter, Amplified Fragment Length
Polymorphism (AFLP) - an invaluable fingerprinting technique for genomic,
transcriptomic and epigenetic studies, PMC 2012 Dec 3.
[7] Penner GA, Bush A, Wise R, Kim W, Domier L, Kasha K, Laroche A, Scoles
G, Molnar SJ, Fedak G., Reproducibility of random amplified polymorphic DNA
(RAPD) analysis among laboratories, NCBI.
[8] Afaf I. Shehata, Haila A. Al- Ghethar, Ali A. Al- Homaidan, Application of
simple sequence repeat (SSR) markers for molecular diversity and
heterozygosity analysis in maize inbred lines, Saudi Journal of Biological
Sciences Volume 16, Issue 2, October 2009, Pages 57–62, science direct
DNA fingerprinting 42