AFU Chitwan Rampur Nepal

1. DNA FINGERPRINTING
(DNA PROFILING)

2. Table Of Content
1. Introduction of DNA Fingerprinting.
2. Stages involved.
3. Principle of DNA Fingerprinting.
4. Importance of DNA Fingerprinting.
5. Diagram.
6. DNA Profiling Process.
7. Applications of DNA Fingerprinting.
8. Advantages and Disadvantages of DNA Fingerprinting.
9. Uses of DNA Fingerprinting.
10. Benefits and limits of DNA Fingerprinting.
11. DNA Fingerprinting in plants.
12. Uses of DNA Fingerprinting in Forensic Science
13. Centers for DNA Fingerprinting.
14. Conclusion.

3. Introduction of DNA Fingerprinting:
• 1980 - American researchers
discovered non-coding regions of
DNA
• 1984 - Professor Alec Jeffreys
developed the process of DNA
profiling
• 1987 - First conviction based on
DNA evidence

4. • A process or technique of analysis
• revealing unique patterns of an individual’s DNA
• involving non-coding regions
Dna profiling

5. DNA FINGERPRINTING
( DNA PROFILING )
STAGES INVOLVED:
•Cells broken down to
release DNA
•DNA strands cut into
fragments
•Fragments separated
•Pattern of fragments
analysed

6. 1. DNA EXTRACTION

7. DNA FINGERPRINTING
2. DNA CUTTING 3. FRAGMENT
SEPARATION
The samples containing the fragments
are pipetted into individual wells in a gel

8. DNA FINGERPRINTING
3. ELECTROPHORESIS
•Fragments separated by
length
•DNA (negatively charged)
•Moves towards +ve
terminal
•Shorter fragments move
faster

9. DNA FINGERPRINTING
4.DNA TRANSFER
•DNA split into single strands
using alkaline solution
•DNA fragments transferred
from gel to filter paper or
nylon membrane
• (This is called Southern
blotting)
•Gel, with filter paper
attached, is removed &
separated

10. Radioactive probe in solution
binds to DNA
Revealing a pattern of bands
X-ray film
5. Analysis

11. DNA FINGERPRINTING
( DNA PROFILING )
Principle of DNA Fingerprinting:
•Base pairing of AT (AU) & GC is the BASIC PRINCIPLE of this procedure.
• Specificity of enzyme activity is the second CRUCIAL principle to understanding DNA
fingerprinting. This refers to the cutting of DNA by specific RESTRICTION ENZYMES at
UNIQUE palindrome sequences.
•The recognition that a CHANGE in a SINGLE BASE PAIR (mutation) can either MAKE
a restriction enzyme-site where one did not exist previously or it can REMOVE or
ELIMINATE a restriction enzyme site from a gene. An analogy would be to "mutate" your
phone number by one letter; callers would get a different person.

12. Importance of DNA Fingerprinting:
1. Lineage
DNA fingerprinting can be used to figure out if two people are related which aids
tremendulously in maternity or paternity tests to show that a person is a mother or father to
a specific child. DNA fingerprinting can also be used to trace heritage back for
generations. This means that not only can you figure out who your mother and father are,
but also siblings, aunts, uncles, grandparents, great grandparents, and even further back.
DNA fingerprinting has been able to show relations to people today with bone remnants of
people that have been dead for centuries, when bones have been preserved.

13. DNA FINGERPRINTING
( DNA PROFILING )
2. Law Enforcement
DNA fingerprinting has aided in law enforcement since its discovery. Forensics teams can
analyze DNA found at a crime scene, whether it be blood, hair, semen, or skin particles, and
compare it with DNA samples found in a DNA database to find out who committed a
crime. DNA fingerprinting can also be used to identify a victim even if the person is
disfigured beyond recognition and dental records cannot be established. DNA
fingerprinting has been used to prove suspects guilty of a crime and has set innocent people
free when earlier evidence has proven them guilty. DNA fingerprinting is not perfect and
there is still much controversy over whether it can be used in a court of law, but regardless,
it remains an essential part of our criminal justice system.

14. DNA FINGERPRINTING
( DNA PROFILING )
3. Personal Identification
DNA fingerprinting has also been thought of as a future method of identification. The way it
would work is that a sample of DNA could be analyzed on spot and compared to a database
of specific DNA belonging to authorized personnel. While DNA is the ultimate bar code, it
would be too expensive and impractical to use DNA fingerprinting for personal
identification while other means of identification such as picture ID and social security
numbers (although both can be faked) are available.

15. DNA FINGERPRINTING
( DNA PROFILING )
DNA Profiling Process:
•Variable Number Of Tandem Repeats(VNTRs)
• Amplified Length Polymorphisms (AFLPs)
•Restriction Fragment Length Polymorphism (RFLP)

16. DNA FINGERPRINTING
( DNA PROFILING )
1.Variable Number Of Tandem Repeats(VNTRs)
•Most of the DNA in a chromosome
does not code for a gene
•These regions contain sequences
that repeat from 20-100 times (ex-
GTCAGTCAGTCAGTCA)
•There are several known variations
of the Short Tandem Repeats (STR)
in
humans
•Ex.- HUMTH01 (repeating AATG)
has
seven different variations

17. DNA FINGERPRINTING
( DNA PROFILING )
•Scientists can look at 4-6
different STR’s in the same person
•The more STR’s identified the higher
the probability of a match
•VNTR is more commonly used
than RFLP
VNTR
Advantages of VNTR
• Less complex patterns in gel
•STR’s are less likely to degrade
Less than 400 base pairs
In the middle of the chromosome
• Can be used on stains that old or have been exposed to decomposition

18. ( DNA PROFILING )

19. ( DNA PROFILING )
2. Amplified Length Polymorphisms (AFLPs)
AFLP definition: Any difference between corresponding DNA fragments from two
organisms A and B that is detected by the amplified restriction length polymorphism
technique (Kahl, 2001).
•Initially, DNA is extracted as for RFLP and then cut with two different restriction
enzymes to produce well defined restriction fragments with sticky ends.
•Synthetic double stranded linkers of approximately 18 to 20 bp with matching sticky ends
are ligated on all the restriction fragments.
•Ligated fragments are subsequently amplified in PCR with 18 to 20 nucleotide length
primers recognizing linkers in each end of the fragments
•A primer used for amplification in AFLP is normally labeled with P33 or some
nonradioactive labeling system.
•The mixture of amplified fragments are separated according to size in a
Polyacrylamide gel and visualized by means of autoradiography or other
procedures for development of non radioactive systems.
•Alternatively, automated sequencing machines can be used to read the sequence
of amplification products directly from the Polyacrylamide gel if primers have been
labeled with special fluorescent dyes.

20. •Also, silver staining of the Polyacrylamide gel after electrophoresis can be used
to stain all amplified DNA fragments in the gel
•To avoid that all restriction fragments from the genome amplify in the same
PCR, which will produce a smear because of too many DNA fragments, a two
step amplification procedure is used.
•Firstly, part of the total number of restriction fragments are amplified in the
"preamplification" with primers containing one extra "selective" nucleotide on
their 3' end.
•This selective nucleotide will allow amplification only of restriction fragments
with a matching nucleotide next to the linker.
•With one selective nucleotide on both primers only 1/16th of all restriction
fragments in the mixture will amplify during the preamplification.
•During the subsequent "selective" amplification, additionally, one or two
selective nucleotides on each primer will further reduce the number of fragments
amplified.
•A good AFLP amplification will show 90 to 100 different fragments in one
analysis.

21. The sequence of adapters and primers used in this study is shown below:
MseI adaptor: 5-GACGA TGA GTCC TGAG-3
3-T ACT CGG ACTCAT-5
EcoRI adaptor: 5-biotin-CTCGTA GAC TGCG TACC-3
3-CTG ACGC ATGGTTAA-5
MseI + 1 primer : 5 - GATGAGTCCTGAGTAAA
MseI + 3 primers : 5 - GATGAGTCCTGAGTAAAAC
EcoRI + 1 primer : 5 - GACTGAGTACCAATTCA
EcoRI + 3 primers : 5 – GACTGCGTACCAATTCACG
Selective amplification is then carried out by PCR using preamplified
DNA fragments with EcoRI and MseI primer with three-selected nucleotide each.
The final PCR products are separated and detected by Polyacrylamide gel
electrophoresis.

22. 3.Restriction Fragment Length Polymorphism (RFLP)
Steps of making an RFLP:
1. Add a restriction enzyme to cut the DNA into fragments - Exact number and
size of fragments produced varies from person to person
2. Fragments are separated by electrophoresis
3. The smaller fragments travel further than the
large fragments
4. This creates a DNA Fingerprint

23. DNA FINGERPRINTING
( DNA PROFILING )

24. ( DNA PROFILING )

25. ( DNA PROFILING )

26. DNA FINGERPRINTING
( DNA PROFILING )
Applications of DNA Fingerprinting:
1. Paternity and Maternity
Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can
be used to establish paternity and maternity. The patterns are so specific that a parental
VNTR pattern can be reconstructed even if only the children VNTR patterns are known
(the more children produced, the more reliable the reconstruction). Parent-child VNTR
pattern analysis has been used to solve standard father-identification cases as well as
more complicated cases of confirming legal nationality and, in instances of adoption,
biological parenthood.

27. DNA FINGERPRINTING
( DNA PROFILING )
2. Criminal Identification and Forensics
DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a
crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to
determine guilt or innocence. VNTR patterns are also useful in establishing the identity of
a homicide victim, either from DNA found as evidence or from the body itself.
3. Personal Identification
The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals
has been discussed, but this is not likely to happen.

28. Advantages And Disadvantages of DNA
Fingerprinting:
Advantages:
•DNA profiling is an ideal method for confirming an identity with absolute
certainty.
•It’s easy and painless to obtain a specimen for testing.
•A thorough, scientific test can be conducted in as little as 48 hours.
•DNA testing is affordable and reliable.

29. DNA FINGERPRINTING
( DNA PROFILING )
Disadvantages:
To be conclusive, a DNA test should be run on multiple samples, at least twice. DNA
People Diagnostics collects four samples and the lab runs every test twice to avoid false
readings. In fact, your samples are sent to the only lab in the country to actually run each
test twice through two separate DNA analyzers, virtually eliminating the chance for
error. The lab also tests up to 29 markers to produce the highest possible probability
indicators. Most labs will only test 10 markers, greatly increasing the need for additional
testing. While most labs charge patients when this occurs, we do not charge when
additional testing is necessary.
Misuse of results can lead to privacy concerns. DNA People Diagnostics guarantees
complete confidentiality. Personal information will not be released to anyone other than
the tested parties and their named representatives without a written order.

30. DNA FINGERPRINTING
( 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.

31. ( 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.
The above picture shows how a paternity test is used to match
a child with their biological father. It shows that the child was
compared with his alleged father, and the test on the right
shows the child's DNA matches the father's DNA. This means
that the father on the right is his biological father, while the one
on the left is not.

32. 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.

33. ( 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.

34. •The more STRs that are compared from each DNA sample, the less likely the chance
will be that they have arisen from the same source. The likelihood of any two specimens
being identical is calculated using the 'product rule'. Here, the probability of the
frequency of occurrence of each STR in a population is multiplied by that of the other
STRs separated from the DNA sample.
•During the O.J. Simpson trial in 1995, for instance, O.J.’s guilt was suggested by STR
tests carried out on his blood and blood from the crime scene, which revealed the
probability of his being innocent was 1 in 240000. Further RFLP tests narrowed this
down to 1 in 57 billion. The main reason he wasn’t convicted was due to the seed of
doubt the defense showed in the minds of the jurors about possible interference with the
evidence by the FBI.
•In addition to its use in providing evidence to incriminate suspects, DNA profiling can
also be of use in exonerating suspects accused of crimes. About 25% of violent crime
cases in the U.S. since 1989 have resulted in the exoneration of suspects because of
DNA profiling procedures. By 1996, over 108 post-conviction exonerations had in fact
occurred in the USA using DNA profiling.
Uses of DNA Fingerprinting in Forensic Science:

35. •Because STRs and VNTRs are inherited from each of our parents, DNA profiling can
also be used to establish paternity in cases involving custody and child support. Other
uses in forensics include the identification of victims of catastrophes such as the
September 11 attacks.
•It must be remembered that these tests are not always foolproof and should be used in
conjunction with other evidence where possible. DNA fingerprinting has never the less
affected the outcome of criminal investigations in a revolutionary way.

36. DNA FINGERPRINTING
( DNA PROFILING )
Centers for DNA Fingerprinting:
•Department of Biotechnology.
•(M/o Science & Technology, Government of India)
CDFD,
Office Block :
Bldg. 7, Gruhakalpa,
5-4-399 / B, Nampally,
Hyderabad - 500 001
Laboratory Block :
Tuljaiguda (Opp MJ Market),
Nampally, Hyderabad
Ph.: 040-27151344
Fax: 040-27155610
Website: www.cdfd.org.in
•The diagnostic screening at CDFD is operated from the Nizam's Institute of Medical
Sciences (NIMS)
Punjagutta, Hyderabad,
Andhra Pradesh, India.
Phone Number : +91-40-23489000
Fax Number: +91-40-23310076

37. 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.