The power point presentation consists of 36 slides explaining about history, principle, different steps involved and applications of DNA fingerprinting. Recent Developments and the Future prospects of DNA profiling have also been mentioned

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DNA FINGERPRITING
DNA PROFILING
DNA TYPING
By
Dr Ichha Purak
University Professor
Department of Botany
Ranchi Women’s College,Ranchi

2. Professor Sir Alec Jeffreys, Kt, FRS: Discovered DNA fingerprinting in
1984
Professor of Genetics and Royal Society Wolfson Research Professor,
Department of Genetics, University of Leicester.
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3. HISTORY
Professor Alec Jeffreys in 1984 developed the DNA(Genetic) fingerprinting
process at Leicester University which is one of his best contributions to human
genetics
Since discovery DNA fingerprints are used for identifying individuals,
establishing family relationships, in medical research, forensic science for
identification of criminals, paternity and immigration evidence.
The purpose of DNA fingerprinting can also be extended to breeding plants
and animals, conserving nature and understanding evolutionary processes.
In 1987 it was used for the first time in law court of England to identify a victim
in Rape case .DNA fingerprinting was first used as forensic evidence in 1988
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4. ORDINARY FINGER PRINTS PRESENT AT FINGER TIPS
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Every person can be identified by his fingerprints and therefore thumb
impression is used in place of signature for those persons who are unable to
read and write. As fingerprints are unique so are being used for identification in
forensic science since long ( 1930’s or even earlier)

5. As DNA of every individual consists of millions of bases and every person
has a different sequence. It is not practicable to analyse the complete genome
sequence so for DNA finger printing some repeated sequences can be
analysed which can be isolated and separated by Gel electrophoresis
The sequence of DNA fragments such as Minisatellite or VNTRs,
Microsatellite or STR or RFLP can be obtained by southern blotting technique
so can be employed for DNA finger printing.
RFLPs (Restriction Fragment Length Polymorphism ) are DNA fragments
produced when DNA is digested by Restriction endonucleases which are able
to cut phospho di-ester bond of DNA at specific sequences
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6. Variable number tandem repeats, or VNTRs represent specific locations
on a chromosome in which tandem repeats of 9-80 or more bases repeat a
different number of times between individuals and also variation in length. Each
variant acts as an inherited allele, allowing them to be used for personal or
parental identification. VNTRS are readily analyzed using the RFLP approach
and a probe specific to a VNTR locus. Their analysis is useful in Genetics and
DNA fingerprinting for comparative analysis in forensics.
Currently the most popular method of DNA fingerprinting is done by using
Short term Repeats (STRs or Microsatelite ) which have repeat sequences of
only 2-5 base pairs . Since the length of DNA fragment being analyzed is short
enough can be amplified by polymerase chain reaction (PCR)
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7. Variable Number of Tandem Repeats (VNTR)
AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA
AGTTCGCGTGA AGTTCGCGTGA
Repeat sequence length: 10-100 base pairs/repeat
_____________________________________________________
______
Short Tandem Repeats (STR)
ATGCC ATGCC ATGCC ATGCC ATGCC
Repeat sequence length 2-9 base pairs/repeat
VNTR AND STR
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8. PRINCIPLE OF DNA FINGER PRINTING
VNTR is a tandem repeat from a single genetic locus in which the number of
repeated DNA segments varies from individual to individual but are inherited
and are used for identification purposes as in DNA fingerprinting.
The VNTRs of two persons may be of same length and sequence at certain
sites, but vary at others.
A child might inherit a chromosome with tandem repeats both from mother and
father, so VNTR alleles of a child resemble to both parents.
Southern Blot is performed to determine if a person has a particular VNTR.
Southern Blot is probed through a hybridization reaction, with a radioactive
version of the VNTR being analysed. The pattern which results from this
process is often referred as a DNA fingerprint
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9. STEPS INVOLVED IN DNA FINGER PRINTING
ISOLATION
RESTRICTION ENDONUCLEASE DIGESTION
GEL ELECTROPHORESIS
 DENATURATION
SOUTHERN BLOTTING USING VNTR/STR/ RFLP
MAKING A RADIOACTIVE PROBE (SSDNA )
CREATING HYBRIDIZATION REACTION
DETECTION OF HYBRIDIZATION BY
AUTORADIOGRAPHY OR OTHER METHODS
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10. STEPS OF DNA FINGER PRINTING
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11. AB
C D
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12. SAMPLE REQUIREMENT FOR DNA FINGERPRINTING
For DNA fingerprinting only a small amount of tissue like blood, semen,
skin, vaginal swab or even follicle root of hair or plant tissue is needed as
only a small amount of DNA is sufficient. DNA can be isolated even from
bone marrow of old bones of victims or blood stains from clothings or
discharge can serve the purpose. Typically DNA content of about 10,000
cells or I microgram is sufficient. If amount is less(less than 1 µg) it can
be amplified by Polymerase Chain Reaction (PCR)
DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION
PCR is an invitro procedure developed by Kary Mullis (1985) that
amplifies enzymatically a particular DNA sequence flanked by two
oligonucleotide DNA primer.
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13. For this a apparatus Thermal cycler is used which has provisions to alternate
temperature in cyclic manner. PCR is based on semi conservative DNA
Replication
PCR involves following steps
Denaturation step takes place at 94-98ᵒC. Double stranded DNA is denatured
by dissolving hydrogen bonds between base pairs resulting in two single
stranded structure which act as template
Annealing step takes place at 50-65ᵒ C for annealing of DNA primers
Extension or Elongation step DNA synthesis takes place at 72ᵒ C by using
Taq DNA polymerase and all the 4 dNTPs resulting in formation of two DNA
double Helices.
These three steps are cycled repeatedly (20-35-40) resulting in making copies
of DNA exponentially as per requirement
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16. STEPS INVOLVED IN DNA FINGER PRINTING
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 DNA is extracted from cells in high speed refrigerated centrifuge using
detergents to remove debries.
 DNA is cut into fragments by using one or more site specific
Restriction Endonucleases and RFLPS( Restriction Fragment Length
Polymorphism or Fragments of different size ) are obtained .
 Choped DNA fragments are passed through Agarose Gel
Electrophoresis or Polyacrylamide Gel Electrophoresis (PAGE)
 The separated fragments can be visualized by staining them with
Ethidium Bromide dye that fluorescence under U-V radiation.
Bromophenol Blue is used as tracking dye.
 Double stranded DNA is then split into single stranded DNA using
alkaline chemicals

17. These separated single stranded DNA fragments are then transferred to
Nitrocellulose or Nylon sheet placed over the gel.
 The adherence ( Blotting ) of single stranded DNA fragments to
nitrocellulose sheet is called Southern Blotting This protocol of
adsorbing DNA was invented by the Scientist E M Southern.( 1976)
 Nitrocellulose sheets are baked at high temperature so as to fix single
stranded DNA to it.
The nitrocellulose sheet is then immersed in a bath and radioactive probe
(DNA ) a synthetic oligonucleotide DNA segment with known sequence are
added
 The probe targets a specific nucleotide sequence which is complementary
(VNTRs Sequence ) and hybridize them
 Finally NC sheet ,containing the SSDNA and Radioactive probe is exposed
to autoradiography . Dark bands develop at the site of hybridization and help in
identifying Hybridization or homology
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18. DEVELOPMENT OF RADIOACTIVE PROBES BY JEFFEREYS
In between the genes which code for protein synthesis are intervening non
coding regions.These are called hypervariable regions because they vary from
person to person.No two people, except identical twins, share same set of
regions.
In the hypervariable regions a simple sequence of 10-15 bases called a core
sequence can be repeated over and over again. In 1984 Jeffreys discovered
that core sequence could be used as genetic markers for the hypervariable
regions. Jeffreys isolated two core sequences of DNA and copied them many
times in the laboratory to produce large quantities of markers which he labeled
with radioactive chemicals. Now these genetic markers could be used as
genetic probes
The probes can be attached to core sequences in a sample of DNA to find
the position of hypervariable regions using photographic film to detect the
radioactivity.
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The probes used for DNA fingerprinting are usually prepared from mini –
satellite DNA which is highly variable mainly due to variation in number
of tandem repeats of short core sequence. These probes hybridize under
condition of low stringency , to a number of polymorphic loci represented
by the mini-satellite DNA. The regions of mini satellite may occur
dispersed throughout the human genome or may be clustered onto a
single chromosome.
A simple universal probe, a tandem repeat of GATA, has been developed
from sex chromosome of branded Krait.
This probe is reportedly very useful in DNA fingerprinting of man and
many other organisms

20. MAKING A RADIOACTIVE PROBE
For making a radioactive probe ,the double stranded DNA to be used
to make probe is put in a tube . A nick is induced in one of the strand and to
this DNA polymerase enzyme and all the four deoxyribonucleotides are
added of which CMP is labeled by having 32P. DNA polymerase strarts repair
at the point of nick and old bases are replaced by new base. Radioactive C
is added where there is G on other strand (Template ) . After repair the
Double stranded DNA is subjected to denaturtion. After denaturtion one
strand has label and other is Non-labeled
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21. 1
2
3 4
5 MAKING A RADIOACTIVE PROBE
Nick
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22. CREATING A HYBRIDIZATION REACTION
Hybridization is the coming together, or binding, of two genetic sequences.
The binding occurs because of the hydrogen bonds between base pairs. (
A & T and C & G )
DNA must first be denatured, usually by using heat or chemicals.
Denaturing is a process by which the hydrogen bonds of the original
double-stranded DNA are broken, leaving a single strand of DNA whose
bases are available for hydrogen bonding.
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23. A single-stranded radioactive probe can be used to see if the denatured
DNA contains a sequence homologous to that of probe . For this the
membrane with denatured DNA strands is put into plastic bag along with
probe and some saline liquid or buffer and is allowed to make base pairs
between complementary sequences
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24. Partial or complete hybridization depends on varying homologous
sequences to the probe sequence .Hydrogen bonds will be established
between A & T by 2 H bonds and G & C by 3 H bonds where the
sequence is homologous
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25. The DNA FINGERPRINTING PROCESS
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26. Applications of DNA Fingerprinting
DNA fingerprints are useful in several applications of human health
care research, as well as in the justice system
DNA Finger Printing can be effectively used in forensics to identify
or match the DNA of suspected individuals in crimes such as
murder, rape and in paternity and immigration disputes
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27. PATERNITY AND MATERNITY
DNA Fingerprinting can be used to establish paternity and maternity on the basis
of VNTR pattern because a person inherits his or her VNTRs from parents. For
this VNTR pattern of Mother ,Father and child or children are compared . By this
disputes of Biological father can be solved
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 of biological parenthood
As VNTR patterns are inherited ,can be used to establish family relations. Many
people choose to trace their heritage by using and tracking DNA fingerprints.
DNA fingerprinting was also used in an effort to identify a missing Russian
princess Anastasia Romanov through remains found in a family grave.
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28. Here is an example of a VNTR. In the diagram, D1 is a biological
daughter, D2 is a step-daughter(daughter of the mother and an x-
husband),S1 is a biological son and S2 is an adopted son.
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29. Criminal identification and Forensic Evidence
DNA isolated from blood, hair root follicles, skin cells, spots on clothing or
other genetic evidences left at the crime scene (murder or rape) can be
compared through VNTR patterns, with the DNA of a criminal suspect (S) to
determine or identify the criminal victim and give relief to non-victim
DNA fingerprinting was first used as forensic evidence in the murder
conviction of Colin Pitchfork in 1988. Since then, crime scene identification,
criminal identification and victim identification is widely done by matching
DNA obtained from hair root follicle, blood, semen, skin and other human
body parts and secretions. Many prisoners have been released based on
new evidence provided through DNA fingerprinting.
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30. DNA fingerprints of 7 suspected victims compared with
that of blood stain obtained from murder scene. On the
basis of DNA bands Number 3 appears as victims
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31. Personal Identification: DNA can be used to identify individual
persons , but this doesn’t seem practical as the process is too complex
and having a DNA database is expensive
DNA Finger printing can be used for personal identification as social
security number, photo identity , Blood group, notable phenotypic traits
etc. The technology of profiling individuals based on VNTR patterns is
very expensive.
Because every organ or tissue of an individual contains the same DNA
fingerprint, the U.S. armed services have just begun a program to collect
DNA fingerprints from all personnel for use later, in case they are needed
to identify casualties or persons missing in action. The DNA method will
be far superior to the ordinary finger prints, dental records, and blood
typing strategies currently in use.
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32. Immigration Evidence
According to the Dolan DNA Learning Center, a mother and son were
separated by immigration officials, The dispute was solved by court in 1985
when DNA fingerprint evidence proved that the two were mother and son and
belong to their own country of England. Unidentified immigrants can be
assigned to their official country .
Medical Research
DNA fingerprinting helps in the early detection of inherited diseases and their
treatment. Through the study of genetic patterns of individuals and groups,
medical research can potentially lead to cures for inherited diseases. Another
medical research purpose for DNA fingerprinting is to match recipients of live
organs from the donors, making transplants more successful.
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33. Developing Cures for Inherited Disorders
Research programs to locate inherited disorders on the chromosomes depend
on the information contained in DNA fingerprints. By studying the DNA
fingerprints of relatives who have a history of some particular disorder, or by
comparing large groups of people with and without the disorder, it is possible
to identify DNA patterns associated with the disease in question. This work is a
necessary first step in designing an eventual genetic cure for these disorders.
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34. Diagnosis of 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. In other
programs, prospective parents use DNA fingerprint information in their
decisions concerning affected pregnancies
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35. Miscellaneous Purposes
Through DNA fingerprinting, selective breeding of animals and plants in
possible. According to a U.S. Department of Agriculture and Cooperative
Extension Service publication, "a scientist who knows which piece of DNA is
associated with a desirable trait can select plants or animals that have that
piece of DNA. It is often easier and less expensive to select plants or animals
that have the DNA marker than to grow them to maturity and see if they
develop the desirable trait.”
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DNA Fingerprinting can be used to identify racial groups to rewrite biological
evolution
It was also used for identification of the body of Nazi physician Joseph
Mengele, the so-called "Angel of Death.”
By DNA fingerprinting a kid lost in Tsunami was handed over to his actual
parents by comparing DNA of other claimants.

36. Recent Developments and the Future prospects of
DNA Testing) (Profiling )
Databases
Many US states have planned to make database of DNA profiles of
individuals having criminal record
New profiling methods
Every cell of human being contain 23 pairs of chromosomes so each gene
has two copies. In addition that each cell has organelles as mitochondria .
Mitochondrial DNA can be used for DNA fingerprinting when amount of
biological samples are limited in case of disaster or accident or burning ,the
bodies becomes badly destroyed.
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END OF PRESENTATION