dna fingerprinting 7 jan 2015

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DNA FINGERPRITING

DNA PROFILING

DNA TYPING

By

Dr Ichha Purak

University Professor

Department of Botany

Ranchi Women’s College,Ranchi

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.


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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ORDINARY FINGER PRINTS PRESENT AT FINGER TIPS


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)

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


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)


Variable Number of Tandem Repeats (VNTR)

AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA

AGTTCGCGTGA AGTTCGCGTGA

Repeat sequence length: 10-100 base pairs/repeat

_____________________________________________________

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Short Tandem Repeats (STR)

ATGCC ATGCC ATGCC ATGCC ATGCC

Repeat sequence length 2-9 base pairs/repeat

VNTR AND STR


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


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


STEPS OF DNA FINGER PRINTING


AB

C D


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.


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


STEPS INVOLVED IN DNA FINGER PRINTING


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

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


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.



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

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


12

3 4

5 MAKING A RADIOACTIVE PROBE

Nick


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.


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


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


The DNA FINGERPRINTING PROCESS


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


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.


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.1/9/2015 DNA fingerprinting 28

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.


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


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.


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.


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.


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


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.”


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.

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.


END OF PRESENTATION

dna fingerprinting 7 jan 2015

Health & Medicine