Dna Fingerprinting 1

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DNA FINGERPRINTING1


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WHAT IS DNA FINGERPRINTING

DNA Fingerprinting is a technique employed

by forensic scientists to assist in the

identification of individuals on the basis oftheir respective DNA profiles

It uses repetative sequences that are highly

variable called Variable Number Tandom

Repeats [VNTR]

It is also called DNA testing, DNA profiling ,

DNA typing or Genetic fingerprinting


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The chemical structure of everyone's DNA is

the same.

The only difference between people (or anyanimal) is the order of the base pairs.

There are so many millions of base pairs in

eachperson's DNA that every person has adifferent sequence


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Using these sequences, every person couldbe identified by the sequence of their base pairs.

Each person has a unique DNA fingerprint,

except monozygous (identical) twins. DNA fingerprint is the same for every cell,

tissue, and organ of a person. It cannot bealtered by any known treatment.

Scientists use a small number of sequences of DNA

that are known to vary among individuals ,and analyze those to get a certain probability of amatching


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HOW IS IT PERFORMED

Performing a southern blot

Making a radioactive probe

Creating a hybridization reaction VNTRs


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Blotting the DNA to permanently attach

DNA to sheet

It is analysed by the use of radioactive geneticprobe in hybridization with the DNA

An X-Ray is taken of the blot after probe

bonds with the denatured DNA on the paper.

Areas where the radioactive probe binds [red]will show up on the film.


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RADIOACTIVE BINDING


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MAKING A RADIOACTIVE PROBE

Obtain some DNA polymerase [pink]. Put the

DNA to be made radioactive (radiolabeled)

into a tube. Introduce nicks, or horizontal breaks along a

strand, into the DNA you want to radiolabel.

At the same time, add individual nucleotides

to the nicked DNA, one of which, *C [lightblue], is radioactive.


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MAKING A RADIOACTIVE PROBE

Add the DNA polymerase [pink] to the tube

with the nicked DNA and the individual

nucleotides. The DNA polymerase willbecome immediately attracted to the nicks in

the DNA and attempt to repair the DNA,

starting from the 5' end and moving toward

the 3' end. The DNA polymerase [pink] begins repairing

the nicked DNA


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Whenever a G base is read in the lower strand, aradioactive *C [light blue] base is placed in thenew strand. In this fashion, the nicked strand, as

it is repaired by the DNA polymerase, is maderadioactive by the inclusion of radioactive *Cbases.

The nicked DNA is then heated, splitting the two

strands of DNA apart. This creates single-stranded radioactive and non-radioactive pieces.The radioactive DNA, now called a probe [lightblue], is ready for use


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CREATING A HYBRIDISATION

REACTION Hybridization is the coming together, or

binding, of two genetic sequences. Thebinding occurs because of the hydrogenbonds [pink] between base pairs. Between aA base and a T base, there are two hydrogenbonds; between a C base and a G base, thereare three hydrogen bonds.

When making use of hybridization in thelaboratory, DNA must first be denatured,usually by using heat or chemicals


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Denaturing is a process by which the

hydrogen bonds of the original double-

stranded DNA are broken, leaving a singlestrand of DNA whose bases are available for

hydrogen bonding

Once the DNA has been denatured, a single-

stranded radioactive probe [light blue] can beused to see if the denatured DNA contains asequence similar to that on the probe.


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The denatured DNA is put into a plastic bagalong with the probe and some saline liquid;the bag is then shaken to allow sloshing. Ifthe probe finds a fit, it will bind to the DNA.

The fit of the probe to the DNA does not haveto be exact. Sequences of varying homologycan stick to the DNA even if the fit is poor; thepoorer the fit, the fewer the hydrogen bondsbetween the probe [light blue] and thedenatured DNA.


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The ability of low-homology probes to still

bind to DNA can be manipulated through

varying the temperature of the hybridizationreaction environment, or by varying the

amount of salt in the sloshing mixture.


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VNTRs

Every strand of DNA has pieces that contain

genetic information which informs an

organism's development (exons) and piecesthat, apparently, supply no relevant genetic

information at all (introns).

Introns contain repeated sequences of base

pairs called VNTRs


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A given person's VNTRs come from the

genetic information donated by his or her

parents; he or she could have VNTRsinherited from his or her mother or father, or

a combination, but never a VNTR either of his

or her parents do not have.


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CASE STUDY

Shown below are the VNTR patterns for Mrs.

Nguyen [blue], Mr. Nguyen [yellow], and their

four children: D1 (the Nguyens' biologicaldaughter), D2 (Mr. Nguyen's step-daughter,

child of Mrs. Nguyen and her former husband

[red]), S1 (the Nguyens' biological son), and

S2 (the Nguyens' adopted son, notbiologically related [his parents are light and

dark green]).


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Because VNTR patterns are inherited

genetically, a given person's VNTR pattern is

more or less unique. The more VNTR probesused to analyze a person's VNTR pattern, the

more distinctive and individualized that

pattern, or DNA fingerprint, will be.


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Use Of PCR

PCR can be used in DNA fingerprinting as a

way to make numerous copies of isolated

DNA. The process can selectively amplify asingle copy of a desired sequence

Dna molecules are melted by raising the

temperature to 95 degrees Celcius.

-Strands separate, temperature is lowered to

60 degrees Celcius.


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Each primer binds specifically to the 3 prime

end of the target sequence on the

appropriate strands of DNA. Primers direct taq polymerase to synthesize

complementary nucleotides.

-Only DNA containing target sequence are

copied by taq polymerase.


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At the end of cycle 1, both strands have been

copied to form 2 partially double-stranded

molecules These steps are repeated during the next

cycles. After two cycles, four partially double-

stranded molecules are produced, all

containing the target sequence


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After many cycles, millions of copies of the

DNA can be produced.

-This is useful in DNA fingerprinting, asresearchers can do many tests on the sameDNA sample.


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PRACTICAL APPLICATIONS OF DNA

FINGERPRINTING Paternity and Maternity

Because a person inherits his or her VNTRs

from his or her parents,P

arent-child VNTRpattern 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


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Criminal Identification and ForensicsDNA isolated from blood, hair, skin cells, orother genetic evidence left at the scene of a

crime can be compared, through VNTRpatterns, with the DNA of a criminal suspectto determine guilt or innocence. VNTRpatterns are also useful in establishing the

identity of a homicide victim, either fromDNA found as evidence or from the bodyitself.


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Personal Identification

The notion of using DNA fingerprints as a sort

of genetic bar code to identify individuals hasbeen discussed, but this is not likely to

happen anytime in the foreseeable future.

The technology required to isolate, keep on

file, and then analyze millions of veryspecified VNTR patterns is both expensive

and impractical.


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Diagnosis of InheritedDisorders

DNA fingerprinting is used to diagnose

inherited disorders in both prenatal andnewborn babies in hospitals around theworld. These disorders may include cystic

fibrosis, hemophilia, Huntington's disease,

familial Alzheimer's, sickle cell anemia,thalassemia, and many others.


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Early detection of such disorders enables the

medical staff to prepare themselves and the

parents for proper treatment of the child. Insome programs, genetic counselors use DNA

fingerprint information to help prospective

parents understand the risk of having an

affected child


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CASE STUDY

In the example shown on the left,DNA

collected at the scene of a crime is

compared withDNA

samples collectedfrom 4 possible suspects. TheDNA has

been cut up into smaller pieces which are

separated on a gel. The fragments from

suspect 3 match those left at the scene ofthe crime, betraying the guilty party.


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Dna Fingerprinting 1

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