DNA Fingerprinting.pdf principles, working

Hematology analyser

1. DNA Fingerprinting

2. What is DNA Fingerprinting?
•DNA fingerprints (DNA testing) identify living organisms at the molecular
level.
•DNA fragments show unique patterns from one person to the next.
•It is also called a DNA profile.
•Uses:
•Analysts use the DNA profile from potential suspects and compare it
against DNA found at a crime scene.
•DNA profiling for paternity tests. These days you can send a sample of DNA
and find out your ancestry to learn more about your origins. This is a very
conclusive test because DNA is specific to each individual(unless you are
an identical twin).

3. Human Genome
About 20% genes
(code for proteins)
About 80% junk
(code for nothing
or unknown…)
Highly repetitive
nucleotides
Random
nucleotides

4. Principle of DNA Fingerprinting
•The DNA of every human being on the planet is 99.9% same.
However, about 0.1% or 3 x 10
6
base pairs (out of 3 x 10
9
bp) of
DNA is unique in every individual.
•Human genome possesses numerous small non-coding but
inheritable sequences of bases which are repeated many times.
They do not code for proteins but make-up 95% of our genetic
DNA and therefore called the ―junk DNA.
•They can be separated as satellitefrom the bulk DNA during
density gradient centrifugation and hence called satellite DNA.
•In satellite DNA, repetition of bases is in tandem. Depending
upon length, base composition and numbers of tandemly re-
petitive units, satellite DNAs have subcategories like
microsatellites and mini-satellites.
•Satellite DNAs show polymorphism. The term polymorphism is
used when a variant at a locus is present with a frequency of
more than 0.01 population.
•Variations occur due to mutations.These mutations in the non-
coding sequences have piled up with time and form the basis of
DNA polymorphism (variation at genetic level arises due to
mutations).

5. The junk DNA regions are thus made-up of length polymorphisms, which show
variations in the physical length of the DNA molecule. At specific loci on the
chromosome the number of tandem repeats varies between individuals. There will
be a certain number of repeats for any specific loci on the chromosome. Depending
on the size of the repeat, the repeat regions are classified into two groups.
•Short tandem repeats (STRs)contain 2-5 base pair repeats and
•variable number of tandem repeats (VNTRs)have repeats of 9-80 base pairs.
•Since a child receive 50% of the DNA from its father and the other 50% from his
mother, so the number VNTRs at a particular area of the DNA of the child will be
different may be due to insertion, deletion or mutation in the base pairs.
•As a result, every individual has a distinct composition of VNTRsand this is the
main principle of DNA fingerprinting.
•As single change in nucleotide may make a few more cleavage site of a given
nucleotide or might abolish some existing cleavage site.
•Thus, if DNA of any individual is digested with a restriction enzyme, fragments
pattern (sizes) will be produced and will be different in cleavage site position. This
is the basics of DNA fingerprinting.

6. 1. Restriction Fragment Length
Polymorphisms, or RFLPs
“Nucleotide sequence variations in a region of DNA that generates
fragment length differences according to the presence or absence of
restriction enzyme recognition sites.”
RFLPs, are generated due to mutations in recognition sites.
What is recognition site?
It’s the specific nucleotide sequence recognized by restriction enzymes.
The restriction enzymes bind to the DNA and cut within the recognition
site.
If the nucleotide sequence has been mutated, then the restriction
enzyme will not bind and therefore will not cut at that site.

7. DNA testing uses the repetitive nucleotides
because these vary among people.

8. As this example shows, the change in the
recognition site will produce different length
fragments in a restriction digest.
Individual 01 has two recognition sites for the
EcoRIenzyme. That means EcoRIwill cut this DNA
fragment twice. What happens when you cut a
piece of string twice? You get three pieces. The
same is true when you cut linear DNA with
molecular scissors like restriction enzymes. Two
cuts generates three fragments labeled here as A,
B, and C.
In individual 2 the first recognition site is not
present. Instead of GAATTC the sequence is now
GAAATTC.
EcoRIwill not bind and cut at this location. That
means in the same restriction digest, the
restriction enzyme will only cut once and generate
two fragments, D and C.

9. The RFLP fragments can be separated by gel
electrophoresis.

10. HOW ARE DNA FINGERPRINTS MADE???
✓The process begins with a sample of DNA
✓Isolate & replicate the DNA

11. ✓Cut the DNA with restriction enzymes (each enzyme
looks for a specific pattern)
✓Place fragmented DNA in the wells of a polyacrylamide
gel and electrophoresis is performed (negative charge on
top by wells and positive charge on bottom)
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12. •DNA has a negative charge,
so the fragments of DNA are
attracted toward the positive
charge (bottom of gel).
•The smaller fragments of
DNA will be able to move
quicker toward the bottom
than the larger pieces.
•Thus, DNA is separated by
size (smaller pieces toward
the bottom and the larger
pieces toward the top).
HOW DOES IT WORK???
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13. Southern Blot
❖Molecular technique where DNA is transferred onto a membrane
from an agarose gel and a probe is hybridized.

14. Southern Blot
❖The first step in preparing a Southern Blot is to cut
genomic DNA and run on an agarose gel.

15. Southern Blot
❖The next step is to blot or transfer single stranded DNA
fragments on to a nylon membrane.

16. ❖The next step is to hybridize a radioactively labeled DNA probe to
specific sequences on the membrane.
Southern Blot

17. Southern Blot
❖The last step is to expose
the radioactively labeled
membrane to a large sheet
of film.
❖You will only visualize bands
where the probe hybridized
to the DNA.

18. Southern Blot