1. “RESTRICTION FRAGMENT
LENGTH POLYMORPHISM”

2. WHAT IS RFLP
• The term Restriction Fragment
Length Polymorphism, or RFLP
refers to a difference between two or
more samples of homologous DNA
molecules arising from differing
locations of restriction sites, and to a
related laboratory technique by which
these segments can be distinguished.

3. Cont….
• Commonly pronounced “rif-lip”.
• Its analysis was the first DNA profiling
technique cheap enough to see widespread
application.
• It is an important tool in genome mapping.
• Localization of genes for genetic disorders.
• Determination of risk for disease, and paternity
testing.

4. WHAT IS DNA
• DNA is a genetic material.
• It is a nucleic acid. The
structure of a DNA is
double helix, two long
strands makes the shape
of double helix.
• Chemically, DNA consist of two long polymers of
simple units, called nucleotides, with backbones
made up of base, sugars & phosphate groups.

5. Restriction Fragment Length
Polymorphism
• A restriction enzyme cuts the DNA molecules at
every occurrence of a particular sequence, called
restriction site.
• For example, HindII enzyme cuts at GTGCAC or
GTTAAC.
• If we apply a restriction enzyme on DNA, it is cut
at every occurrence of the restriction site into a
million restriction fragments each a few
thousands nucleotides long.

6. Cont…
• Any mutation of a single nucleotide may destroy
or create the site(CTGCAC or CTTAAC for
HindII) and alter the length of the corresponding
fragment.
• The term polymorphism refers to the slight
differences between individuals, in base pair
sequences of common genes.
• RFLP analysis is the detection of the change in the
length of the restriction fragments.

7. ANALYSIS TECHNIQUE
• The basic technique for detecting RFLPs involves
fragmenting a sample of DNA by a restriction
enzyme, which can recognize and cut DNA
wherever a specific short sequence occurs, in a
process known as a restriction digestion.
• The resulting DNA fragments are then separated
by length through a process known as agarose gel
electrophoresis.
• Then observed the DNA fragments in UV
illuminator

10. DNA EXTRACTION PROCEDURE
Bogenvillia leaf
Crush the leaves
with the motor
and pestle
Add CTAB
buffer
solution
in it
Transfer it
into clean
eppendrof
and incubate
at 55 C for 20
min in dry
bath
Centrifuge it at 8000
rpm for 10 min
Collect
supernatant &add
chloroform, phenol
& isoamylalcohol
(25:24:1)
Mix it by
inversion
Centrifuge at
10000 rpm
for 5 min
Collect upper
layer & add
chilled ethanol
in equal volume
Incubate at
-20 C for 24
hours & than
centrifuge for
1min at 10000
rpm
Collect pallet
&resuspended
in TE buffer &
than use for
RFLP analysis

11. PREPARATION OF RFLP MIXTURE
Assay buffer 10x = 20 micro liter
Template DNA or phase DNA = 100 micro liter
EcoR I = 3 micro liter
Hind III = 3 micro liter
BSA(Bovine Serum Albumin) = 5 micro liter
Mix it and incubate at 37 C for 1 hour and now prepare the
agarose gel and TAE buffer.
Preparation of TAE buffer ,Take 2ml
of TAE & add 98ml distilled water

12. Gel-Electrophoresis
• DNA is cut into fragments using an enzyme
• The cut DNA is put on a Gel material
• An electric current is applied on the Gel
• DNA fragments will start moving towards the
positively charged side
• It shows that DNA is negatively charge
• Smaller fragments move faster
• After some time, we have a separation of the
different fragment lengths

13. DNA Sample
• Some cells are
obtained by DNA
extraction technique
Restriction Enzyme
• A restriction enzyme is
used to cut the DNA into
fragments
• Hind III restriction site
is AAGCTT

14. RESTRICTION ENZYME
• EcoR I restriction site
is GAATTC
EcoR I
• BSA is used in
restriction digest to
stabilize some enzymes
during digestion of DNA
and to prevent adhesion
of the enzyme to
reaction tube.
BSA

15. Apply Enzyme
• RFLP mix are put
together in a tube.
• The tube is shaken by
rotation for DNA,
Hind III ,BSA,EcoRI
& assay buffer to mix.

16. Water Bath
• The tube is put on a
plate floating on
water at 37 C.
• It is left for 60
minutes.
• This is needed for the
apply enzymes
reaction to take
place

17. After incubation collect the
eppendrof and add gel loading dye
Preparation of DNA dye(gel loading dye)
3ml = 30% glycerol
0.025gm = bromophenol blue
7ml distilled water
Now prepare the agarose gel for
loading the sample

18. Preparing the Agarose Gel
• In the meantime, we
prepare the Gel.
• Agarose powder is the
basic substance for
making the Gel.
• For 2% agarose gel
take 2gm agarose
powder and dissolved
in 100ml boil distilled
water.

19. • The powder is mixed
with water in a
container.
• The container is
heated until the
powder completely
dissolves in the water.
• The solution becomes
clear.

20. • Now add Etbr.The DNA
is visualized in the gel
by addition of ethidium
bromide. This binds
strongly to DNA by
intercalating between
the bases and is
fluorescent meaning that
it absorbs invisible UV
light and transmits the
energy as visible orange
light.

21. • The liquid Gel is poured
into the inner box.
• A comb like piece is put at
the edge of the inner box.
• The liquid Gel is left to cool
and solidify.
• When the Gel solidifies, the
comb will create wells for
the DNA samples to be put.

22. Gel casting
• Fill the H shaped
container with TAE
buffer solution
• Remove comb

23. Putting DNA on the Gel
• DNA samples mixed with
colored solution and UV
reactive solution i.e.is
DNA dye which we added.
• DNA samples inserted
into wells
• A sample DNA containing
only specific fragments
(called ladder) can be
used for comparison
original
uncut DNA
DNA cut by
enzymes
DNA with rflp mix ladder

24. Run the Gel
• Apply electric current
• the DNA Fragments will
migrate towards the
positive charge which
means that the DNA is
negatively charged
• Small fragments move
faster

25. DNA Fragments Move
• The colored solution
provides an indication to
how much the DNA has
traveled on the Gel.

26. Observation
• Gel can be viewed
under UV light in
UV illuminator.

27. Observation
• Original uncut DNA sample
makes a sharp band at the
beginning (one big fragment)
• DNA sample cut with enzymes
makes s smear (lots of
fragments of all sizes)
• Ladders are used for
comparison (they contain
specific fragments)
• We could run it for a longer
time to achieve better
separation
Hind III band
EcoR I
band
Original
uncut DNA

28. Original
uncut DNA
Hind III bandEcoR I
band
RFLP
mix

29. Restriction fragment length polymorphism
(RFLP) is most suited to studies at the
intraspecific level or among closely related
taxa. Presence and absence of fragments
resulting from changes in recognition sites
are used identifying species or populations.