RFLP (Restriction Fragment Length Polymorphism) is a DNA profiling technique that involves using restriction enzymes to cut DNA into fragments of varying lengths, separating the fragments via gel electrophoresis, and comparing fragment patterns to detect differences between individuals' DNA. It was an early and important method for genome mapping, disease risk determination, and forensic identification but has been replaced by newer techniques as it is expensive, time-consuming, and requires large DNA amounts. RFLP analysis cuts DNA with restriction enzymes, separates fragments by size via gel electrophoresis, and compares patterns to detect length polymorphisms indicating sequence differences between individuals.

1. SUBMITTED BY: SUBMITTED TO:
V. MAHARASI DR. G
RAMANATHAN
II MSC MICROBIOLOGY
ASSISTANT PROFESSOR
REG NO: 20201232516109
DEPARTMENT OF MICROBIOLOGY
SRI PARAMAKALYANI COLLEG
SEMESTER-VI
SUBJECT – BIOTECHNOLOGY
SEMINAR TOPIC-RFLP- RESTRECTION FRANGMENT LENGTH
POLYMORPHISM

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

3. CONT…
•Commenly pronounced rif-lip
•Its analysis is 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

4. 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

5. CONT…
•Any mutation of a single nucleotide may
destroy or geat 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 commen genes .
•RFLP analysis is the detection of the change in
the length of the restriction fragments.

6. •RFLP analysis may be subdivided into
single (SLP) and multi –locus probe(MLP)
paradigms.
•Usually, the SLP method is preferred over
MLP because it is more sensitive, easier to
interpret and capable of analyzing mixed
–DNA sample.

7. ANALYSIS TECHNIQUE:
•The basic technique for detecting RFLPs involves
fragment 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 transferred to a membrane via the southern

8. ANALYSIS TECHNIQUE :
•Hybridization of the membrane to labeled DNA
probe then determines the length of the
fragments which are complementary to the
probe
•Each fragment length is considered an allele,
and can be used in genetic.

9. •How to works:

11. 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 is negatively charge.
• DNA fragments will start moving towards the positively
charged side.
• Smaller fragments move faster
• After some time we have a separation of the different
fragment lengths.

12. DNA SAMPLE :
•Some cells are
obtained by DNA
extraction
technique.

13. RESTRICTION ENZYME:
•A restriction enzyme is
used to cute the DNA
into fragments
•Hind III restriction site
is a AGCTT.

14. APPLY ENZYME:
•DNA sample and Hind
III are put together in
a tube
•The tube is shaken by
rotation for DNA and
Hind III to mix

15. WATER BOTH:
•The tube is put on a
plate floating on water
at 37°C.
•It is left for 30
minutes
•This is needed for the
Hind III reaction to

16. PREPARING THE GEL:
•In the meantime, we
prepare the gel.
•Agrose powder is the
basic substance for
making the gel.

17. PREPARING GEL:
•The powder is
mixed with water
in a container.

18. PREPARING THE GEL :
•The container is heated
until the powder
completely dissolves in
the water.
•The solution becomes
clear.

19. PREPARING GEL:
•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

20. GEL READY:
•Fill the H shaped
container with
water.
•Remove comb.

21. PUTTING DNA ON THE GEL:
•DNA samples mixed with colored solution
nand UV reactive solution.
•DNA samples inserted into wells.
•A sample DNA cotaining only specific
fragments (called ladder) can be used for
comparison.

22. RUN THE GEL:
•Apply electric current .
•DNA is negatively
charged.
•Fragments will migrate
toward the positive
charge.
•Samll fragments move

23. DNA FRAGMENTS MOVE :
•The colored solution
provides an indication
to how much the DNA
has traveled on the
gel.

24. VIEWING :
•Get can be
viewed under
UV light.

25. ADVANTAGES OF RFLP:
• Some of the advantages of RFLP are as follow.
• It is a simple process
•It is an accurate process
•No sequence information required.

26. DIS ADVANTAGES OF RFLP:
• Some of the dis advantages of RFLP as follow.
•It is an expensive process
•It is slow and time consuming process
•Labour intensive
•Requires very large amount of DNA.
•It is difficult to automate.