Restriction Fragment Length Polymorphism

1. R F L P
Restriction Fragment Length Polymorphism

2. CONTENTS
● Introduction
● Principle
● Methodology
● Uses & Application
● Limitations

3. Introduction
Restriction Fragment Length Polymorphism (RFLP) is a technique by which
organisms may be differentiated by analysis of patterns derived from
cleavage of their DNA. If two organisms differ in the distance between sites
of cleavage of a particular restriction endonuclease, the length of the
fragments produced will differ when the DNA is digested with a restriction
enzyme. The similarity of the patterns generated can be used to
differentiate species (and even strains) from one another.
Restriction fragment length polymorphism (RFLP) is a technique invented in
1984 by the English scientist Alec Jeffreys during research into hereditary
diseases.

4. Principle
RFLP is an enzymatic procedure for
separation and identification of desired
fragments of DNA. Using restriction
endonuclease enzymes, fragments of DNA
are generated and the desired fragments
are detected using restriction probes. An
example of RFLP is Southern hybridization
which isolates desired length of DNA
fragments.

5. Methods outline

6. Methodology
Step I: Restriction digest
● Extraction of desired fragments of DNA using restriction endonuclease(RE).
● The enzyme has specific restriction sites on the DNA, so it cuts the DNA
into fragments. Different sizes of the fragments are generated along with
thespecific desired fragments.
Step II: Gel electrophoresis
● The digested fragments are run in Agarose gel electrophoresis to separate
the fragments on the basis of their lengths, sizes or molecular weights.
● Different sizes of fragments form differentbands.

7. cont...
Step III: Denaturation
The gel is placed in sodium hydroxide (NaOH) solution for
denaturation so that single stranded DNA are formed.
Step IV: Blotting
The single stranded DNA obtained are transferred into charged
membrane (nitrocellulose paper) by the process called capillary
blotting or electro-blotting.

8. cont...
Step V: Baking and blocking
● The nitrocellulose paper to which the DNA was transferred is fixed by
autoclaving.
● Then the membrane is blocked by using bovine serum albumin or casein
to prevent binding of labelled probe nonspecifically to the charged
membrane.
Step VI: Hybridization and visualization
● The labelled RFLP probe is hybridized with DNA on the nitrocellulose
paper.
● The RFLP probes are complimentary as well as labelled with radioactive
isotopes so they form coloured bands when visualized by
autoradiography.

10. Applications
1. In paternity cases or criminal cases to determine the
source of a DNA sample. (i.e. it has forensic
applications).
2. Determining the disease status of an individual. (e.g. it
can be used for detection of mutations)
3. To measure recombination rates which can lead to a
genetic map with the distance between RFLP loci.
4. In the characterization of genetic diversity or breeding
patterns in animal populations.
5. RFLP has been developed for chromosome mapping
of humans, mice, maize, tomato, rice, etc.

11. Limitations
● Slow
● Cumbersome
● Requires a large amount of sample DNA.
● Automation not possible
● Low levels of polymorphism in some species
● Need a suitable probe library
● Needing the combined process of probe labeling, DNA
fragmentation, electrophoresis, blotting, hybridization, washing,
and autoradiography.

12. Example case
Paternity Case
Let's use RFLP technology to determine if Jack is the father of Jill's
child named Payle. In this scenario, DNA was extracted from white blood
cells from all the three individuals and subjected to RFLP analysis. The
results are shown below.

13. In this case, it appears that Jack could be
the father, since Payle inherited the 12.4
kb fragment from Jill and the 4.3 fragment
from Jack. However, it is possible that
another man with similar RFLP pattern
could be as well.To be certain, several
more RFLP loci would be tested. It would
be highly unlikely that two men (other than
identical twins) would share multiple RFLP
patterns and so paternity could be
confirmed.

14. Thank you!