This document discusses Restriction Fragment Length Polymorphism (RFLP), a genetic marker technique. RFLP involves digesting genomic DNA with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and detecting variations in fragment lengths between individuals. The key steps are isolating DNA from samples, restriction enzyme digestion of the DNA, electrophoresis to separate fragments, Southern blotting to transfer DNA to a membrane, and autoradiography to detect polymorphisms. RFLP has advantages like using reliable genotypic characteristics and being co-dominant, but also disadvantages like being time-consuming and multi-step. RFLP can be applied to mapping chromosomes, assessing genetic diversity, and distinguishing

1. Dr. T. Ramesh
Assistant Professor
PG & Research Department of Zoology
Vivekananda College
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM

2. Definition
Post Graduate and Research Department of Zoology
RFLP - RESTRICTION FRAGMENT LENGTH POLYMORPHISM
RFLP & is very useful study in Genomic DNA Sequence.
RFLP has been developed for chromosomes mapping of
humans, mice, maize, tomato, rice, etc.
RFLP is a non-PCR based method .
RFLP based Genetic Marker.
In this Method DNA is digested with restriction Enzymes.
RFLP is 1-10 loci detected

3. Post Graduate and Research Department of Zoology
“The Variations in the Restriction DNA Fragments length between
individuals of a species is called RFLP.”
1. BOTSTEIN.et. al.(1980)-The DNA sequence variation Detected
by this method was termed RFLP.
2. GRODZICKER first Described this phenomenon for mutant
strain of adenoviruses.
Definition and History

4. Analysis
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STEPS:
1.Genomic DNA isolation.
2. Digestion with Restriction enzyme.
3. Fractions on a Agarosegel.
4. DNA in millions of restriction fragments fractionated in the gel
by Molecular marker weight .
4. DNA transferred out of the Gel on to a membrane filter southern
Hybridization.
5. Autoradiography
6. RFLP pattern with positive Bands.

6. Procedure
SAMPLE– COLLECTIONS
Tissue are cells of individual are collected to extract their DNA.
The sample are collected separately.
ISOLATION OF DNA
DNA of the collected samples is isolated separately by standard
procedure& purified to get genomic DNA.
RESTRICTION – DIGESTION
Genomic DNA of each samples is cut with a restriction enzyme separately
To generate variable length of DNA fragments
Restriction enzyme such as EcoR1, HINDIII, Pst, etc,are of much use for
RFLP.
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7. Procedure
ELECTROPHORESIS
The Digested genomic DNA of the samples are loaded into separate cells in
agarose or polyacrelamide gel &subjected to electrophoresis.
BLOTTING OF DNA
As in southern blotting the DNA is transferred to a nitrocellulose filter by
using a blotting set up
AUTOREDIOGRAPHY
Images of radioactive probes are captured on an x- Ray film using
autoradiography.
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8. Procedure
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9. Action of Restriction Enzymes
Total DNA Size 6.5 kb
EcoR1 3kb 3.5kb Two Fragments
Hind III 2kb 4.5kb Two fragments
EcoR1+HindIII 2kb 1kb 3.5kb Three Fragments
A B C
EcoR1- A+B (2+1 kb)
EcoR1- C (3.5kb)
Hind III- A (2kb)
Hind III- B+C (1+3.5 kb)
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A B C

10. Advantages and Disadvantages
Results are based on reliable genotypic characteristics' rather than on
phenotypes.
RFLP based Genetic Marker
RFLP is the co dominant marker
RFLP & is very useful study in Genomic DNA Sequence
ADVANTAGES
Time consuming.
Multistep procedure.
Post Graduate and Research Department of Zoology
DISADVANTAGES

11. 1. RFLP technique can be used in
Forensic Medicines.
2. To characterize Germplasm
resources .
3. Use of genotype specific RFLP
patterns can be done to
distinguish verities in crop like
maize, rice, soya bean, potato
etc.
4. To assess genetic diversity.
5. To identify Breeding lines &
varieties
Applications
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12. THANK YOU.