RFLP is a technique that uses restriction enzymes to cut DNA into fragments of varying lengths that can then be used to differentiate between organisms. It involves digesting DNA with restriction enzymes, separating the fragments via gel electrophoresis, transferring the fragments to a membrane, and detecting specific fragments with labelled probes. RFLP has applications in paternity testing, disease detection, genetic mapping, and studying genetic diversity in populations. However, it is also a slow and cumbersome technique that requires a large DNA sample.

1. RFLP
Dileep.R
2nd Msc Biochemistry

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

3. Introduction
Restriction Fragment Length Polymorphism (RFLP) is a technique in 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
is obtained and the desired fragment is
detected by using restriction probes.
Southern hybridization using restriction
endonuclease enzymes for isolation of
desired length of DNA fragments is an
example of RFLP.

5. Methods outline

6. Methodology
Step I: Restriction digest
● Extraction of desired fragments of DNA using restriction endonuclease (RE).
● The enzyme RE has specific restriction site on the DNA, so it cut DNA into
fragments. Different size of fragments are generated along with the specific
desired fragments.
Step II: Gel electrophoresis
● The digested fragment are run in polyacrylamide gel electrophoresis or
Agarose gel electrophoresis to separate the fragments on the basis of length
or size or molecular weight.
● Different size of fragments forms different bands.

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 charge
membrane ie. Nitrocellulose paper by the process called capillary
blotting or electro-blotting.

8. cont...
Step V: Baking and blocking
● The nitrocellulose paper transferred with DNA 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 color band under visualization 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 in the 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 chromosomes 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 three individuals and subjected to RFLP analysis. The
results are shown below, 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!