Restriction Fragment Length Polymorphism (RFLP) is a DNA analysis technique used to differentiate organisms based on DNA fragment patterns created by restriction enzymes. It has applications in forensics, disease prognosis, genetic counseling, pedigree analysis, and varietal identification, providing a method to create unique DNA profiles. While RFLP is robust and effective, it has limitations including the need for a large DNA sample and time-consuming procedures.

1. Molecular Marker
RFLP
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Synopsis
• INTRODUCTION
• RFLP
• ANALYSIS TECHNIQUE
• APPLICATION OF RFLP
• FORENSIC
• DISEASE PROGNOSIS
• GENETIC COUNSELING
• PEDIGREE
• VARIETAL
• ADVANTAGES OF RFLP
• DISADVANTAGES OF RFLP
• CONCLUSIONS
• REFERENCES

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 from one another .
First developed in the 1970s, it still has many uses.

4. RFLP
RFLP stands for "restriction fragment length
polymorphism."
The first two words refer to the process of DNA
fragmenting with specially applied enzymes.
Once this takes place, the DNA strands break up into
various lengths.
Polymorphism in Greek word means "many shapes."
This word references the multiple shapes, or lengths, that
can result in a definitive match distinctive to each human
being.

5. Analysis technique
1. Extraction
The first step is extraction of the DNA from the
sample, be it blood, saliva, semen or some other
biological sample .
2. Production of Restriction Fragments
The purified DNA is then cut into fragments by
RESTRICTION ENZYMES.

6. 3. Electrophoresis
• The restriction fragments have negative charge and can be
separated by a technique called GEL ELECTROPHORESIS, which
separates the pieces of DNA based on their size.
• with the smaller fragments migrating farther than the larger
fragments.

7. 4. DNA transfer by Southern blotting
• DNA transfer is called ‘Southern blotting’. In this method, the
gel is first denatured in a basic solution and placed in a tray.
• A porous nylon or nitrocellulose membrane is laid over the gel,
and the whole weighted down. All the DNA restriction
fragments in the gel transfer as single strands by capillary
action to the membrane.
• All fragments retain the same pattern on the membrane as on
the gel.

8. 5. DNA hybridization
• The membrane with the target DNA is incubated with the
DNA probe.
• Incubation conditions are such that if strands on the
membrane are complementary to those of the probe,
hybridization will occur and labelled duplexes formed.

9. 6. AUTORADIOGRAPHY
• An X-ray of the blot provides a picture of the
sequencing.

10. Analysis technique

11. APPLICATIONS OF RFLP
APPLICATION OF RFLP IN FORENSIC
APPLICATION OF RFLP IN DISEASE PROGNOSIS
APPLICATION OF RFLP IN GENETIC COUNSELING
APPLICATION OF RFLP IN PEDIGREE
APPLICATION OF RFLP IN VARIETAL

12. APPLICATION OF RFLP IN FORENSIC
 RFLP was one of the first applications of DNA analysis
to forensic investigation.
 Any type of organism can be identified by examination
of DNA sequences unique to that species.
 To identify individuals, forensic scientists scan 13 DNA
regions, that vary from person to person and use the data
to create a DNA profile of that individual.
 There is an extremely small chance that another person
has the same DNA profile for a particular set of 13
regions.
 Using samples from blood, bone, hair, and other body
tissues and products.

13. Some Examples of RFLP Uses for Forensic Identification
• In criminal cases, this generally involves obtaining samples from
crime-scene evidence and a suspect, extracting the DNA, and
analyzing it for the presence of a set of specific DNA regions.
• A series of probes bound to a DNA sample creates a distinctive
pattern for an individual.
• Forensic scientists compare these DNA profiles to determine
whether the suspect's sample matches the evidence sample.
• If the sample profiles don't match, the person did not contribute
the DNA at the crime scene.
• If the patterns match, the suspect may have contributed the
evidence sample.
• determine paternity; that is, who the father of the child really
is.

14. APPLICATION OF RFLP IN
DISEASE PROGNOSIS
• RFLP allows an investigator to a potentially defective
DNA sequence, it can be used effectively in the
diagnosis of diseases in which a specific mutational site.
• Diagnoses utilizing RFLP linkage analysis can be
extended further by comparing genotypes between a
known whose status is unknown, the approach is
applicable to single gene disorders inherited as
autosomal dominant , or x-linked traits .
• Cloned genes can be modified with specific mutational
changes at specific sites by using the technique of site
directed mutagenesis.

15. EX. (1) SICKLE CELLANEMIA

16. APPLICATION OF RFLP IN
GENETIC COUNSELING
• If a particular RFLP is usually associated with
a particular genetic disease, then the presence
or absence of that RFLP can be used to counsel
people about their risk of developing or
transmitting the disease.

17. APPLICATION OF RFLP IN PEDIGREE
• In humans, one mating (one pair of parents) rarely has enough
children to give reliable ratios. In that case one can still do pedigree
analysis. Modern pedigree analysis is much more sophisticated than
anything we can do.
• The pedigree shows the inheritance of a RFLP marker through three
generations in a single family. A total of 8 alleles (numbered to the
left of the blots) are present in the family. The RFLPs of each
member of the family are placed directly below his (squares) or her
(circles) symbol and RFLP numbers.

18. APPLICATION OF RFLP IN VARIETAL
• RFLP analysis is now a standard, well-tested procedure for
estimation of genetic diversity.
• We have evaluated RFLP markers as tools for assessing genetic
variation and determining the relationships among different
populations from a wide range of geographical origins and
representing Species.
• RFLP uses in Verify varietal identity and stability.

19. ADVANTAGES OF RFLPS
Highly robust methodology with good transferability between
laboratories.
Co-dominantly inherited and, as such, can estimate
heterozygosity.
No sequence information required.
Because based on sequence homology, highly recommended
for phylogenetic analysis between related species.
Well suited for constructing genetic linkage maps .

20. DISADVANTAGES OF RFLPs
Large amounts of DNA required.
Low levels of polymorphism in some species.
Need a suitable probe library.
Time consuming, especially with single-copy
probes.
Costly
Distribution of probes to collaborating
laboratories required.

21. CONCLUSIONS
• The actual analysis time is lengthy and
complicated, but is also less prone to error.
• RFLP analysis is most common techniques
used in a variety of scientific areas, including
genetics and forensics.
• Takes advantage of differences in DNA
between individuals that result in different
fragments when digested with restriction
enzymes.

22. REFERENCES
• BOOKS
Cell and molecular biology by Gerald karp 6th
edition.
• PDF FAILS ( Date- 140313,Time -08:00 PM )
1. 18591_restriction fragment length polymorphism(1).pdf
2. Mol Markers Vol1III RFLPs(1).pdf