The document provides an overview of the Restriction Fragment Length Polymorphism (RFLP) technique used for DNA analysis, which differentiates organisms based on DNA cleavage patterns. It outlines the process of RFLP, including the steps of DNA digestion, gel electrophoresis, and hybridization, as well as its applications in forensics, genetic mapping, and disease detection. The advantages and disadvantages of RFLP are also discussed, highlighting its effectiveness and challenges.

1. K. Narayanapura, Kothanur (PO), Bengaluru 560077
Tel+91 80 – 68737777 / 28465770 /28465353 Fax. 080- 68737799
e-mail:info@kristujayanti.com, www.kristujayanti.edu.in
Dr. Manikandan Kathirvel
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Bengaluru
Restriction fragments length
polymorphism(RFLP)

2. Introduction to RFLP:
 Definition: it is a technique in which organism may be
differentiated by analysis of pattern derived from cleavage
of their dna.
 It exploits variations in homologous chromosome.
 RFLP is an enzymatic procedure for separation and
identification of desired fragments of DNA.
 Using restriction endonuclease enzymes fragments of Dna
obtained and the desired fragments is detected by using
restrictions probes.
 An rflp occurs when the length of a detected fragments
varies between individuals.
 RFLP analysis technique involves cutting a particular region
of DNA with known variability, with restriction enzymes,
then separating the DNA fragments by agarose gel
electrophoresis and determining the number of fragments
and relative sizes.

3. Principle :
 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 and differences of the patterns thus generated
can be used to differentiate species (and even strains) from one
another.
Purpose:
RFLP test is used in identification and differentiation of organisms by
analyzing unique patterns in genome. It is also used in identification of
recombination rate in the loci between restriction sites.

4. Pattern generated depends mainly
on…
 Difference in dna of selected strains.
 Restriction enzymes used.
 Dna probe employed for southern hybridization.
 Point and Frameshift mutations.
 Difference in alleles for a particular sequence.

5. Restrictions enzymes used… ..
Endonuclease that cleaves the double stranded
dna at specific 4-8 nucleotide long palindromic
sequence.
They recognize specific sequences in DNA and
then cut the DNA and then cut the DNA to
produce fragments, called restriction fragments.

6. Steps:
Step 2. Restrictions digest
The DNA in each sample is digested with the same restriction enzyme(s). 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 3.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.

7. STEP 4. DENATURATION
THE GEL IS PLACED IN SODIUM HYDROXIDE (NAOH) SOLUTION FOR
DENATURATION SO THAT SINGLE STRANDED DNA ARE FORMED.
STEP 5. BLOTTING.
THE SINGLE STRANDED DNA OBTAINED ARE TRANSFERRED INTO CHARGE
MEMBRANE I.E. NITROCELLULOSE PAPER BY THE PROCESS CALLED CAPILLARY
BLOTTING OR ELECTRO-BLOTTING.
STEP 6.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 Labeled Probe Nonspecifically To The
Charged Membrane.

9. Step 7: Hybridization and visualization
The labeled RFLP probe is hybridized with DNA on the
nitrocellulose paper. The RFLP probes are complimentary as well as
labeled with radioactive isotopes so they form color band under
visualization by autoradiography.

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

12. Rflp results

13. Advantages
 Produces semi-dominant markers, allowing
determination of homozygosity, or heterozygosity.
 • Stable and Reproducible, gives constant results over
time, and location.
 No prior information on DNA sequence is required.
 Relatively simple technique.

14. Disadvantages
Very long methodology before results are gained.
•High labour requirements.
High quality, and large quantities of DNA must be used.
•Must frequently work with radio isotopes.
•Many probes are not available depending on species.
•Too many polymorphisms may be present for a short probe.
•Cost of development is very high due to time, and labour
requirements
•Low frequency of desired polymorphisms in polyploid plants (eg.
Wheat).

15.  Thank you