Restriction Fragment Length

Polymorphism (RFLP)
 Restriction Fragment Length Polymorphism
(RFLP)
• is a molecular method of genetic analysis that allows
individuals to be identified based on unique patterns
of restriction enzyme cutting in specific regions of
DNA.
• RFLP Analysis, the technique takes advantage of the 
polymorphisms in individual people's genetic codes.
• Even though all members of a species have
essentially the same genetic makeup, these slight
differences account for variations in phenotype, such
as appearance or metabolism, between individuals.
 Restriction enzyme
• an enzyme, a protein molecule, that cuts DNA
at restriction sites.
• restriction endonuclease enzymes that cleave
deoxyribonucleic acid (DNA) at specific sites
has made it possible to identify the presence
of polymorphic regions in the isolated
fragments.
• The discovery of enzymes in bacteria
• that cleave the DNA of a foreign intruder
• is a m echanism by which bacteria protects itself from outside attack, such as
• invasion by virus. W hile these enzymes,
• called restriction nucleases, can recognize specific sites in foreign DNA and
• cleave them , they are, how ever, p re ­
• vented from attacking the DNA of the
• bacterial cell. This is accomplished by
• another enzym e which m ethylates the
• specific bacterial DNA sequence that
• would otherwise have been recognized
• by the restriction enzyme, thereby providing a m echanism for the restriction
• enzymes to distinguish betw een the bacterial and foreign DNA. These restriction
• nucleases can recognize specific DNA
• sequences four to six bases long.
Types of RE
 RFLP Analysis Technique
• RFLP analysis technique involves cutting a
particular region of DNA with known variability,
with restriction enzymes, then separating the
DNA fragments by agarose/polyacrylamide gel
electrophoresis and determining the number of
fragments and relative sizes.
• The full RFLP process requires
• probe labeling,
• DNA fragmentation,
• electrophoresis, blotting,
• hybridization,
• washing, and
• autoradiography. The detected RFLP is visualized using X-
ray film in autoradiography, where DNA fragments can be
viewed and analyzed after they are separated from one
another by electrophoresis.
 Application for RFLP Use
• DNA Fingerprinting: Forensic scientists may use RFLP
analysis to identify suspects based on evidence samples
collected at scenes of crimes. 
• Paternity: RFLP is also used in the determination
of paternity or for tracing ancestry. 
• Genetic Diversity: The technique can be used in studying
evolution and migration of wildlife, studying breeding
patterns in animal populations and the detection and
diagnosis of certain diseases.
• Genetic diseases status: reveal a carrier /disease status of the
mutant genes 
• Genetic counceling
 Genetic disease status/mutation screening
RES
Allele 1 Wildtype
Allele 2 Mutant (mutation causes loss of restriction site)

Mutation causing loss of RES

FP
Allele 1 Wildtype
208 bp RP
262 bp

Allele 2 Mutant
470 bp
 If
“No gain or loss of restriction enzymes site”
Design primer with mismatch to artificially create a restriction site in PCR amplified product