Restriction Fragment Length Polymorphism
(RFLP)
Presented by
Salina pudasaini
[Link] 7th semester
Introduction
• RFLP is a molecular technique used to detect variations in DNA
sequences by cutting DNA with restriction enzymes and analyzing the
different lengths of the resulting fragments on agarose gel
electrophoresis.
• RFLP analysis is now mostly obsolete because modern DNA
sequencing is cheaper and faster.
However, it was the first affordable DNA profiling method to be widely
used.
• In the past, RFLP played an important role in:
-Genome mapping
-Locating genes responsible for genetic disorders
-Determining the risk of inherited diseases
-Paternity testing and early DNA fingerprinting
History
✓RFLP was developed in 1984 by Alec Jeffreys, an English scientist,
while studying hereditary diseases.
✓ He discovered that people have unique patterns in their DNA
fragments, which can be used to differentiate individuals.
✓ These patterns are called VNTRs (Variable Number of Tandem
Repeats).
✓Genetic polymorphism means the normal inherited differences in
DNA seen in more than 1% of a population.
✓RFLP uses these natural DNA differences to study variation within
a species (intraspecies) and between species (interspecies).
Principle
• RFLP is an enzymatic method used
for the separation and identification
of specific DNA fragments.
Restriction endonuclease enzymes
are used to cut DNA into fragments,
and the desired fragment is
detected using DNA probes.
Southern hybridization with
restriction enzymes to isolate DNA
fragments of specific lengths is a
common example of RFLP.
Restriction
endonuclease
Restriction Enzymes (RE) are enzymes that cut DNA at
specific sequences.
Also called “molecular scissors” because they can
precisely cut DNA.
Naturally found in bacteria as a defense against viruses
(they destroy foreign DNA).
Key tool in molecular biology, genetic engineering, and
DNA analysis.
Hundreds of restriction enzymes are now available
commercially.
Most labs use Type II REs(eg: EcoRI, HindIII,
BamHI)because of high specificity and simplicity.
.
1. Recognition
➢Restriction enzymes recognize specific
DNA sequences (usually 4–8 bp).
➢These sequences are called
recognition sites.
➢Most recognition sites are palindromic
(read the same forwards & backwards).
➢Example: EcoRI recognizes GAATTC
and cuts between G and A.
2. Binding
➢The enzyme binds to DNA at its
Cont... specific recognition site.
➢Binding is highly specific, ensuring only
the target sequence is cut.
3. Cleavage
➢The enzyme cuts DNA at or near the recognition site, producing
fragments.
➢Can produce sticky ends (overhanging single strands) or blunt ends
(straight cuts).
• Blunt Ends – where the DNA strands are cut straight across, producing flush
ends.
• Sticky Ends – where the cuts leave overhanging single-stranded DNA,
facilitating easier recombination.
➢Fragments are then used in RFLP analysis.
4. Detection in RFLP
➢DNA fragments are separated by gel electrophoresis.
➢Southern blotting with labeled probes detects specific fragments.
➢Differences in fragment lengths between individuals indicate
polymorphisms.
Steps/procedure
1. DNA extraction
2. Restriction enzyme
digestion
3. Gel electrophoresis
4. Southern blotting
5. Probe hybridization
6. Detection of fragment
pattern
Steps...
1. DNA Extraction
➢DNA is isolated from cells or tissue samples (blood, saliva, plant/animal
tissue).
➢High-quality, intact DNA is required for accurate analysis.
➢Common methods: phenol-chloroform extraction, spin column kits, or
magnetic beads.
2. Restriction Enzyme Digestion
➢DNA is cut into fragments using restriction enzymes (molecular scissors).
➢Each enzyme recognizes a specific sequence in DNA.
➢The cutting generates fragments of variable lengths, depending on the DNA
sequence.
[Link] Electrophoresis
.
➢DNA fragments are separated by size using agarose gel
electrophoresis.
➢Smaller fragments move faster through the gel than larger fragments.
➢This produces a pattern of bands corresponding to fragment sizes.
4. Southern Blotting
➢DNA fragments are transferred from the gel to a nylon or nitrocellulose
membrane.
➢This preserves the fragment pattern and makes them easier to probe.
➢Involves DNA denaturation so fragments can bind to the membrane.
.
5. Probe Hybridization
➢A labeled DNA probe complementary to a specific fragment is added.
➢A radioactive or fluorescent DNA probe is added.
➢Probe binds (hybridizes) only to its matching DNA sequence.
➢Labels can be radioactive, fluorescent, or chemiluminescent for detection.
6. Detection of Fragment Pattern
➢After hybridization, the bound probe reveals specific fragments.
• If radioactive probe → X-ray film shows bands.
• If fluorescent probe → UV or special scanner shows bands.
• Each band represents a DNA fragment of a specific length, and the pattern is
compared .
➢Differences in fragment length indicate polymorphisms.
➢Patterns can be used for genetic fingerprinting, disease gene mapping, or paternity
testing.
Applications
1. Forensic and Paternity Testing 4. Genetic Diversity and Breeding
➢Used in criminal investigations to identify the ➢ Characterizes genetic diversity in
source of a DNA sample. populations.
➢Helps in paternity testing by comparing DNA ➢ Useful in breeding programs for animals or
fragment patterns. plants.
2. Genetic Disease Detection 5. Chromosome Mapping in Various
➢Can detect mutations in specific genes. Organisms
➢ Applied in humans, mice, maize, tomato,
➢Helps determine the disease status of an
individual. rice, and other species.
➢ Important in studying inheritance patterns
and gene localization.
3. Genome Mapping
➢Measures recombination rates between RFLP
markers.
➢Creates genetic maps showing distances
between genes or loci.
USES
RFLP in Microbiology:
• RFLP analysis is a powerful tool in microbiology, offering several key
uses:
• It aids in identifying and differentiating microbial strains, which is
essential for tracking outbreaks and studying microbial diversity.
• RFLP helps detect genetic variations and mutations in microorganisms,
contributing to understanding antibiotic resistance and pathogenicity.
• This technique supports the construction of genetic linkage maps in
microbes, facilitating research on gene organization and recombination
events.
Limitations
➢Slow and labor-intensive process.
➢Cumbersome; requires multiple steps.
➢Needs a large amount of DNA sample.
➢Not easily automated.
➢Some species have low levels of polymorphism, limiting
usefulness.
➢Few loci are detected per assay.
➢Requires a suitable probe library.
.
Thank you