Restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences to distinguish between individuals. It was invented in 1984 and works by digesting DNA with restriction enzymes, which cut the DNA at specific recognition sites. This produces DNA fragments of different lengths depending on the number of recognition sites. These fragments are then separated via gel electrophoresis based on their size and can be used to generate unique fingerprints to identify individuals. RFLP analysis has applications in determining genetic diseases, paternity testing, genetic mapping, and identifying carriers of disease-causing mutations.

1. Restriction Fragment Length
Polymorphism & DNA
Fingerprint

2. Restriction fragment length polymorphism
(RFLP)
• It is a technique that exploits variations in homologous DNA sequences,
known as polymorphisms, in order to distinguish individuals, populations, or
species or to pinpoint the locations of genes within a sequence.
• This technique was invented in 1984 by the English scientist Alec Jeffreys
during research into hereditary diseases.
• It is used for the analysis of unique patterns in DNA fragments in order to
genetically differentiate between organisms.
• RFLP analysis requires investigators to dissolve DNA in an enzyme that
breaks the strand at specific points.

3. PRINCIPLE
• Restriction endonucleases are enzymes that cut lengthy DNA into short
pieces. Each restriction endonuclease targets different nucleotide sequences
in a DNA strand and therefore cuts at different sites.
• The distance between the cleavage sites of a certain restriction endonuclease
differs between individuals. Hence, the length of the DNA fragments
produced by a restriction endonuclease will differ across both individual
organisms and species.

4. How does it Work?
• RFLP is performed using a series of steps:
DNA Extraction
• To begin with, DNA is extracted from blood, saliva or other samples and purified.
DNA Fragmentation
• The purified DNA is digested using restriction endonucleases. The recognition sites of these
enzymes are generally 4 to 6 base pairs in length. The shorter the sequence recognized, the
greater the number of fragments generated from digestion.
• For example, if there is a short sequence of GAGC that occurs repeatedly in a sample of
DNA. The restriction endonuclease that recognizes the GAGC sequence cuts the DNA at
every repetition of the GAGC pattern.
• If one sample repeats the GAGC sequence 4 times whilst another sample repeats it 2 times,
the length of the fragments generated by the enzyme for the two samples will be different.

6. Gel Electrophoresis
• The restriction fragments produced during DNA fragmentation are analyzed using
gel electrophoresis.
• The fragments are negatively charged and can be easily separated by electrophoresis,
which separates molecules based on their size and charge. The fragmented DNA
samples are placed in the chamber containing the electrophoretic gel and two
electrodes.
• When an electric field is applied, the fragments migrate towards the positive
electrode. Smaller fragments move faster through the gel leaving the larger ones
behind and thus the DNA samples are separated into distinct bands on the gel.
• The gel is treated with luminescent dyes in order to make the DNA bands visible.

8. Applications of RFLP
• To determine the status of genetic diseases such as
Cystic Fibrosis in an individual.
• To determine or confirm the source of a DNA
sample such as in paternity tests or criminal
investigations.
• In genetic mapping to determine recombination
rates that show the genetic distance between the loci.
• To identify a carrier of a disease-causing mutation
in a family.

10. DNA FINGERPRINTING
• DNA fingerprinting also known as genetic fingerprinting, DNA typing, and DNA
profiling is a molecular genetic method that enables identification of individuals
using hair, blood, semen, or other biological samples, based on unique patterns
(polymorphisms) in their DNA.
• Different DNA fingerprinting methods exist, using either restriction fragment length
polymorphism (RFLP), polymerase chain reaction (PCR), or both.
• Each method targets different repeating polymorphic regions of DNA, including
single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs).
• The odds of identifying an individual correctly depend on the number of repeating
sequences tested and their size.

12. STEPS IN DNA FINGERPRITING
• Extracting the DNA from cells
• Cutting up the DNA using an enzyme
• Separating the DNA fragments on a gel
• Transferring the DNA onto paper
• Adding the radioactive probe
• Setting up the X-ray film

13. Extraction of DNA
• All cells (except red blood cells) in all living creatures contain DNA.
• The first step in DNA fingerprinting is getting DNA in a pure form. The blood is treated with a series of chemicals
until pure DNA emerges as a white solid. The DNA is stored, dissolved in essentially water, in a small plastic tube
and kept in a fridge until ready for the next stage.
Cutting up the DNA
• Freshly extracted DNA in water is quite sticky, because the DNA strands are very long. They are too long to be
separated in the gel in the next stage. The next step is to cut up the DNA strands using a restriction enzyme. This
restriction enzyme doesn’t cut randomly in the DNA, but at specific letter sequences. This stage involves adding the
restriction enzyme (colourless liquid) to the DNA (another colourless liquid), using a pipette. The enzyme takes a
few hours to cut at all the places it can in the DNA strands.
Separating the DNA fragments on a gel
• Gel electrophoresis

14. Transferring the DNA onto paper
• The gel-separated DNA fragments are converted to single stranded fragments by dunking the gel in weak acid, so
that the DNA letters are exposed, rather than being in the middle of the double helix. The gel-separated DNA
fragments are then transferred to white nitrocellulose paper, so the paper now carries an exact replica of the DNA
on the gel. This is called “Southern blotting”.
Adding the radioactive probe
• Probe determines which DNA fragments can be seen at the end of experiment. It is a small chunk of radioactive
DNA of a particular sequence of letters. The probe sticks to the fragments of the DNA that has the matching
sequence, but only those fragments that have the matching sequence of letters, no other fragments.
• In DNA fingerprinting the probe is a sequence of 33 letters that is found in the repeated “stutters” of the genome.
Therefore, only the DNA fragments that contain these repeated “stutters” are seen at the end of the experiment.
They are seen as the dark bands
• The nitrocellulose paper and the probe (colourless, radioactive liquid) are placed together in a glass tube in a
hybridisation oven at 65 degrees Celsius(think a rotisserie) for an hour or two, so that the probe covers the paper
and can stick to the DNA fragments with the matching sequence. The nitrocellulose paper is then rinsed to remove
any radioactive probe liquid that has not stuck.

16. Fields Where DNA Fingerprinting Is Beneficial
The most common uses for DNA fingerprinting include:
Forensics: DNA fingerprinting can be accomplished with a very small quantity of DNA and is a sure-fire way to
"finger" a culprit in a crime. Similarly, DNA fingerprinting can and does exonerate innocent people of crimes—
sometimes even crimes committed years ago. DNA fingerprinting also can be easily used to identify a decomposing
body.
Noncriminal Identification: Is Joe really Billy's father? DNA fingerprinting can answer that question quickly and
accurately. In addition to identifying adoptive children and settling paternity suits, DNA fingerprinting also has been
used to establish a relationship in the case of inheritance. More than once, DNA fingerprinting has made it possible for
people separated as a result of natural disaster or war to find their children and parents.
Medicine: One important instance is identifying good genetic matches for organ or marrow donation. Doctors also are
beginning to use DNA fingerprinting as a tool for designing personalized medical treatments for cancer patients.
Moreover, DNA fingerprinting has been used to ensure that a tissue sample has been correctly labeled with the right
patient's name.
Agriculture: All living things have DNA, which means that DNA fingerprinting can be used to identify genetically
modified plants or plants that are likely to have therapeutic value. It also can be used to prove pedigree in valuable
animals such as racehorses.

17. THANK YOU FOR YOUR ATTENTION