RFLP analysis involves using restriction enzymes to cut DNA into fragments of different lengths at specific nucleotide sequences. These fragments are then separated by gel electrophoresis based on their size and can be used to identify differences between DNA samples. DNA is extracted from samples, digested with restriction enzymes, and the fragments are run on a gel and either detected directly or transferred to a membrane through Southern blotting for detection using DNA probes. This allows analysis of genetic variation and has applications in forensics, disease detection, and genetic mapping.

1. “RESTRICTION FRAGMENT
LENGTH POLYMORPHISM
AND ITSANALYSIS”

2. PRINCIPLE
• The term Restriction Fragment Length
Polymorphism, or RFLP refers to a
difference between two or more samples
of homologous DNA molecules arising
from differing locations of restriction
sites, and to a related laboratory technique
by which these segments can be
distinguished.

3. RESTRICTION
ENDONUCLEASES(principle condt.)
Restriction endonucleases are enzymes that cleave DNA
molecules at specific nucleotide sequences depending on the
particular enzyme used. Enzyme recognition sites are usually 4
to 6 base pairs in length. Generally, the shorter the recognition
sequence, the greater the number of fragments generated. If
molecules differ in nucleotide sequence, fragments of different
sizes may be generated. The fragments can be separated by gel
electrophoresis. Restriction enzymes are isolated from a wide
variety of bacterial genera and are thought to be part of the cell's
defenses against invading bacterial viruses. These enzymes are
named by using the first letter of the genus, the first two letters
of the species, and the order of discovery.

4. APPLICATIONS OF RFLP:
RFLPs can be used in many different settings to accomplish different
objectives:
• 1- RFLPs can be used in paternity cases or criminal cases to
determine the source of a DNA sample. (i.e. it has forensic
applications).
• 2- RFLPs can be used determine the disease status of an individual.
(e.g. it can be used in the detection of mutations particularly known
muations)
• 3- RFLPs can be used to measure recombination rates which can lead
to a genetic map with the distance between RFLP loci measured in
centiMorgans.
• It is most suited to studies at the intraspecific level or
among closely related taxa. Presence and absence of
fragments resulting from changes in recognition sites are
used identifying species or populations.

5. ANALYSIS TECHNIQUE:
• The basic technique for detecting RFLPs involves fragmenting a
sample of DNA by a restriction enzyme, which can recognize and cut
DNA wherever a specific short sequence occurs, in a process known
as a restriction digestion.
• The resulting DNA fragments are then separated by length through a
process known as agarose gel electrophoresis.
• Then transferred to a membrane via the Southern blot procedure.
• Hybridization of the membrane to a labeled DNA probe then
determines the length of the fragments which are complementary to
the probe.
• Each fragment length is considered an allele, and can be used in
genetic analysis.

6. RESTRICTION DIGESTION
• Extracted DNA is digested with specific, carefully chosen,
restriction enzymes. Each restriction enzyme, under appropriate
conditions, will recognise and cut DNA in a predictable way,
resulting in a reproducible set of DNA fragments (‘restriction
fragments’) of different lengths.

7. ISOLATING DNA
• Total DNA is extracted from plant cells. Alternatively,
chloroplast and mitochondrial DNA can be used.
• DNA must be clean and of high molecular weight
• Complications:
• • Breakage during isolation
• • DNA degraded by nucleases
• • Joint isolation of polysaccharides
• • Isolation of secondary plant metabolites

9. GEL-ELECTROPHORESIS:
• DNA is cut into fragments using an enzyme.
• The cut DNA is put on a Gel material.
• An electric current is applied on the Gel.
• DNA is negatively charge.
• DNA fragments will start moving towards the positively
charged side.
• Smaller fragments move faster.
• After some time, we have a separation of the different fragment
lengths.

10. Preparing the Gel:
• Agarose powder is the basic substance for making the Gel.
• The powder is mixed with water in a container.
• The container is heated until the powder completely dissolves in
the water and the solution becomes clear.
• The liquid Gel is poured into the inner box.
• A comb like piece is put at the edge of the inner box.
• The liquid Gel is left to cool and solidify.
• When the Gel solidifies, the comb will create wells for the DNA
samples to be put.

11. Gel Ready:
• Fill the H shaped container
with water.
• Remove comb.

12. Adding DNAon the Gel
• DNA samples mixed with
colored solution and UV
reactive solution.
• DNA samples inserted into
wells.
• A sample DNA containing
only specific fragments
(called ladder) can be used
for comparison.
original
uncut DNA
DNA cut by
Hind III
ladder 1 ladder 2

13. Run the Gel:
• Apply electric current.
• DNA is negatively charged,
fragments will migrate toward
the positive charge.
• Small fragments move faster.
• The colored solution provides an
indication to how much the DNA
has traveled on the Gel.

14. Viewing:
• Gel can be viewed under UV
light.

15. Viewing
• Original uncut DNA sample makes
a sharp band at the beginning (one
big fragment).
• DNA sample cut with Hind III
makes a smear (lots of fragments of
all sizes).
• Ladders are used for comparison
(they contain specific fragments).
• We could run it for a longer time to
achieve better separation.

16. DNATRANSFER BY SOUTHERN
BLOTTING
• DNA transfer is called ‘Southern blotting’, after E.M.
Southern (1975), who invented the technique. 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.

17. DNAHYBRIDIZATION
• 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, hybridisation will occur and labelled
duplexes formed. Where conditions are highly stringent, hybridisation with
distantly related or non-homologous DNA does not happen.
• Thus, the DNA probe picks up sequences that are complementary and
'ideally‘ homologous to itself among the thousands or millions of undetected
fragments that migrate through the gel.
• Desired fragments may be detected after simultaneous exposure of the
hybridised membrane and a photographic film.
• To detect the subset of DNA fragments of interest from within all the
fragments generated by restriction digestion, a probe is needed. The DNA
probe usually comes from a DNA library (either genomic or cDNA), which
is a collection of vectors (e.g. plasmids) that contain a representation of an
original DNA molecule cut into pieces.
• Vectors may be transformed into bacteria and may multiply the piece of
DNA they contain many times.

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