RFLP: Restriction Fragment Length Polymorphism
• A restriction enzyme cuts the DNA molecules at every occurrence of
a particular sequence, called restriction site.
For example, HindII enzyme cuts at GTGCAC or GTTAAC
• Any mutation of a single nucleotide may destroy or create the site
(AACCTT instead of AAGCTT for Hind III)
• RFLP analysis is the detection of the change in the length of the
restriction fragments.
RFLPs associated with base changes that alter restriction endonuclease
sites. After the cleavage we will obtain DNA fragments of different
molecular weight
Restriction endonucleases cut DNA molecules at defined positions
If a mutation occurs in X chromosome in a
restriction site an individual may appear as
• Normal GAATTC
• Mutant GAGTTC
The internal EcoRI site is missing in the second individual
• sequence recognized by EcoRI
GAATTC
CTTAAG
• Mutated Sequence not recognized by the enzyme
GAGTTC
CTCAAG
Sickle Cell anemia with RFLP
• Sickle Cell anemia is caused by a mutation in the
beta-globin gene.
• The difference between the normal BA allele and
the sickle-cell BS allele is a single-nucleotide
substitution (A to T) .
• The sequence of the standard BA allele (CCTGAGG)
is cut with MstII restriction enzyme .This sequence
is altered in the BS allele (CCTGTGG).
• In the genetic test for the BS allele, total DNA from the
individual tested is digested with MstII and run in a
southern blot.
• If the normal BA allele is present, the probe produces
two smaller bands.
• If the sickle-cell BS allele is present, the probe produces
one larger band.
• Thus, a standard AA homozygote shows the two-band
pattern, a SS homozygote (with sickle-cell anemia)
shows the one-band pattern, and an AS heterozygote
(with sickle-cell trait) shows all three bands.
normal BA = two bands
sickle-cell BS allele = one larger band
AS heterozygote = all three bands
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 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
Restriction Enzyme
• A restriction enzyme is used
to cut the DNA into fragments
• Hind III restriction site
is A AGCTT
Apply Enzyme
DNA sample and Hind III are
put together in a tube
• The tube is shaken by
rotation for DNA and Hind III
to mix
Water Bath
The tube is put on a plate floating on
water at 37oC
• It is left for 30 minutes.
• This is needed for the Hind III reaction
to take place
Preparing the Gel
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
Loading DNA on the Gel
DNA samples mixed with colored solution and
UV reactive solution
• DNA samples inserted into wells
• A sample DNA containing size marker for
comparison
Run the Gel
•Apply electric current
• DNA is negatively charged
• Fragments will migrate toward the positive
charge
• Small fragments move faster
Viewing
• Original uncut DNA sample makes a sharp
band at the beginning (one big fragment)
• DNA sample cut with Hind III makes smear
(lots of fragments of all sizes)
• Ladders are used for comparison.
• We could run it for a longer time to achieve
better separation
Use of restriction enzymes in forensics –
DNA fingerprinting
• DNA is subjected to restriction
enzyme, each individual will
generate different sizes of DNA
fragments .
• The restricted DNA fragments
are then exposed to gel
electrophoresis
• Gel electrophoresis separates the
DNA fragments depending on
the size.