RESTRICTION

FRAGMENT
LENGTH
POLYMORPHISM
 What is rflp
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.
Commonly pronounced rif-lip.
Its analysis was the first DNA profiling technique
cheap enough to see widespread application.
It is an important tool in genome mapping.
Localization of genes for genetic disorders.
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.
If we apply a restriction enzyme on DNA, it is
cut at every occurrence of the restriction site into
a million restriction fragments each a few
thousands nucleotides long.
Any mutation of a single nucleotide may destroy
or create the site(CTGCAC or CTTAAC for HindII)
and alter the length of the corresponding
fragment.
The term polymorphism refers to the slight
RFLP analysis may be subdivided into single-
(SLP) and multi-locus probe (MLP) paradigms.
Usually, the SLP method is preferred over MLP
because it is more sensitive, easier to interpret
and capable of analyzing mixed-DNA samples.
 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.
DNA Sample
Restriction 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.
The tube is put on a plate floating on water at
37C.
It is left for 30 minutes.
This is needed for the Hind III reaction to take
place.
Preparing the Gel
Putting DNA on the Gel
 Viewing
Original uncut DNA sample makes a sharp band
at the beginning (one big fragment).
DNA sample cut with Hind III makes s 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.