Restriction Fragment

Length Polymorphism
(RFLP)
Dr. Sundus Akhtar
Assistant Professor
School of Botany
Minhaj University, Lahore.
What is RFLP ?
 Restriction Fragment Length Polymorphism is a variation
in the length of a DNA fragment produced by a
specific restriction enzyme acting on a DNA from
different individuals that usually results from a genetic
mutation.

 If two organisms differ in the distance between site of

cleavage of a particular restriction endonuclease, the
length of the fragments produced will be different when
the DNA is digested with a restriction enzyme.

 RFLP analysis is the detection of the change in the

length of the restriction fragments.
 The similarity of the patterns generated can be used to
differentiate species (and even strains) from one another.
 Polymorphisms are inherited differences
found among the individuals in more than 1%
of normal population.
 A restriction enzyme cuts the DNA molecule at every
occurrence of a particular sequence, called restriction
site.

 For Example, HindIII enzyme cuts at AAGCTT.

 If we apply a restriction enzyme on DNA, it is cut every

occurrence of the restriction site into a million
restriction fragments each a few thousands nucleotides
long.
 Any mutation or polymorphism of a single nucleotide may
destroy (AAGCTT for HindIII) and change the length
of the fragment.

 The term polymorphism refers to the slight differences

between individuals, in base pair sequences of genes
or
 A polymorphism is a clinically harmless DNA variation
that does not affect the phenotype.

 So RFLP analysis is a technique which is used to detect

the change in the length of the restriction fragments.
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.
Restriction Endonucleases
 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.
RFLP Technology
 RFLP detection depends on the possibility of comparing
band profiles generated after restriction enzyme
digestion of target DNA. These differences in fragment
lengths can be seen after gel electrophoresis,
hybridization and visualization. The basic steps involved
are as follows:

1. Isolation of DNA
2. Restriction Digestion & Gel Electrophoresis
3. DNA transfer by Southern blotting
4. DNA hybridization
(1) Isolation of DNA
 Isolating DNA is the first step for many DNA-based
technologies. DNA is found either in nuclear
chromosomes or in organelles (mitochondria and
chloroplast).

 To extract DNA from its location, several laboratory

procedures are needed to break the cell wall and nuclear
membrane, and so appropriately separate the DNA from
other cell components.

 When doing so, care must be taken to ensure the

process does not damage the DNA molecule and that it
is recovered in the form of a long thread.
 Lysis of cells:
 Through liquid nitrogen
 Through broken glass
(2) Restriction Digestion & Gel
Electrophoresis

 Extracted DNA is digested with specific, carefully

chosen, restriction enzymes.

 Each restriction enzyme, under suitable conditions, will

recognize and cut DNA resulting in a restriction
fragments of different lengths.

 The thousands of restriction fragments produced are

commonly separated by Gel Electrophoresis on agarose
gels.
 For visualization of DNA bands on the gel, it is stained
with ethidium bromide. But staining alone cannot detect
polymorphisms.

 Hybridization must therefore be used to detect specific

fragments.
(3) DNA transfer 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 of NaOH and placed in a tray. A porous Nylon or
nitrocellulose membrane is laid over the gel.

 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.
(4) DNA Hybridization

 The membrane with the target DNA is exposed to the

DNA probe (radioactively labelled). On the basis of
availability and complementarity, hybridization will
occur.

 The DNA probe is a singe-stranded molecule,

conveniently labelled, using any standard method (e.g. a
radioisotope), and hybridize with the target DNA, which
is stuck to the membrane.
 Thus the DNA probe binds with the sequences that are
complementary to it among the thousands or millions
of undetected fragments that migrate through the
gel.

 Desired fragments maybe detected after simultaneous

exposure of the hybridized probe to X-rays, which on
exposure appear as black spots on the X-ray film.
1. The first step in this process is to isolate the DNA from the target.
2. Once the the DNA is isolated from the sample it is subjected to
restriction digestion using restriction enzymes.
3. The digested DNA sample is then subjected to gel electrophoresis,
in which the DNA is separated based on its size. Many DNA
fragments with slight differences in length are produced.
4. The gel is then exposed to a chemical to denature double-stranded
DNA to become single- stranded.
5. This is followed by southern blotting where DNA is transferred
from gel to nylon membrane.
6. The nylon membrane is then exposed to solution with radioactive
complementary nucleotide probes that hybridize to specifically
chosen DNA sequences on nylon membrane.
7. The membrane is then placed against X- ray film, where hybridized
radioactive probes cause exposure of X-ray film, producing an
autoradiogram.
8. RFLP analysis is carried out to detect differences in pattern to
confirm polymorphisms.
 Isolation of sufficient DNA for RFLP analysis is time-
consuming and labor intensive. However, PCR can be
used to amplify very small amounts of DNA, usually in 2-
3 hours, to the levels required for RFLP analysis.
Therefore, more samples can be analyzed in a shorter
time.
Applications of RFLP

 The important applications of RFLP are:

1. RFLP can be used paternity cases or criminal cases to

determine the source of a DNA sample (i.e. it has
forensic applications).

2. RFLP can be used to detect mutations.

Advantages of RFLP

 Some of the advantages of RFLP are as follow:

1. It is a simple process.

2. It is an accurate process.

3. No sequence information required.

 Disadvantages of RFLP
 Some of the disadvantages of RFLP are as
follow:

1. It is an expensive process.

2. It is slow and time consuming process.

3. Labour intensive.

4. Requires very large amount of DNA.