This document discusses Restriction Fragment Length Polymorphism (RFLP) analysis. RFLP is a technique used to detect genetic mutations and variations between individuals. It works by digesting DNA with restriction enzymes, which cut the DNA into fragments of varying lengths. These fragments are then separated via gel electrophoresis and analyzed to detect any length polymorphisms between individuals, indicating genetic differences. RFLP has applications in forensics, mutation detection, and requires isolating DNA, restriction digestion, gel electrophoresis, Southern blotting, and DNA hybridization.

2. Restriction Fragment
Length Polymorphism
(RFLP)
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Basit Ali (38)
Muhammad Saleh

3. 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.

4. Continuous
 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.

5. Continuous
 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.

6. 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

7. (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.

8. (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.

9. Continuous
 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.

10. (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.

11. (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.

12. Continuous
 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.

14. 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.

15. 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.

16. 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.
5. It is difficult to automate.