FOR MBBS

1. Dr. Vishnu Kumar
Professor, Department of
Biochemistry, SRMSIMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
DNA Finger & DNA Foot Printing
Restriction fragment
length polymorphism ( RFLP)

2. Learning Objectives
After completion of this lecture learner
should be able to define :
 DNA Finger Printing
 Restriction fragment length
polymorphism ( RFLP)
 Applications of RFLP
 DNA Foot Printing

3. Restriction fragment length
polymorphism ( RFLP)
• Traditionally the most suitable method
to detect a criminal in the scene of
crime is matching of finger prints. With
discovery of RDT a more useful tool is
now RFLP or DNA finger printing.
• Polymorphism Genetic diversity in a
population is called polymorphism.

4. RFLP
• It implies that the DNA sequence of one
individual differs from that of the other.
Some of these changes may affect the
recognition site of RE. So the size of
the restriction fragments will be
different in different individual if their
genome is subjected to the same RE.
Such variation in the pattern of
restriction fragments in the population
is referred to as RFLP.

5. Restriction Fragment Length Polymorphism (RFLP) is
a technique in which organisms may be differentiated
by analysis of patterns derived from cleavage of their
DNA.
A restriction fragment length polymorphism (RFLP) is
a genetic variant that can be examined by cleaving
the DNA into fragments (restriction fragments) with a
restriction enzyme.

6. Seven steps to understanding DNA
fingerprinting:
1. Extracting the DNA from cells.
2. Cutting up the DNA using an enzyme.
3. Separating the DNA fragments on a gel.
4. Transferring the DNA onto paper.
5. Adding the radioactive probe.
6. Setting up the X-ray film.
7. Yes - we've got the result!

7. Any mutation of a single nucleotide may destroy
(CTGCAC or CTTAAC for HindII) and alter the length
of the corresponding fragment.
 The term polymorphism refers to the slight
differences between individuals, in base pair sequences
of common genes.
or
 A polymorphism is a clinically harmless DNA variation
that does not affect the phenotype.
RFLP analysis is the detection of the change in the
length of the restriction fragments.

9. Isolating DNA is the first step for many DNA-based
technologies. DNA is found either in nuclear
chromosomes or in organelles (mitochondria and
chloroplasts).
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.

10. Extracted DNA is digested with specific, carefully
chosen, restriction enzymes.
Each restriction enzyme, under appropriate
conditions, will recognize and cut DNA in a
predictable way, resulting in a reproducible set of
DNA fragments (‘restriction fragments’) of different
lengths.
The millions of restriction fragments produced are
commonly separated by electrophoresis on agarose
gels. Because the fragments would be seen as a
continuous ‘smear’ if stained with ethidium bromide,
staining alone cannot detect the polymorphisms.
Hybridisation must therefore be used to detect
specific fragments.

11. 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.

12.  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 hybridized membrane and a photographic film.

13. RFLP Technology Pictures
After agarose has been
poured into the gel
mould, combs are
immediately inserted to
form wells and left until
the gel hardens.
The combs are then
removed and the gel
placed in an
electrophoresis
chamber.

14. Samples of
digested DNA,
with bromophenol
blue dye added,
are loaded into
the wells
with a pipette.

15. After electrophoresis,
the gel is treated with
NaCl to break the
DNA double helix
bonds and make it
single-stranded.
This allows later
hybridisation with a
single-stranded DNA
probe.

16. The blotting tray is
first prepared by
saturating
sponges with
NaOH.
Safety glasses
and gloves are
required, and a
laboratory coat
recommended.

17. Absorbent paper is placed on top of
the sponges to prevent direct contact
with the gel.

18. Bubbles between the absorbent paper and sponges are removed
by rolling a pipette or a glass rod across the paper. This ensures
a complete transfer of the solution all through
the gel.

19. The treated agarose gel is
placed on top of the
absorbent paper.

20. Membrane is cut
into the appropriate
size.
The membrane is
placed on top of the
gel, then covered
with a piece of
absorbent paper.

21. The entire set-up is
topped with a weight
(here, a bottle of water
standing on a piece of
glass) to promote good
transfer.
After some hours the
transfer is complete, the
blotting
paper is taken away, and
the membrane stored
until hybridisation with
the probe.

22. The process of hybridisation begins. A DNA is
boiled to denature it to single strands.

23. The labeled probe is
added to the container
with the hybridisation
solution and membrane,
and incubated overnight
in an oven.
The following day, the
membrane is removed
from the hybridisation
set up, and washed with
the appropriate
stringency solution.

24. The membrane is then
blotted dry and put into
a cassette for holding
X- ray film.
The cassette is
wrapped, or sealed
with tape, and stored in
a freezer until the film
is
sufficiently exposed,
usually 1 to 4 days.

25. Clinical application of RFLP.
• 1) Diagnosis of genetic diseases e.g.
sickle cell anemia.
• 2) Forensic medicine e.g. identifying
a criminal.
• 3) Tracing of chromosome from parent
s to offspring. e.g. paternity test.

27. 4. RFLPs can be used determine the disease
status of an individual. (e.g. it can be used in
the detection of mutations particularly known
mutations).
5. In human population genetics, geographical
isolates and comparison of genetical makeup
of related species.

28. Highly robust methodology with good
transferability between laboratories.
No sequence information required.
highly recommended for phylogenetic analysis
between related species.
Well suited for constructing genetic linkage
maps.
Simplicity—given the availability of suitable
probes, the technique can readily be applied to
any plant.

29. Large amounts of DNA required.
Automation not possible.
Low levels of polymorphism in some
species.
Time consuming, especially with single-
copy probes
Costly.
Moderately demanding technically.

30. DNA
FOOT PRINTING

31. INTRODUCTION
• DNA footprinting is a method of investigating
the sequence specific of DNA – binding protein
in vitro.
• This technique can be used to study protein –
DNA interactions both outside and within cell.
• Techniques like DNA footprinting help elucidate
which protein bind to these associated region
of DNA and unravel the complexities of
transcriptional control.

32. HISTORY
• In 1978,David galas and Albert Schmitz
developed the DNA footprinting technique
to study the binding specificity of the lac
repressor protein .
• It was originally a modification of the
maxam – gilbert chemical sequencing
technique .

33. PRINCIPLE
• In this technique, nucleases like DNase I is
used which will degrade DNA molecule.
• Nucleases cannot degrade DNA if it is
bounded by a protein. Thus that region is
protected from degradation by nucleases.
• This protected DNA region is called the foot
print.

35. METHOD
The simplest application of this technique is to
assess whether a given protein binds to a region of
interest within a DNA molecule
•Polymerase chain reaction(PCR) amplify and label
region if interest that contain a potential protein-
binding site.
•Add protein of interest to a portion of the labelled
template DNA ; a portion should remain separate
without protein ,for later comparison
•Now, add a cleavage agent to both the portion of DNA
template (cleavage agent is a chemical or enzyme
that will cut at random locations in a sequence
independent manner).

36. • Run both the samples side by side on a
polyacrylamide gel electrophoresis .
• The portion of DNA template without protein bill
will be cut at random locations , and thus when
it is run on a gel ,will produce a ladder- like
distributions.
• The DNA template with a protein will result in
ladder distribution with a break in it , the
“footprint” , where the DNA has been protected
from the cleavage agent .

37. Labeling
• The DNA template can be labeled at the 3’ or
5’ end , depending on the location of binding
sites .
• Labels that can be used are : radioactivity and
fluorescence .
• Radioactivity has been traditionally used to
label DNA fragments for footprinting analysis.
• Radioactive labelling is very sensitive and is
optimal for visualising small amount of DNA .
• Fluorescence is a desirable advancement due
to the hazards of using radio-chemicals .

38. • However, it has been difficult to optimize
because it is not always sensitive enough to
detect the low concentrations
of
the target DNA strands used in a DNA
footprintings experiments.
• Electrophoretic sequencing gels or capillary
electrophoresis have been successfully in
analysing foot printing of fluorescent tagged
fragments .

39. Cleavageagent
• A variety of cleavage agent can be chosen
.
• Ideally a desirable agent is one that is
sequence neutral , easy to use , and is easy to
control .
• The following cleavage agent are described in
detail : DNase I is a large protein that function
as a double – strand endonuclease .
• It binds the minor group of DNA and cleaves
the phospodiester backbone .it is good
cleavage agent for footprinting because its size
makes it easily physically hindered .thus is
more likely to have to its action blocked by a

40. • Hydroxyl radicals are created from the
Fenton reaction , which involves reducing
Fe2+ with H2o2 to form free hydroxyl
molecules
.These hydroxyl molecules react with the DNA
backbone resulting in a
break. Due to their small size , the resulting DNA
footprint has a high resolution .
• Ultraviolet irradiation can be used to excite
nucleic acid and create photoreactions , which
result in damaged bases in a DNA strand.
Advantages of uv are that it reacts with very
quickly and therefore capture the interactions

41. Applications
• DNA foot printing is often used to identify
the binding sites of proteins in a DNA
molecule.
• Researchers often use this technique to
identify whether a particular protein can
activate or inhibit transcription.
• In addition, scientists also use this method
to detect where proteins bind to DNA in a
living cell.

42. LONG ANSWER QUESTIONS
1. Describe the Restriction fragment
length polymorphism ( RFLP)
2. DNA Foot printing

43. SHORT ANSWER QUESTIONS
1. Clinical Application of RFLP
2. DNA Foot printing