Southern Blotting Technique

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1. SOUTHERN BLOTTING
M.PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.

2. OUTLINE
DNA
SPECIMEN COLLECTION AND
STORAGE
PROCEDURE
WATCHPOINTS
USES

3. DNA
Each individuals unique genetic
blueprint is stored in material known
as DNA.
DNA is found in all cells containing a
nucleus.
DNA can be extracted for analysis
from
hair, bones, saliva, sperm, skin, organ
s, all body tissues and blood.

4. DNA
The deoxyribonucleic acid, DNA, is a
long chain of nucleotides which
consist of:
1. Deoxyribose(sugar with 5 carbons)
2. Phosphate groups
3. Organic(nitrogenous)bases

5. Nitrogenous Bases
Two classes:
Purines
◦Adenine
◦Guanine
Pyrimidines
◦Cytosine
◦Thymine

6. DNA
DNA molecules are arranged in a
double helix which resembles a tightly
coiled twisted ladder.
The sides of the ladder have
alternating units of phosphate and
deoxyribose sugar.

7. DNA
The rungs of the ladder are formed by
the nitrogenous “base pairs”.
Hydrogen bonds hold the strands
together.
The bases bind together in a
complementary fashion.

8. DNA
The base adenine (A) always pairs
with thymine (T).
The base guanine (G) always pairs
with cytosine (C).

9. DNA
Example
First strand GGGTTTAAACCC
Second strand CCCAAATTTGGG

10. DNA STORAGE AND
COLLECTION
I. Temperature Storage for
DNA
◦Purified DNA may be
refrigerated at 4°C for up to 3
years.
◦Samples kept over 3 years
should be frozen at -70°C.

11. DNA STORAGE AND
COLLECTION
II. Specimens used in DNA testing
◦Whole blood
◦Solid tissue
◦Serum and plasma
◦Urine
◦Bone marrow
◦and many others

12. DNA STORAGE AND
COLLECTION
III. Specimen Collection
Requirements
◦A. Blood and Bone Marrow
Collection tubes are EDTA or ACD
5-15 ml
Samples should not be frozen for
transport
4-25°C

13. DNA STORAGE AND
COLLECTION
B. Serum
◦Collection tubes with no additives
◦100 µl to 1 ml
◦Transported at 20-25°C

14. DNA STORAGE AND
COLLECTION
Spin the samples to separate the
plasma, RBC, and buffy coat.
Extract the buffy coat
The buffy coat is used because the
WBC are nucleated and contain DNA.

15. DNA STORAGE AND
COLLECTION
C. Tissue
◦A sterile container with no formalin or
paraffin must be used for collection.
◦30 mg
◦Dry ice should be used for transport.

16. DNA STORAGE AND
COLLECTION
D. Urine
◦Urine container should be used for
collection.
◦At least 1 ml should be collected.
◦Transported at 4-25°C

17. SOUTHERN BLOTTING
The technique was developed by E.M.
Southern in 1975.
The Southern blot is used to detect
the presence of a particular piece of
DNA in a sample.
The DNA detected can be a single
gene, or it can be part of a larger
piece of DNA such as a viral genome.

18. SOUTHERN BLOTTING
The key to this method is
hybridization.
Hybridization-process of forming a
double-stranded DNA molecule
between a single-stranded DNA probe
and a single-stranded target patient
DNA.

19. SOUTHERN BLOTTING
There are 2 important features of
hybridization:
◦The reactions are specific-the probes will
only bind to targets with a complementary
sequence.
◦The probe can find one molecule of target
in a mixture of millions of related but non-
complementary molecules.

21. SOUTHERN BLOTTING
Steps for hybridization
◦1. The mixture of molecules is separated.
◦2. The molecules are immobilized on a matrix.
◦3. The probe is added to the matrix to bind to the
molecules.
◦4. Any unbound probes are then removed.
◦5. The place where the probe is connected
corresponds to the location of the immobilized
target molecule.

22. SOUTHERN BLOTTING
I. DNA Purification
◦Isolate the DNA in question from the
rest of the cellular material in the
nucleus.
◦Incubate specimen with detergent to
promote cell lysis.
◦Lysis frees cellular proteins and
DNA.

23. SOUTHERN BLOTTING
Proteins are enzymatically degraded
by incubation with proteinase.
Organic or non-inorganic extraction
removes proteins.
DNA is purified from solution by
alcohol precipitation.
Visible DNA fibers are removed and
suspended in buffer.

24. SOUTHERN BLOTTING
II. DNA Fragmentation
◦Cut the DNA into different sized
pieces.
◦Use restriction endonucleases (RE)
◦Bacterial proteins
◦In vivo, they are involved in DNA
metabolism and repair or in bacterial
host defense.

25. SOUTHERN BLOTTING
Nucleases hydrolyze the bonds
that connect bases within the
strand, resulting in cleavage of
the strand.
They cleave the double stranded
nucleic acid only at specific
points.

26. SOUTHERN BLOTTING
This allows for specific sequences
to be identified more readily.
Fragments are now easily
separated by gel electrophoresis.

27. SOUTHERN BLOTTING
III. Gel Electrophoresis
◦Sorts the DNA pieces by size
◦Gels are solid with microscopic
pores
◦Agarose or polyacrimide
◦Gel is soaked in a buffer which
controls the size of the pores
◦Standards should also be run

28. SOUTHERN BLOTTING
Nucleic acids have a net negative charge and will
move from the left to the right. The larger molecules
are held up while the smaller ones move faster. This
results in a separation by size.

29. SOUTHERN BLOTTING
Gels can be stained with ethidium
bromide.
This causes DNA to fluoresce under
UV light which permits photography of
the gel.
You can tell the exact migration of
DNA standards and the quality of the
RE digestion of the test DNA.

30. SOUTHERN BLOTTING
High quality intact DNA should give
the appearance of a single band.
Degraded material will smear
downwards.
Only a small amount of degradation is
tolerable.

31. SOUTHERN BLOTTING
IV. Blotting
◦Transfer the DNA from the gel to a
solid support.
◦The blot is usually done on a sheet
of nitrocellulose paper or nylon.

32. SOUTHERN BLOTTING
DNA is partially depurinated with dilute
HCL which promotes higher efficiency
transfer by breaking down fragments
into smaller pieces.
DNA is then denatured with an
alkaline solution such as NAOH.
This causes the double stranded to
become single-stranded.

33. SOUTHERN BLOTTING
DNA is then neutralized with NaCl
to prevent re-hybridization before
adding the probe.
Transferred by either electrophoresis
or capillary blotting.

34. SOUTHERN BLOTTING
1) Electrophoresis-takes advantage of the
molecules negative charge.

35. SOUTHERN BLOTTING
2) Capillary blotting-fragments are eluted from the gel
and deposited onto the membrane by buffer that is
drawn through the gel by capillary action.

36. SOUTHERN BLOTTING
The blot is made permanent by:
◦Drying at ~80°C
◦Exposing to UV irradiation

37. SOUTHERN BLOTTING
V. Blocking
◦Buffer binds to areas on the blot not
occupied by patient DNA.
◦Blocks the empty sites from being
bound during hybridization.

38. SOUTHERN BLOTTING
VI. Preparing the probe
◦Small piece of DNA used to find
another piece of DNA
◦Must be labeled to be visualized
◦Usually prepared by making a
radioactive copy of a DNA fragment.

39. SOUTHERN BLOTTING
The DNA fragment is labeled by the
Random Hexamer Labeling Process:
◦1. The template DNA is denatured by
boiling.
◦2. A mixture of hexamers (6
nucleotides) containing all possible
sequences is added and allow to
base pair.

40. SOUTHERN BLOTTING◦3. DNA polymerase is added
with radioactive nucleotides.
◦4. The mixture is boiled to
separate the strands and is
ready for hybridization.

41. SOUTHERN BLOTTING
The Random Hexamer Labeling
Process produces a radioactive
single-stranded DNA copy of both
strands of the template for use as
a probe.

42. SOUTHERN BLOTTING

43. SOUTHERN BLOTTING
VII. Hybridization
◦The labeled probe is added to the
blocked membrane in buffer and
incubated for several hours to allow
the probe molecules to find their
targets.

44. SOUTHERN BLOTTING
VIII. Washing
◦Excess probe will have bound
nonspecifically to the membrane despite
the blocking reagents.
◦Blot is incubated with wash buffers
containing NaCl and detergent to wash
away excess probe and reduce
background.

45. SOUTHERN BLOTTING
IX. Detection
◦Radioactive probes enable
autoradiographic detection.

46. SOUTHERN BLOTTING
If the probe is radioactive, the particles
it emits will expose X-ray film.
By pressing the filter and film, the film
will become exposed wherever probe
is bound to the filter.
After development, there will be dark
spots on the film wherever the probe
bound.

47. SOUTHERN BLOTTING
Summary of procedure
◦1. Extract and purify DNA from cells
◦2. DNA is restricted with enzymes
◦3. Sort by electrophoresis
◦4. Denature DNA
◦5. Transfer to nitrocellulose paper
◦6. Block with excess DNA
◦7. Wash off unbound probe
◦8. Autoradiograph

48. Watch points
Using too little DNA-compromise the
sensitivity of the test
Using too much DNA-poor restriction
enzyme digestion
Using too high voltage setting for
electrophoresis-gel to melt or
appearance of artifacts

49. Watch points
Improper blocking-high background
and uninterpretable results.
Insufficient washing-high background
and uninterpretable results.
Excess washing-dissociate the
specific hybrids.

50. USES
Identify mutations, deletions, and gene
rearrangements
Used in prognosis of cancer and in
prenatal diagnosis of genetic diseases
Leukemias
Diagnosis of HIV-1 and infectious
disease

51. USES
Every person has repeated sequences
of base pairs which are called Variable
Number Tandem Repeats (VNTRs)
To find a particular VNTR we use a
radioactive version of the one in
question.
This pattern is known as a DNA
fingerprint.

52. USES
Applications of DNA fingerprinting
include:
◦Paternity and Maternity Testing
◦Criminal Identification and Forensics
◦Personal Identification

53. THANK
YOU