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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