Southern blot kanchana

SOUTHERN BLOTTING
BY
KANCHANA. A.S

BLOTTING TECHNIQUES
 Analytical tool - used for specific identification of
desired DNA or RNA fragments
 Process of immobilisation of sample nucleic acids
on solid support ( nitro cellulose or nylon
membrane )
 Blotted nucleic acid - targets
hybridisation experiment

TYPES OF BLOTTING TECHNIQUES:
 Southern blotting - for DNA
 Northern blotting - for RNA
 Dot blotting - for DNA / FOR RNA
 Western blotting - PROTEIN OF INTEREST

OUTLINE OF NUCLEIC ACID BLOTTING
TECHNIQUES:

SOUTHERN BLOTTING:
 First nucleic acid blotting procedure
 Developed by E.M. Southern (in 1975)
 detect the presence of a particular piece of DNA in a
sample.
 DNA detected can be a single gene OR part of a
larger piece of DNA such as a viral genome

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

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.
 Proteins are enzymatically degraded by incubation with
proteinase.
 Organic or non-inorganic extraction removes proteins.
 DNA is purified from solution by alcohol precipitation.

II. DNA FRAGMENTATION
 Cut the DNA into different sized pieces  use
restriction endonucleases (RE)
 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.
 This allows for specific sequences to be identified
more readily.
 Fragments are now easily separated by gel
electrophoresis.

III. GEL ELECTROPHORESIS
 Agarose or polyacrylamide
 Gel is soaked in a buffer which controls the size of the
pores
 Standards should also be run
 Gels can be stained with ethidium bromide.
 This causes DNA to fluoresce under UV light which
permits photography of the gel.
 Sorts the DNA pieces by size
 The desired DNA fragments is separated by gel
electrophoresis

STEP IV: BLOTTING
 The separated strands of DNA is then transferred to
positively charged membrane nylon membrane
(Nitrocellulose paper) by the process of 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.

STEP V: BAKING AND BLOCKING
 After the DNA of interest bound on the membrane,
it is baked on autoclave to fix in the membrane.
 The membrane is then treated with casein or
Bovine serum albumin (BSA) which saturates all
the binding site of membrane

STEP VI: HYBRIDIZATION WITH LABELED PROBES
 The DNA bound to membrane is then treated with
labeled probe
 The labeled probe contains the complementary
sequences to the gene of interest
 The probe bind with complementary DNA on the
membrane since all other non-specific binding site
on the membrane has been blocked by BSA or
casein.
 Blot is incubated with wash buffers containing NaCl
and detergent to wash away excess probe and
reduce background.

STEP VII: VISUALIZATION BY AUTORADIOGRAM
 The membrane bound DNA labeled with probe can
be visualized under autoradiogram which give
pattern of bands.

APPLICATION OF SOUTHERN BLOTTING:
 To detect DNA in given sample.
 DNA finger printing.
 Used for paternity testing, criminal identification,
victim identification
 To isolate and identify desire gene of interest.
 Used in restriction fragment length polymorphism
 To identify mutation or gene rearrangement in the
sequence of DNA
 Used in diagnosis of disease caused by genetic
defects
 Used to identify infectious agents

Southern blot kanchana

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