Blotting A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier. The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Types of blotting techniques Southern Blotting Northern Blotting Western Blotting A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Mellor Southern. - Methods in Southern blotting - Advantages and disadvantages

1. PRESENTED BY: NUSRAT SHEIKH

2. Content
 Introduction to Blotting
 Types of blotting
 Southern blotting
 Principle
 Methods involved in southern blotting
 A schematic view of southern blotting
 Application
 Advantages and Disadvantages
 Reference

3. Blotting
 A blot, in molecular biology and genetics, is a method of
transferring proteins, DNA or RNA, onto a carrier.
 The term "blotting" refers to the transfer of biological
samples from a gel to a membrane and their subsequent
detection on the surface of the membrane.

4. Types of blotting techniques
 1 ) Southern blotting ( to detect DNA )
 2 ) Northern blotting ( to detect RNA )
 3 ) Western blotting ( to detect protein )

5. Southern Blotting
 A Southern blot is a method used
in molecular biology for detection of a
specific DNA sequence in DNA samples.
 Southern blotting combines transfer
of electrophoresis -separated DNA fragments
to a filter membrane and subsequent
fragment detection by probe hybridization.
 The method is named after its inventor,
the British biologist Edwin Mellor
Southern.

6. Principle
 This method Involves separation, transfer and hybridization.
 The key to this method is hybridization.
Hybridization: It is the process of forming a double stranded
DNA molecule between a single-stranded DNA probe and a single-
stranded target DNA.
 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.

7. Methods in Southern blotting
 Restriction endonucleases cut
high-molecular-weight DNA
strands into smaller fragments.
 The mixture of fragmented DNAs fig: Restriction endonuclease
is separated in agarose get
electrophoresis.
 The restriction fragments present
in the gel are denatured with
alkali and transferred onto a
nitrocellulose filter or nylon
membrane by blotting.
fig: Gel Electrophoresis

8.  This procedure preserves the
distribution of the fragments in
the gel creating a replica of the
gel on the filter.
 A sheet of nitrocellulose or nylon
membrane is placed on top of the Fig: DNA denaturation by NaOH
gel. Pressure is applied evenly to
the gel to ensure good and even
contact between gel and
membrane.
Fig: Membrane is placed on top of gel
Cont..

9. Cont..
 Membrane is then baked in a vacuum or regular oven at
80°C for 2 hours or exposed to ultraviolet radiation
(nylon membrane) to permanently attach the transferred
DNA to the membrane.
 The probe is added to the matrix to bind to the
molecules.
 Any unbound probes is washed away and the probe
bound to the filter is detected by autoradiography, which
reveals the DNA fragment to which the probe hybridized.

10. Prehybridization
 This step involved in closing the binding site of
membrane other than DNA fragments.
For this a special prehybridization solution is used.
 Such as-
1.polyvinylpyrrolidone
2.bovine serum albumin
3.dried milk
 After this, membrane is washed and ready
for hybridization

11. A Schematic view of southern blotting

12. Applications
• To identify specific DNA in a DNA sample.
• To Isolate desired DNA for construction of rDNA.
• Identify mutations, deletions, and gene rearrangements.
• Used in prognosis of cancer and in prenatal diagnosis of
genetic diseases.
• In RFLP, gene mapping and discovery.
• Diagnosis of HIV-1 and infectious disease.
• In DNA fingerprinting:
-Paternity and Maternity Testing
-Criminal Identification and Forensics
-Personal Identification

13. Advantages
• Effective way to detect a specific DNA sequence in a
large, complex sample of DNA.
• Can be used to quantify the amount of the present DNA.
• Cheaper than DNA sequencing
Disadvantages
• More expensive than other tests
• Complex and labour intensive
• Time consuming and cumbersome
• Require large amount of DNA

14. Reference
 S.P. Vyas., Pharmaceutical Biochemistry, CBS
Publishers, New Delhi,2008.
 U. Satyanarayana, U. Chakrapani, Biochemistry,
Arunabha Sen Books Pvt.Ltd., 2010.
 Robert K. Murray, Daryl K. Granner, Peter A. Mayer,
Victor W. Rodwell.,
 Harper’s Illurstrated Biochemisry, Lange Medical
publications, 2005
 http://sciencing.com

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