The document provides an overview of blotting techniques in molecular biology, focusing on Southern blotting for DNA detection. It details the principles, methods, applications, advantages, and disadvantages of Southern blotting. Additionally, it includes references for further reading on the topic.

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

15. THANK YOU