This document discusses various blotting techniques used to identify specific DNA or RNA fragments, including Southern blotting for DNA, Northern blotting for RNA, and Dot blotting for DNA and RNA. It describes the process of Southern blotting, which involves digesting genomic DNA with restriction enzymes, separating fragments via gel electrophoresis, transferring DNA to a membrane, then using labeled cDNA probes to detect complementary DNA bands through autoradiography. Northern blotting follows a similar process to detect specific RNA molecules. These techniques allow for the identification and analysis of genes.

2.  Analytical tools for the specific identification
of desired DNA or RNA fragments from
thousands of molecules.
 Blotting is a process of immobilization of
sample nucleic acids on solid support
(nitrocellulose or nylon membranes).
 Blotted nucleic acids are used as targets in
the hybridization experiments.

3.  Southern blotting (for DNA)
 Northern blotting (for RNA)
 Dot blotting (DNA/RNA)

4.  It is technique for blotting DNA.
 The genomic DNA isolated from cell/tissues is
digested with one or more restriction enzymes.
 This mixture is loaded into a well in an agarose or
polyacrylamide gel & subjected to electrophoresis.
 The negatively charged DNA migrates to wards the
anode (positively charged electrode).
 Smaller DNA fragments move faster.

5.  The separated DNA molecules are denatured
by exposure to a mild alkali & transferred to
nitrocellulose or nylon paper.
 This results in an exact replica of the pattern
of DNA fragments on the gel.
 The DNA can be annealed to the paper on
exposure to heat (80˚C).
 Nitrocellulose or nylon paper is exposed to
labeled cDNA probes.

6.  These probes hybridize with complementary
DNA molecules on the paper.
 The paper after thorough washing is
exposed to X-ray film to develop
autoradiograph.
 This reveals specific bands corresponding to
DNA fragments recognized by cDNA probe.

8.  It is an invaluable method in gene analysis.
 Important for the confirmation of DNA
cloning results.
 Highly useful for the determination of
restriction fragment length polymorphism
(RFLP) associated with pathological
conditions.

9.  It is a technique for specific identification of
RNA molecules.
 RNA molecules are subjected to
electrophoresis, followed by blot transfer,
hybridization & autoradiography.
 RNA molecules do not easily bind to
nitrocellulose paper or nylon membrane.

10.  Blot – transfer of RNA molecules is carried
out by using a chemically reactive paper
prepared by diazotization of
aminobenzyloxymethyl to create
diazobenzyloxymethyl (DBM) paper.
 The RNA can covalently bind to DBM paper.
 A good technique for determining the
number of genes (through mRNA) present on
a given DNA.

12.  It is a modified southern & northern
blotting technique.
 It is particularly useful in obtaining
quantitative data for the evaluation of
gene expression.

13.  It involves the identification of proteins.
 It is very useful to understand the nucleic acid functions,
particularly during the course of gene manipulations.
 It involves transfer of electrophoresed protein bands
from polyacrylamide gel to nylon or nitrocellulose
membrane.
 These proteins can be detected by specific protein-ligand
interactions.
 Antibodies or lectins are commonly used for this purpose.

14.  Autoradiography is the process of
localization & recording of a radiolabel
within a solid specimen, with the production
of an image in a photographic emulsion.
 These emulsions are composedo f silver
halide crystalss uspended in gelatin.

15.  β-particle or a γ-ray from a radiolabel passes
through the emulsions, silver ions are
converted to metallic silver atoms.
 This results in the development of a visible
image which can be easily detected.
 Applications:
 Autoradiography is closely associated with
blotting techniques for the detection of DNA,
RNA & proteins.

16.  Textbook of Biochemistry – U Satyanarayana