southern and northern blotting

1. TECHNIQUES OF
GENE ANALYSIS
Submitted by :RAFA ZUBAIR N.V
I.MPHARM
Department:PHARMACOLOGY

2. CONTENTS
 Blotting
 Types of blotting
 Southern blotting
• Principle
• Apparatus
• Steps involved in southern blotting
• Application
• Advantages and Disadvantages
 Northern blotting
• Steps involved in northern blotting
• Applications
• disadvantages

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.
Technique for transferring DNA ,RNA and Proteins onto
a carrier so they can be separated, and often follows the
use of a gel electrophoresis.

4. TYPES OF BLOTTING

5. 1.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
• 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. STEPS INVOLVED IN SOUTHERN
BLOTTING
1. Extract and purify DNA from cells;
2. DNA is restricted with enzymes;;
3. Separated by electrophoresis;
4. Denature DNA;
5. Transfer to nitrocellulose paper;
6. Add labeled probe for hybridization to take
place;
7. Wash off unbound probe;
8. Autoradiograph.

8. APPARATUS

9. 1.Extract and purify DNA from cells
• 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.
• Visible DNA fibers are removed and suspended in buffer.
2.DNA is restricted with enzymes.

10. 3.Separated by electrophorosis.
• The complex mixture of fragments is subjected to gel
electrophoresis to separate the fragments according to size.

11. 4. Denature DNA.
• The restriction fragments present in the gel are denatured
with alkali.
• This causes the double stranded to become single-stranded.
• DNA is then neutralized with NaCl to prevent re-hybridization
before adding the probe.

12. 5.Transfer to nitrocellulose paper.
• Transfer the DNA from the gel to a solid support, ie,
blotting.
• The blot is made permanent by:
– Drying at ~80°C
– Exposing to UV irradiation
6. Add labeled probe for hybridization.
• The filter is incubated under hybridization conditions with
a specific radiolabeled DNA probe.

13. • The probe hybridizes to the complementary DNA
restriction fragment.
7. Wash off unbound probe.
• Blot is incubated with wash buffers containing NaCl and
detergent to wash away excess probe and reduce
background.

14. 8. Autoradiograph.
• If the probe is radioactive, the particles emits when
expose to X-ray film.
• There will be dark spots on the film wherever the probe
bound.

16. 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.
 Diagnosis of HIV-1 and infectious disease.
 In DNA fingerprinting:
Paternity and Maternity Testing
Criminal Identification and Forensics
Personal Identification

17. 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 expansive than
most other tests.
Complex and labor-
intensive.
Time consuming and
cumbersome.

18. 2.Northern Blotting
• Northern blotting is a technique for detection of
specific RNA sequences. Northern blotting was
developed by James Alwine and George Stark
at Stanford University (1979) and was named
such by analogy to Southern blotting.

19. Steps involved in Northern
blotting
1. RNA is isolated from several
biological samples (e.g. various
tissues, various developmental
stages of same tissue etc.)
* RNA is more susceptible to
degradation than DNA.

20. 2. Sample’s are loaded on
gel and the RNA
samples are separated
according to their size on
an agarose gel .
• The resulting gel
following after the
electrophoresis run.

21. 3. The gel is then blotted on
a nylon membrane or a
nitrocellulose filter paper
by creating the sandwich
arrangement.

22. 4. The membrane is placed
in a dish containing
hybridization buffer with a
labeled probe.
• Thus, it will hybridize to
the RNA on the blot that
corresponds to the
sequence of interest.
5. The membrane is washed
to remove unbound
probe.

23. 6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is used). In both
cases this results in the formation of a
dark band on an X-ray film.
• Now the expression patterns of the
sequence of interest in the different
samples can be compared.

24. APPLICATIONS
• A standard for the study of gene expression at the
level of mRNA (messenger RNA transcripts).
• Detection of mRNA transcript size .
• Study RNA degradation .
• Study RNA splicing .
• Study RNA half-life.
• Often used to confirm and check transgenic /
knockout mice (animals) .

25. Disadvantage of Nourthern
Blotting
1.The standard northern blot method is relatively
less sensitive than nuclease protection assays
and RT-PCR.
2. Detection with multiple probes is a problem.
3. If RNA samples are even slightly degraded by
RNAses, the quality of the data and
quantitation of expression is quite negatively
affected.