Souther and northern blotting systems

1. Southern Blotting and Northern
Blotting
Presented by :
Chaudhary Ankit R.
Submitted to :
Dr. Kapil K Tiwari
Department of Genetics & Plant Breeding
C .P . College of Agriculture
Sardarkrushinagar, Dantiwada
Reg. No. : 04-AGRMA-01571-2017

2. • Blotting are techniques for transferring DNA,
RNA and proteins onto a carrier so they can be
separated, and often follows the use of a gel
electrophoresis. The Southern blot is used for
transferring DNA, the Northern blot for RNA and
the western blot for Protein.
• These powerful techniques allow us to identify
and characterize specific molecules in a complex
mixture of related molecules
What is blotting?

3. The key to these methods is Hybridization.
Hybridization: It is the process of forming a double
stranded DNA molecule between a single-stranded DNA
probe or 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.
PRINCIPLE

4. Hybridization
technique
Southern Blot
It is used to detect
DNA.
Northern Blot
It is used to detect
RNA.
Western blot
It is used to detect
protein.
Dot Blot
used to detect sexually
transmitted diseases
In Situ Hybridization
Used to detect Location of
specific DNA sequences.

5. • Nitrocellulose membrane
• Poly vinylidene difluoride (PVDF) membrane
• Nylon membrane
• Diazo benzyloxy methyl(DBM) and Diazo phenyl
thioether(DPT)
Blotting membranes

6. • 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.
Southern blot

7. 1. Extract & purify DNA from cells
2. DNA is restricted with enzymes
3. Separated by electrophoresis
4. Denature DNA
5. Transfer to nitrocellulose paper (blotting)
6. Add labeled probe for hybridization
7. Wash off unbound probe
8. Autoradiograph
Steps in Southern Blotting

8. Step 1 :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, cell lysis frees cellular protein and DNA.
•Proteins are enzymatically degraded by
incubation with proteinase.
•DNA is purified from solution by alcohol
precipitation.
•Visible DNA fibers are removed and suspended in
buffer.
PROCEDURE

9. Step 2:RESTRICTION DIGEESTION
• Cut the DNA into different sized fragments using
restriction endonucleases (RE).

10. Step 3 : Gel electrophoresis
• Nucleic acid has a net negative charge and will
move from the left to right. The larger molecules
are held up while the smaller ones move faster.
This results in a separation by size.
• Gels are Agarose or polyacrylamide With
microscopic pores.
• Gel is soaked in a buffer which Controls the size of
the pores.

11. •Gels can be stained with ethidium bromide, this
cause DNA to fluoresce under UV light which
permits photography of the gel.
•This will help us to know the exact migration of
DNA strands and the quality of the RE digestion
of the test DNA.

12. • Cover gel with nitrocellulose paper…then…
• Cover nitrocellulose paper with thick layer of
paper towels.
• Compress apparatus with heavy weight.
• ssDNA binds to nitrocellulose at same position it
had on the gel.
• Vacuum dry nitrocellulose at 80 0C to
permanently fix DNA in place.
Step 3. Nitrocellulose Blot

14. • Incubate nitrocellulose sheet with
a minimal quantity of solution
containing 32P-labeled ssDNA
probe.
• Probe sequence is complementary
to the DNA of interest.
• Incubate for several hours at
suitable temperature that will
permit probe to anneal to its
target sequence(s).
• Wash & dry nitrocellulose sheet.
Step 4. Hybridization

15. • Place nitrocellulose
sheet over X-ray film.
• X-ray film darkens
where the fragments
are Complementary
to the radioactive
probes.
Step 5. Autoradiography

16. General Scheme for Southern Blot

17. • To identify specific DNA in a DNA sample.
• To isolate desired DNA for construction of rDNA.
• Identify mutations , deletions ,and gene
rearrangements.
• Diagnosis for HIV and infectious disease.
APPLICATIONS

18. Modern Blotting Techniques
• Using nylon instead of nitrocellulose
membrane is speed up the movements of
DNA. (membrane = 18h, nylone = 2h)
• Electro blotting using electrophoresis between
gel to nylon speed up the DNA movements.
• Vacum blotting

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

20. • Technique was developed in 1977 by James
Alwine, David Kemp and George Stark at
Stanford University.
• No need to digest RNA with REs.
• SS RNA can form the secondary structure during
running.
• Use formaldehyde to break H-bonds and
denature RNA because single-stranded RNA will
form intramolecular base pairs and "fold“ on
itself.
Northern Blot: RNA

21. Isolate RNA & treat with formaldehyde
Gel electrophoresis
Transfer single-stranded RNA to nitrocellulose or nylon
membrane
Incubate membrane with labeled DNA or RNA probe
Autoradiography
Steps

22. Isolate RNA:
• To detect rare mRNA, isolate the polyA+ mRNA.
• RNA is both biologically and chemically more labile
than DNA. Thus eliminate RNases.
Step 1
Electrophoresis:
• Performed in formaldehyde agarose gel to prevent RNA
from folding on itself.
• Stain with EtBr to visualize the RNA bands.
• PAGE can be used with Urea.
Step 2

23. Transfer ssRNA to nitrocellulose or nylon membrane:
• Transfer buffer contain formamide (it lowers the
annealing temperature of the probe-RNA
interaction)
• Nitrocellulose typically has a binding capacity is
lesser (of about 100μg/cm) than nylon (has a
binding capacity of about 500μg/cm.)
Covalently link RNA to membrane:
UV cross linking is more effective in binding RNA to
the membrane than baking at 80 0C
Step 3

24. Pre-hybridize before hybridization:
• Blocks non-specific sites to prevent the single-stranded
probe from binding just anywhere on the membrane.
Incubate membrane with labeled DNA or RNA probe with
target sequence:
• Probe could be 32P, biotin / streptavidin or a
bioluminescent probe.
Autoradiography:
• Place membrane over X-ray film.
• X-ray film darkens where the fragments are
complementary to the radioactive probes
Step 4 & 5

25. • To determine the conditions under which specific
genes are being expressed (mRNA level).
• 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
Application

26. Advantages
• Simple method
• Highly specific
• Quality and quantity of gene can be measured
Disadvantages
• Detect small number of genes at a time
• Use of ethidium bromide and UV light needs
special training
• Detection with multiple probes is difficult