In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.

1. SOUTHERN AND
NORTHERN
BLOTTING
Presented by: Sagarika Sahoo(BT-17001)
Anjali Mandal(BT17008)
Dipti Mundhe(BT-17010)
Subject: Recombinant DNA Technology
Submitted to: Dr. Mayank Kumar

2. WHAT IS BLOTTING
• Biochemistry studies molecules such as DNA, RNA
and proteins.
• Blotting techniques is use to separate these types of
molecules. In cells, they exist as a mixture.
• Blotting allows researchers to find one protein among
many, like a needle in a haystack.
• This technique immobilizes the molecule of interest
on a support
• It uses hybridization techniques for the identification
of the specific nucleic acids and genes.
• Blotting is used in molecular biology for the
identification of proteins and nucleic acids and is
widely used for diagnostic purposes.

3. GENERAL PROCEDURE OF
BLOTTING
https://microbionotes.com/wp-content/uploads/2019/03/Blotting.jpg

4. TYPES OF BLOTTING
https://image.slidesharecdn.com/sayanti-140423040236-phpapp01/95/immunoblotting-techniques-5-
638.jpg?cb=1398225926

5. TYPES OF BLOTTING
https://upload.wikimedia.org/wikipedia/en/3/33/Blotting_Compass_for_Molecular_Probes.png

7. WHAT IS SOUTHERN BLOT?
• It is a method routinely used in
molecular biology for detection of a
specific DNA sequence in DNA
sample. The DNA detected can be a
small gene or part of an entire genome.
https://lh3.googleusercontent.com/proxy/Hugp0QaP7pI7VpH9mkzLRovkZkCbAfM
m-1zQU2_MzLo3QElKdA0Bl3zEu-
sq_PWo3reXzhOMrrxHA5wsuojr7DM6DZK53fb1CTMcZ_NDHPzcYg

8. ABOUT
• Southern blotting was named after
“Edward M. Southern”, Prof. of
Biochemistry and Fellow of Trinity
• He developed this technique 30 years ago,
in 1975, at the Edinburgh University
• He won the Laskar Award of Clinical
Medical Research for this technique

9. PRINCIPLE
• The key to this method is “Hybridisation”
• Hybridisation: It’s the process of forming a double-
stranded DNA molecule between a single-stranded
DNA probe and a single-stranded target DNA
• The ability of the DNA strands to hybridize come from
its complementary nature clearly depicted in Watson-
Crick’s DNA structure model
https://classificationsystemsccc.weebly.com/dna-dna-
hybridisation.html

10. PRINCIPLE
• The 2 important features of hybridisation:
i. The specificity- The specific binding of the probes to their target with a
complementary sequence
ii. These probes can “find one target molecule from millions of related but non-
complementary molecules”

11. DNA
extraction
Fragmentation
Separation by
electrophoresis
Denaturing
DNA
Transfer to
membrane
Add probe
for
hybridisation
Detection by
autoradiograph
Analysis of
data

12. PROCEDURE

13. DNA EXTRACTION
http://www.bio-helix.com/products/193
• To extract the DNA present inside the nucleus of a cell, we must first lyse the entire
cell to enable the expulsion of the DNA.
• Incubating the cell culture with detergent lyses the entire cell.
• Now the lysed sample contains DNA, protein, and debris.
• Protein is lysed by adding the proteinase enzyme and incubated.
• DNA is purified and separated by alcohol precipitation and fibers are removed by using
a buffer.

14. FRAGMENTATION
• The long nucleotide sequences obtained from the
extraction should be broken into smaller fragments
for the purification or identification process.
• This is done by the restriction endonuclease enzyme.
• Restriction Endonuclease: These are enzymes that
recognize and bind specific DNA sequences and
cleave at specific nucleotides either within the
recognition sequence or outside of the recognition
sequence
https://www.neb.com/applications/cloning-and-synthetic-
biology/dna-preparation/restriction-enzyme-digestion

15. ELECTROPHORESIS
• It is a common laboratory technique used to
identify, quantify, purify nucleic acid fragments.
• The DNA sequences cut by restriction enzymes
are loaded into the wells of the
agarose/polyacrylamide gel.
• They are subjected to an electric field, causing
the negatively charged nucleic acids to move
towards the positively charged anode.
• This movement across the gel causes the strands
to separate on the basis of their mol size.
https://ocw.mit.edu/courses/biological-engineering/20-109-laboratory-
fundamentals-in-biological-engineering-fall-2007/labs/mod1_2/

16. ELECTROPHORESIS
• Various kinds of electrophoresis can be used here:
i. Agarose Gel Electrophoresis (AGE)
ii. Polyacrylamide Gel Electrophoresis (PAGE)
• The mobility of the nucleic acids is influenced by:
i. Agarose/ polyacrylamide concentration
ii. Molecular size
iii. Molecular conformation
https://www.genome.gov/genetics-
glossary/Electrophoresis

17. AGAROSE GEL ELECTROPHORESIS
• It’s a type of gel electrophoresis in which the gel is
made up of agarose.
• The DNA obtained after digestion is a mixture of ds-
DNA fragments
• On the basis of their sizes,
i. If the DNA fragments are of 1-10kb, they’re run on
a gel of 0.3-2% agarose
ii. If the fragments are >15kb then they’re treated
with dil. HCl to depurinate them
• Longer gels are generally needed in the case of
separation of genomic DNA or multiple fragments
that are having similar sizes in order to guarantee
appropriate separation.
https://www.sigmaaldrich.com/technical-
documents/articles/biology/nucleic-acid-electrophoresis.html

18. POLYACRYLAMIDE GEL
ELECTROPHORESIS
• It’s a kind of electrophoresis where the gel is made up
of polyacrylamide
• Polyacrylamide is a co-polymer of acrylamide and bis-
acrylamide
• It forms a highly cross-linked mesh
• It can separate DNA smaller than 500 bp long
• It gives a better resolution of the separated fragments
https://capricorn.bc.edu/wp/pathways/protein-
expression/sds-page/

19. VISUALISATION
• The separated nucleic acids are visualised by
staining with fluorescent dyes- ethidium bromide
• EtBr binds to ds-DNA more strongly, hence gives
a stronger fluorescence
• Under UV it gives a strong orange-ish
fluorescence
https://orbitbiotech.com/analysis-of-nucleic-acids-by-gel-
electrophoresis/orbit-biotech-analysis-of-nucleic-acids-by-gel-
electrophoresis/

20. DENATURING
• DNA thus attained are double stranded in nature, for our purpose of probe hybridization,
we need a single stranded DNA.
• DNA is therefore denatured in an alkaline solution. The gel is soaked in 0.5M NaOH
solution, to obtain ss-DNA
• This is done to improve binding of the negatively charged thymine residues of DNA to a
positively charged amino groups of membrane, and destroys any residual RNA that may
still be present in the DNA

21. BLOTTING
• Blotting is the transfer of the
fragmented DNA sequence to a
membrane
• There are 2 ways:
- Capillary action
- Electro blotting
https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-
biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/western-blot-transfer-methods.html

22. MEMBRANES USED
• Nitrocellulose membrane: Is used commonly in
southern blot because:
- small pore size (0.1–0.45 μm) and
- high binding capacity (80–100 μg/cm2),
- along with its high frequency performance,
• All these attributes make it a good substrate to
be used for the characterization of DNA
molecules.
https://www.fishersci.ca/shop/products/sartorius-cn-
membrane-filters-36/p-4301121

23. CAPILLARYACTION
• The gel is placed on a wet pad of buffer soaked
filter paper and nitrocellulose membrane is placed
on the gel
• Buffer is then drawn through the gel by placing a
pad of dry absorbent material followed by a heavy
weight on top of the sheet
• Passage of buffer by capillary action through the
gel carries the separated nucleic acid on the
nitrocellulose membrane, on which they bind
irreversibly by hydrophobic interactions
• The DNA molecule is saturated using a NaCl
solution and permanently fixed using either UV
radiation or drying. https://www.gbiosciences.com/image/pdfs/protocol/BE-
315_protocol.pdf

24. ELECTRO-BLOTTING
• Quicker and more efficient way of transfer
• Sandwich of gel and the membrane compressed in a
cassette and immersed in buffer between two
electrodes
• Current is passed through this cassette causing the
nucleic acid to electrophorese out of gel and into the
nitrocellulose membrane
https://en.wikipedia.org/wiki/Electroblotting

25. HYBRIDISATION
• A nucleic acid with a homologous sequence to the target
sequence is labelled with:
- radioactively,
- fluorescent dyes,
- enzymes that can generate a chemiluminescent signal
when incubated with appropriate substrate
• The choice of label depends on:
- Nature of probe/template
- Quantification requirements
- Sensitivity needed
https://www.slideshare.net/RaviKantAgrawal/norther
n-southern-and-western-blotting

26. WHAT IS PROBES?
• The membrane is then treated with a small
piece of DNA or RNA called a probe,
which has been designed to have a
sequence that is complementary to a
particular DNA sequence in the sample;
this allows the probe to hybridize, or bind,
to a specific DNA fragment on the
membrane and produce an appropriate
signal.
https://www.youtube.com/watch?v=XKErwQAvj9w

27. PROBES USED
• Radioactively labelled, mostly 32P because of its ease
of incorporation in dNTPs
• Colorimetric detection:
- Alkaline phosphatase- acts on BCIP to produce purple
product
- HRP (Horse Radish Peroxidase)- acts on H2O2 to
produce brown product
• Chemiluminescent detection:
- Luminol
- Isoluminol
https://biologicresearch.blogspot.com/2014/

28. WASHING
• Excess probe will have bound nonspecifically to the membrane despite the blocking
agents.
• Blot is incubated with wash buffers containing NaCl and detergent to wash away the
excess probe and reduce background

29. DETECTION
• In the detection step, the bound, labeled probe is detected using the method required for
the particular label used.
• For example, radiolabeled probes may be detected using X-ray film or a phosphor
imaging instrument.
• Enzymatically labeled probes are typically detected by incubating with a
chemiluminescent substrate and exposing the blot to X-ray film.
https://www.mun.ca/biology/scarr/F12-18smc3.jpg

30. AUTORADIOGRAPH
• Autoradiography is a class of techniques in which the positions of radioisotope-labelled
biological material makes an autoradiogram ("self-picture").
• Radioisotopes such as 14C, 32P, 35S emit radiation that exposes photographic or X-Ray
film.
• A Southern Blot filter is placed inside a light-proof casette box and overlain with a sheet
of X-ray film.
• The cassette is closed and left for several hours to several days.
• The radioisotope-labelled DNA exposes the film, which when developed shows a pattern
of black bands that indicate the positions of labelled DNA in the blot.
http://www.mun.ca/biology/scarr/Autoradiography.html

31. CHEMILUMINESCENCE
• Chemiluminescence (also chemoluminescence) is the emission of light (luminescence), as
the result of a chemical reaction.
• There may also be limited emission of heat.
• It used for detection of biotin-labeled nucleic acid probes in various membrane blotting
applications.
• It is done using luminol substrate for horseradish peroxidase (HRP) with optimized
blocking and wash buffers that provide fast, trouble-free detection on film or by CCD
camera.
https://www.thermofisher.com/order/catalog/product/89880#/89880

32. COLORIMETRIC DETECTION
• Colorimetric detection generally involves the production of a colored precipitate which
can be seen with the naked eye.
• One commonly used example is alkaline phosphatase which will act on substrates NBT &
BCIP to produce a dark purple product
Blot
incubated
with DIG
probe
Probe
detected by
Anti-DIG Ab
conjugated
with Alkaline
phosphatase
Phosphatase
reacts with
BCIP to form
a purple
substrate
Incubate with
substrate BCIP
Wash to remove
excess

33. NORTHERN BLOTTING

34. NORTHERN BLOTTING
• The northern blot, or RNA blot, is a technique used in molecular biology research to study gene
expression by detection of RNA (or isolated mRNA) in a sample by using DNA probes.
• It was developed in 1977 by David Kemp, James Alwine, and George Stark et al. Stanford
University.
• It was named after the Southern blot technique
David Kemp James Alwine George Stark
http://www.chimicare.org/curiosita/wp-content/uploads/2015/06/inventori-del-
Northern-Blotting.jpg

35. ISOLATION OF RNA
https://www.neb.com/-
/media/catalog/long%20description/s1550_fig2.gif

36. PROCEDURE OF NORTHERN
BLOTTING
https://www.mun.ca/biology/scarr/F12-18smc3.jpg

37. WHY DENATURATION OF RNA IS
IMPORTANT?
• RNA always exists in secondary structure.
• Probe is not able to bind to the secondary structure of RNA
• To resolve this problem Denaturation of RNA is done.
• Formaldehyde added in the Agarose gel.
• Formaldehyde denatures the RNA and forces it to adopt a linear arrangement, making it
run more like a dsDNA fragment.

38. WHY THERE IS NEED OF NORTHERN
BLOTTING
• For finding the gene expression we need to check RNA sequence.
• By knowing the amount of RNA we can find how much the gene has expressed.
• High amount of RNA means gene is over expressed
• Low amount of RNA indicates that the gene is not so much expressed.
https://i.pinimg.com/originals/0e/76/7b/0e767b3858116480884580f505b35262.png
STEPS OF GENE
EXPRESSION

39. DIFFERENCE BETWEEN SOUTHERN
AND NORTHERN BLOTTING
• Southern blotting is used to detect DNA sequence while Northern is used for RNA
detection.
• Membrane used in southern blotting is different form northern blotting.
• By southern blotting you can find if the gene is present in the genome or not.
• But by northern blotting you will find if the gene is going to express or not.
• In northern blotting sample is used in its native state while in southern blotting sample has
to be denature.
• In southern blotting there is DNA-DNA hybridization while in northern blotting there is
RNA-DNA hybridization.

40. DIFFERENCES
Northern blotNorthern blot Southern blotSouthern blot

41. REFERENCES
• https://wwhttps://en.wikipedia.org/wiki/Northern_blot
• https://biologywise.com/northern-blot-vs-southern-blot-technique
• https://www.slideshare.net/mprasadnaidu/southern-32910096
• https://en.wikipedia.org/wiki/Autoradiograph
• http://www.mun.ca/biology/scarr/Autoradiography.html
• https://en.wikipedia.org/wiki/Chemiluminescence
• https://www.thermofisher.com/order/catalog/product/89880#/89880
• https://sciencing.com/biochemistry-blotting-techniques-20171.html
• https://www.mybiosource.com/learn/testing-procedures/northern-blotting/
• https://www.majordifferences.com/2013/12/difference-between-southern-and.html#.Xo3jgUBuLIU
• w.slideshare.net/rgrg507/blotting-techniques-in-biotechnology
• https://www.thermofisher.com/in/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-
electrophoresis/southern-blotting.html

42. CONTD…
• https://www.nature.com/articles/srep29089
• https://www.sigmaaldrich.com/technical-documents/articles/biology/nucleic-acid-electrophoresis.html
• https://en.wikipedia.org/wiki/Southern_blot
• https://www.biologyexams4u.com/2013/12/southern-blotting-procedure-steps.html#.Xo3wIcgzbIU
• https://giphy.com/gifs/dna-zBBXQLtqIvGxi
• https://www.mybiosource.com/learn/southern-blotting/

43. THANK YOU!!!