NAME:
VINISHA LAWRENCE
COURSE/SEM:
BSc Hons Bt-5
ENROLL.NO:
A80404421018
SUBJECT:
RECOMBINANT DNA TECHNOLOGY
SUBMISSION DATE:
31.10.2023
Southern blotting =>
This is basically a technique performed in a laboratory which is used to detect specific DNA
molecules from amongst many other varied DNA molecules. The name of this technique was after
Edward M. Southern, it’s inventor at Edinburgh University during 1970s. In short here the DNA
is being digested with a single or more restriction enzymes, and the resulting fragments are then
separated in accordance to their respective sizes with electrophoresis through a standardized agarose
gel.
1Kg WEIGHT
Stack of
blotting
paper
DNA MOVES UP
NYLON FILTER Gel
support
TANK CONTAINING
TRANSFER SOLUTION
The original Southern Blotting method doesn’t require any
special equipment to carry out the DNA transfer. As the
procedure carry on, the blotting paper in the stack becomes wet
which can be replaced by then for complete DNA transfer to the
nylon filter.
FACTUAL HISTORY: Prefatorily, the experimental procedures were being carried out in
order to study the renaturing ability of the DNA and the formation of DNA/RNA hybrids to
understand tissue or cell specific expression. After which the concept of hybridization came into
action carried on with the development of probes which detects specific sequences within the target.
Objective: To analyze and identify specific DNA sequences in a given sample using the southern
blotting technique.
Materials:
DNA sample (e.g., Genomic DNA or PCR product)
Restriction enzymes
Agarose gel
Ethidium bromide
20x SSC buffer (saline-sodium citrate)
Denaturation solution (e.g., NaOH)
Neutralization solution (e.g., Tris-HCL)
Nylon membrane
Blotting paper
UV transilluminator
Hybridization solution
Autoradiography film
Procedure:
DNA Digestion-
Digest the DNA sample with a suitable restriction enzyme.
i. Run the digested DNA on an agarose gel with a DNA ladder for size reference.
ii. Stain the gel with ethidium bromide and visualize the DNA bands under UV light.
iii. Photograph the gel for documentation.
DNA Transfer-
i. Prepare a nylon membrane by cutting it to the same size as the gel.
ii. Soak the membrane in 20x SSC buffer.
iii. Assemble the gel, blotting paper, and nylon membrane on a blotting apparatus.
iv. Apply pressure and allow the DNA to transfer from the gel to the membrane by
capillary action. This might take several hours or even overnight.
DNA fixation-
After the transfer, fix the DNA onto the membrane by UV cross-linking or baking it at a low
temperature.
Prehybridization-
i. Prepare the hybridization solution according to the probe’s requirements (e.g.,
Temperature, buffer)
ii. Preheat the membrane in the hybridization solution at the specified temperature.
Hybridization-
i. Add the labeled hybridization probe to the prehybridization solution.
ii. Incubate the membrane with the probe solution at the specified temperature
for several hours or overnight.
Washing-
Wash the membrane to remove unbound probe following the specific protocol
for your probe type (radioactive or non-radioactive).
Detection-
Expose the membrane to autoradiography film (for radioactive probes) or perform
chemiluminescent detection (for non-radioactive probes)
Analysis-
Develop the autoradiography film or use an appropriate detection system to visualize
the hybridized DNA bands.
Compare the band patterns with your known DNA samples or standards to identify
specific DNA sequences.
Documentation-
Document the results, including the size and intensity of the bands, for
your analysis.
Data Interpretation-
Analyze the results to draw conclusions about the presence or absence of specific
DNA fragments in your sample.
Troubleshooting and Optimization-
If necessary, troubleshoot and optimize the experiment
for better results.
Applications-
Genetic research
Gene sequencing
Mutation detection
Clinical diagnostics
Advantages-
Specificity
Quantification
Genomic analysis
Mutation detection
Disadvantages-
Labor-intensive
Sensitivity limitations for non-radioactive probes
Contamination risks
Safety concerns
RADIOACTIVE PROBES:>
Radioactive probes are labeled with radioactive isotopes such as
phosphorus-32 (P32) or tritium-3 (H3). These probes emit radioactive decay particles
that can be detected using X-ray film or a phosphorimager.
LABELLING THE PROBE:
Radioactive probes are typically labeled during their
synthesis. For example, you can incorporate P32 labeled nucleotides during
DNA or RNA probe synthesis using enzymes like polynucleotide kinase
(PNK).
HYBRIDIZATION:
Incubate the labeled radioactive probe with the
membrane containing the transferred DNA fragments. Ensure that the
hybridization conditioned (temperature, buffer, etc.) are optimized for your
specific probe and target DNA.
WASHING:
After hybridization, wash the membrane to remove any unbound
radioactive probe. This is crucial to remove any unbound radioactive
probe. This is crucial to reduce background signal.
AUTORADIOGRAPHY:
Expose the membrane to an autoradiography
film or a phosphorimager screen for a specific amount of time, depending on
the radioisotope used. The emitted radioactive particles expose the film or
phosphorimager screen.
ANALYSIS:
Develop the autoradiography film or scan the
phosphorimager screen to visualize the bands. The bands represent the presence
of the specific DNA fragments in our sample.
SAFETY PRECAUTIONS:
Always handle radioactive materials in a designated
area.
NON-RADIOACTIVE PROBES:>
Non-radioactive probes are
labeled with molecules that can be detected using non-radioactive methods
like chemiluminescence or fluorescence.
LABELING THE PROBE:
Non-radioactive probes are typically labeled
during their synthesis with molecules like biotin, digoxigenin (DIG), or
fluorescent dyes (e.g., FITC or Cy3). You can incorporate these labels into the
probe during synthesis.
HYBRIDIZATION:
Incubate the labeled non-radioactive probe with the
membrane containing the transferred DNA fragments, following optimized
hybridization conditions.
DETECTION:
Detection methods for non-radioactive probes include-
Chemiluminescence: Use substrate such as luminol that emit light upon
reacting with an enzyme-conjugate antibody or streptavidin.
Fluorescence: Directly visualize the fluorescence signal from a
fluorescently labeled probe under an appropriate excitation source and
filter.
ANALYSIS:
Capture and analyze the signal using a chemiluminescent or
fluorescent imaging system, depending on the labeling method used. The
bands represent the presence of specific DNA fragments in your sample.
SAFETY PRECAUTION:
Non-radioactive probes are generally safer to handle than radioactive probes
but still require adherence to laboratory safety guidelines.
References:
Southern Blotting: -
• https://www.bio.davidson.edu/genomics/method/Southern
blot.html
• https://www.cytivalifesciences.com/en/us/solutions/geno
mics/knowledge-center/northern-and-southern-blotting
• https://www.cambridge.org/core/services/aop-cambridge-
core/content/view/77A318CD82229BDD86867045C9DD77
4D/S0029665196000912a.pdf/southern-blotting.pdf
• https://cshprotocols.cshlp.org/content/2021/7/pdb.prot10
0487.full
• https://www.sigmaaldrich.com/IN/en/technical-
documents/protocol/protein-biology/gel-
electrophoresis/southern-and-northern-blotting
• https://askabiologist.asu.edu/southern-blotting
• Weinberg, R.A. (2013). The Biology of Cancer. Garland
Science.
• Sambrook, J. Fritsch, E.F., & Maniatis, T. (1989). Molecular
Cloning: A Laboratory Press.
• Southern blotting, E.M. (1975).
Radioactive and Non-radioactive probes: -
• Molecular Cloning: A Laboratory Manual by Sambrook, J., &
Russell, D.W.
• Molecular Biology of the cell by Alberts, B., et al.
• DIG Application Manual for Filter Hybridization by Roche
Applied Science.
• Biotin and Digoxigenin as labels for Light Microscopical
Detection of Nucleic Acids by Avrameas, S.
