southern and northern blotting - wikispaces · southern and northern blotting . comparison of...
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Southern and Northern blotting
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Comparison of Southern, Northern,
and Western analyses of Gene X
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SOUTHERN BLOTTING
The technique was developed by E.M. Southern in 1975.
The Southern blot is used to detect the presence of a particular piece of DNA in a sample.
The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.
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Southern hybridization
Transfer buffer
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Flow chart of Southern hybridization Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope
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1) DNA Fragmentation
The DNA sample is digested with one or more restriction enzymes.
Nuclease hydrolyze the bonds connect bases within the strand.
If large amount of restriction fragments
faint smears rather than discrete bands
Same or nearly the same length
migrate together during electrophoresis
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2) Gel Electrophoresis
Separate the fragments according to size to be detected using ethidium bromide.
Smaller fragments are migrating faster than larger fragments.
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Gel Treatment
Double stranded DNA denatured using NaOH (base). separating it into single DNA strands for later hybridization to the single stranded probe.
If too large, treated with HCL (acid) to depurinate & break it into smaller fragment & neutralized before proceed.
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Gel treatment
Acid treatment
• 0.2 N HCl solution
Denaturation
• NaOH solution
Neutralization
• Tris-Cl buffer (pH8.0)
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Depurination
Optional
Occurs before neutralization
When DNA fragments is > 15 kilobases, it is
too hard to be transferred to filter
The gel is treated with dilute acid (0.2 M HCl
for 15 minutes)
• depurinate DNA fragment into smaller pieces
and promote higher efficiency transfer to filter.
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Neutralization
DNA is placed into an alkaline solution containing
0.5mM NaOH to denature the dsDNA into ssDNA and
neutralize the acid in previous step.
Function:
(1) improve binding of the –vely charged DNA to
+vely charged filter
(2) ssDNA strands for hybridization
(3) destroy any remaining RNA present in the sample
Only ssDNA can be
transferred to filter
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3. Blotting
Exert pressure
evenly to a gel to
ensure even contact
between gel and
membrane
Transfer is done by
capillary action which
draws buffer (binds ssDNA)
up onto the membrane
The binding of DNA
to membrane is due
to ion exchange
interactions
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Southern transfer
Measure gel and set up transfer
assembly:
• Wick in tray with 20x SSC
• Gel
• Nitrocellulose or Nylon filters
(soaked in H2O and 20x SSC)
• 3MM Whatman filter paper
• Paper towels
• Weight
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Blotting
Transferred the gel onto a membrane which is a sheet of nitrocellulose filter or nylon membrane to carry out blotting.
Enable the DNA fragments accessible by the label probes required for analysis by autoradiography or other detection methods depend on your probes.
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Blotting
Gel is placed on the filter paper dipped in a reservoir containing transfer buffer. The membrane is laid on top of the gel wit paper towels placing on top of the membrane. Buffer lift up by capillary action to move the DNA from the reservoir to the membrane.
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Blot permanently
Binding of the DNA to the membrane is weak after blotting (unstable).
Easily to washed off during washing.
A) Treated the DNA fragments with UV light
B) Oven baked it around 80°C.
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4) Pre Hybridization- Blocking
To block non specific sites.
To ensure the specificity of the binding of the probe to the sample DNA.
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5) Probe Labeling
The probe DNA is labeled so that it can be detected.
A)Radioactive labeling
B)Tagging with non radioactive probes
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6) Hybridization
Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA. The reactions are specific where 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.
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7) Post Hybridization- Washing
Excess unbound probe is washed away with buffers containing NaCl and detergent.
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8) Detection
Labeled probe bound to the membrane is detected by autoradiography. Reveals the position of DNA fragment to which the probe hybridized. Autoradiograph is an image on an x-ray film which will visualized the discrete bands directly caused by beta emissions from the 32P radioactivelabeled probe. Hybridization of the probe to the sample DNA fragment on the membrane indicates that the fragment contains DNA sequence that is complementary to the probe.
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Autoradiography
Exposure to x-ray
film
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SOUTHERN BLOTTING
The key to this method is hybridization.
Hybridization-process of forming a
double-stranded DNA molecule between
a single-stranded DNA probe and a
single-stranded target patient DNA.
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Step 3. DNA Denaturation
T G A A T C
A C A T T G
• Eliminate hydrogen bonds with sodium
hydroxide (NaOH)
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Step 4. Transfer DNA to Membrane
• Two methods for transferring DNA to a
membrane
– capillary
– electrophoretic
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Other Applications
DNA fingerprinting
• RFLP of VNTRs
Dot or slot blot
Colony or plaque lifts
Microarray analysis
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Other Applications
DNA fingerprinting
• RFLP of VNTRs
Dot or slot blot
Colony or plaque lifts
Microarray analysis
![Page 28: Southern and Northern blotting - Wikispaces · Southern and Northern blotting . Comparison of Southern, Northern, and Western analyses of Gene X. ... Comparison of Southern and Northern](https://reader034.vdocuments.us/reader034/viewer/2022050711/5ac5b7917f8b9aae1b8e36c6/html5/thumbnails/28.jpg)
Other Applications
DNA fingerprinting
• RFLP of VNTRs
Dot or slot blot
Colony or plaque lifts
Microarray analysis
![Page 29: Southern and Northern blotting - Wikispaces · Southern and Northern blotting . Comparison of Southern, Northern, and Western analyses of Gene X. ... Comparison of Southern and Northern](https://reader034.vdocuments.us/reader034/viewer/2022050711/5ac5b7917f8b9aae1b8e36c6/html5/thumbnails/29.jpg)
Other Applications
DNA fingerprinting
• RFLP of VNTRs
Dot or slot blot
Colony or plaque lifts
Gene Expression
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Other Applications
Microarray technology
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Northern Blotting
Almost the same with Southern blot. Northern blotting is used for detecting RNA fragments. Southern blotting is used for detecting DNA fragments. Technique for detecting specific RNA separate by electrophoresis using RNA probe.
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Northern blotting
Technique for detecting specific RNAs
separated by electrophoresis by
hybridization to a labeled DNA probe.
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Application
Advantages
Northern blotting is able
to detect small changes in
gene expression that
microarrays cannot.
Disadvantages
Thousand of genes
visualized at a time with
microarray, while northern
blotting is usually looking at
one or small number of
genes
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The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope
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An example of Northern blotting
Northern blot
RNA gel 28 S
18 S
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Comparison of
Southern and Northern Blot
Southern blotting Northern blotting
Molecule detected
DNA mRNA
Gel electrophoresis
Agarose gel Formaldehyde agarose gel
Gel pretreatment
Depurination, denaturation, and
neutralization
-
Blotting method
Capillary transfer Capillary transfer
Probes DNA Radioactive or nonradioactive
cDNA, Radioactive or nonradioactive
Detection system
Autoradiography Chemiluminescent
Colorimetric
Autoradiography Chemiluminescent
Colorimetric