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Transcript of bismillah - elearning.med.asu.edu.eg
Blotting
Techniques
Prof. Dr. Hanan Shehata
Medical Biochemistry Department
1.Define hybridization and blotting techniques
2.Elaborate the objectives of blotting techniques
3.Recognize the procedure of blotting techniques
4.Differentiate between different blotting types.
3
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Southern Blot DNA fragments
Northern Blot RNA fragments
Western Blot Proteins
Visualization (Isolation) of a specific DNA (or RNA or PROTEIN) fragments among many contaminating molecules.
(the first was named after the person who screened the library)
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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.
Specific base pairing properties(A T) & (C G)
in complementary nucleic acid strands( in case of southern & northern)
OrPerfect Ag Antibody
(in western )
Hybridization.
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Hybridization reactions are specific
the probes will only bind to targets
with complementary sequence (or,
in the case of antibodies, sites with
the correct 3-d shape)
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Southern Blot
The technique was developed by E. 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
Steps1) Digest the DNA with a given restriction Enzyme.
2) Separate the resultant DNA fragments on an agarose gel.
3)Treat the gel with NaOH to denature the DNA.
4) Transfer the DNA from the gel to nitrocellulose or nylon filter
Paper (by either capillary diffusion or under electric current ) .
5) Fix the DNA to the filter by baking or ultraviolet light treatment.
6) Probe the filter for the presence of a given fragment of DNA
by various radioactive or non-radioactive means.
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Steps
1. Digest DNA
2. Gel electrophoresis
3. Transfer to solid support
4. Blocking
5. Preparing the probe
6. Hybridization
7. Washing
8. Detection of Probe-Target hybrids
SOUTHERN BLOTTINGDNA Fragmentation
– Cut the DNA into different sized pieces.– Use restriction endonucleases (RE)
• Nucleases hydrolyze the bonds that connect bases within the strand, resulting in cleavage of the strand.
• They cleave the double stranded nucleic acid only at specific points.
• Fragments are now easily separated by gel electrophoresis.
SOUTHERN BLOTTING
Gel Electrophoresis
– Sorts the DNA pieces by size
–Gels are solid with microscopic pores
–Agarose or polyacrimide
–Gel is soaked in a buffer
– Standards should also be run
SOUTHERN BLOTTING
•Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones
move faster. This results in a separation by size.
SOUTHERN BLOTTING
• Gels can be stained with ethidium bromide.
• This causes DNA to fluoresce under UV light which permits photography of the gel. High quality intact DNA should give the appearance of a single band.
• Degraded material will smear downwards.
• Only a small amount of degradation is tolerable
SOUTHERN BLOTTING
DNA Denaturation• DNA is then denatured with an alkaline
solution such as NaOH.
• This causes the double stranded to become single-stranded in order for the probe to hybridize to them..
SOUTHERN BLOTTING
Blotting
–Transfer the DNA from the gel to a solid support.
–The blot is usually done on a sheet of nitrocellulose paper or nylon.
–Transferred by either electrophoresis or capillary blotting.
SOUTHERN BLOTTING
• The blot is made permanent by:
Drying at ~80°C.
Exposing to UV irradiation
SOUTHERN BLOTTING
Blocking–Blocking buffer binds to areas on the blot
not occupied by patient DNA.
–Blocks the empty sites from being bound during hybridization.
SOUTHERN BLOTTING
Preparing the probe– Small piece of DNA used to find another
piece of DNA
–Must be labeled to be visualized
–Usually prepared by making a radioactive copy of a DNA fragment.
SOUTHERN BLOTTING
Hybridization–The labeled probe is added to the
blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets.
SOUTHERN BLOTTING
Washing–Excess probe will have bound
nonspecifically to the membrane despite the blocking reagents.
–Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe and reduce background.
SOUTHERN BLOTTING
Detection▪ Radioactive probes enable autoradiographic
detection.
▪ If the probe is radioactive, the particles it emits will expose X-ray film.
▪ After development, there will be dark spots on the film wherever the probe bound.
SOUTHERN BLOTTING
• Summary
–1. Extract and purify DNA from cells
–2. DNA is restricted with enzymes
–3. Sort by electrophoresis
–4. Denature DNA
–5. Transfer to nitrocellulose paper
–6. Hybridization with labelled probe
–7. Wash off unbound probe
–8. Autoradiograph
Apparatus
APPLICATIONS
•Southern blots are used in gene discovery ,mapping, evolution and development studies, diagnostics and forensics.
•In regards to genetically modified organisms, Southern blotting is used for testing to ensure that a particular section of DNA of known genetic sequence has been successfully incorporated into the genome of the host organism.
APPLICATIONS
•Southern blots allow investigators to determine the size of a restriction fragment and to measure relative amounts in different samples.
•Southern blot is used to detect the presence of a particular bit of DNA in a sample
•Analyze the genetic patterns which appear in a person's DNA.
•Analyze restriction digestion fragmentation of DNA or a biological sample
Restriction Fragment Length Polymorphism (RFLP)
• Polymorphism refers to the DNA sequence variation between individuals of a species.
• If the sequence variation occurs at the restriction sites, it could result in RFLP.
• The most well known example is the RFLP due to b globin gene mutation.
• Restriction Fragment Length Polymorphism
(RFLP) resulting from b-globin gene mutation.
• In the normal cell, the sequence corresponding to 5th to
7th amino acids of the b-globin peptide is
CCTGAGGAG, which can be recognized by the
restriction enzyme MstII.
• In the sickle cell, one base is mutated from A to T,
making the site unrecognizable by MstII.
• Thus, MstII will generate 0.2 kb and 1.2 kb fragments in
the normal cell, but generate 1.4 kb fragment in the
sickle cell.
• These different fragments can be detected by southern
blotting, by using a probe spanning the polymorphic site.
Northern blotting
•Analysis of RNA following its attachment
to a solid support.
•The RNA is sized by gel electrophoresis.
•Transfer the RNA to nitrocellulose filter
paper as for Southern blotting.
•Probe the filter for a particular RNA
similarly to probing of Southern blots
APPLICATIONS
• A standard for the direct study of gene
expression at the level of mRNA (messenger
RNA transcripts).
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing - can detect alternatively
spliced transcripts
• Often used to confirm and check transgenic
/knockout mice (animals)
Western blotting
•Involves the analysis of proteins following attachment to a solid support.
•The proteins are separatedby size SDS-PAGE.
• Then electrophoretically transferred to nitrocellulose or nylon filters.
•The filter is then probed with antibodies raised against a particular protein.
Applications• The confirmatory HIV test employs a Western
blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added.
• The stained bands then indicate the proteins to which the patient's serum contains antibody.
