Recombinant DNA2 kỹ thuật tái tổ hợp DNA bản dịch đính kèm
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Heat shock Transformation
Heat shock Transformation
Electrophoration Transformation
Southern blot
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‘Southern’ hybridization named after Sir Edwin
Southern
Developed in 1975
•
One of the most highly cited scientific
publications
•
Earned Sir Southern a Lasker Award in 2005
Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)
Southern blot
(DNA)
Southern Blot: DNA-DNA*
Uses gel electrophoresis together with hybridization probes to
characterize restriction fragments of genomic DNA (or DNA
from other sources, such as plasmids).
Identifies DNA with a specific base sequence.
Can be done to detect specific genes present in cells.
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Goals of Southern Hybridization
• Immobilize DNA onto a permanent
substrate
• Membrane
– paper-like matrix
– nylon or nitrocellulose
– usually has a slight positive charge
• Identify DNA sequence (gene) of interest
General Scheme for Southern Blot
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Southern Steps
1.
DNA to be analyzed is digested to completion with a
restriction endonuclease.
2.
Electrophoresis to maximally separate restriction fragments
in the expected size range. A set of standards of known size
is run in one lane of the gel.
3.
Blot fragments onto a nitrocellulose membrane.
4.
Hybridize with the 32P probe.
5.
Autoradiography.
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Step 2
Gel electrophoresis
• Separates DNA fragments.
Soak gel in 0.5 M NaOH
• Converts dsDNA to ssDNA
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• Cover gel with nitrocellulose
paper…then…
Step 3. Nitrocellulose Blot
• 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.
• Vacum dry nitrocellulose at
80C to permanently fix DNA in
place or cross link (via covalent
bonds) the DNA to the
membrane.
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Step 4. Hybridization
• 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 renaturation
temperature that will permit
probe to anneal to its target
sequence(s).
• Wash & dry nitrocellulose sheet.
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Step 5. Autoradiography
• Place nitrocellulose sheet over Xray film.
• X-ray film darkens where the
fragments are complementary to
the radioactive probes.
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Southern Application:
• Identification the number of copies
• Isolation mutated
• Variation
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Northern Blots
1.
Isolate RNA & treat with formaldehyde.
2.
Electrophorese RNA in denaturing agarose gel (has
formaldehyde). Visualize RNA in gel using Ethidium bromide
stain and photograph.
3.
Transfer single-stranded RNA to nitrocellulose or nylon
membrane. Covalently link RNA to membrane.
4.
Incubate membrane (RNA immobilized on membrane) with
labeled DNA or RNA probe with target sequence.
5.
Development.
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Total RNA on agarose gel
Northern Blot Results
Northern Blot Results (cont.)
Northern Application
• Northern blots are particularly useful for determining the conditions
under which specific genes are being expressed, including which
tissues in a complex organism express which of its genes at the
mRNA level.
• For instance:
When trying to learn about the function of a certain protein, it is
sometimes useful to purify mRNA from many different tissues or cell
types and then prepare a Northern blot of those mRNAs, using a
cDNA clone of the protein of interest as the probe.
Only mRNA from the cell types that are synthesizing the protein will
hybridize to the probe.
Example:
Expression sybII gene
at different life stages in the
frog Xenopus laevis
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/>/CNS/sybII.html
Summary
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Southern
DNA on membrane.
Digest DNA.
Convert dsDNA to
ssDNA.
Probe with DNA or RNA.
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Northern
RNA on membrane.
No need to digest DNA.
Denature “folded” RNA
with formaldehyde.
Probe with DNA or RNA.
Western Blot encyclopedia
