Professional Documents
Culture Documents
Southern Northern and Western Blotting
Southern Northern and Western Blotting
HARITHA
Comparison of Southern,
Northern, and Western analyses
of Gene X
Southern hybridization
Transfer buffer
Detection of an RFLP by
Southern blotting
Flow chart of Southern
hybridization
Preparing the samples and running the gel
Southern transfer
Hybridization
Post-hybridization washing
Signal detection
Preparing the samples and
running the gel
Digest 10 pg to 10 g of desired DNA samples
to completion.
Prepare an agarose gel, load samples
(remember marker), and electrophorese.
Stain gel ethidium bromide solution (0.5
g/ml).
Photograph gel (with ruler).
Critical parameters (I)
Note the complexity of DNA
Genomic DNA
A single-copy of mammalian gene, 3 Kb
average in length
10 g x 3 Kb/3 x 106 Kb = 10 g x 1/106 = 10
pg
Plasmid DNA or PCR products
0.1 g of a 3 Kb plasmid DNA 100 ng
Gel treatment
Acid treatment
0.2 N HCl solution
Denaturation
NaOH solution
Neutralization
Tris-Cl buffer (pH8.0)
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
After Southern transfer
Dissemble transfer
pyramid and rinse
nitrocellulose in 2x SSC
Bake nitrocellulose at 80C
for 2 hr or UV-crosslink
Nylon membrane for
seconds
Preparation of probes
Remove prehybridization
solution and add
hybridization solution
Add 500,000 cpm of the
probe/ml hybridization
solution.
Hybridize overnight at
appropriate temperature.
Post-hybridization washing
NC Nylon
Hydrophobic binding Covalent binding
Fragile Durable
Probe length > 200 ~ < 200 ~ 300 bp is
300 bp O.K.
Lower background Higher background
Cannot be exposed Can be exposed to
to basic solution basic solution
Not easily Can be reprobed
reprobed several times
Signals detection
Autoradioragraphy
Non-isotope detection system
- Chemiluminescent detection
- Colorimetric detection
- Multicolor detection
Autoradiography
Northern transfer
Probe preparation
Isotope
Prehybridization Non-isotope
Hybridization
Post-hybridization washing
Signal detection
Preparation of
agarose/formaldehyde gel
E.g. Prepare a 350 ml 1.2%
agarose/formaldehyde gel
4.2 g agarose in 304.5 g water. Microwave, then
cool to 60C. Add 35 ml 10x MOPS running buffer
and 10.5 ml 37% formaldehyde
Preparation of RNA samples
Prepare a premix:
5 l of 10x MOPS running buffer
8.75 l of 37% formaldehyde
25 l of formamide.
Prepare RNA samples:
38.75 l of premix
RNA (0.5 to 10 g)*
water to 50 l
*If the mRNA species of interest makes up a relatively high percentage of
the mRNA in the cell (>0.05% of the message), total cellular RNA can be
used. If the mRNA species of interest is relatively rare, however, it is
advisable to use poly(A)+ RNA.
Incubate 15 min at 55C
Running the RNA gel
RNA gel 28 S
18 S
Western blotting, or
immunoblotting
Protein bands
detected by
specific antibody
There are different VNTRs, as there are different plant species, number and location
of restriction enzyme-recognition sites.
PCR amplification of DNA is not required for this method. The routine southern blot
experiment can be used.
The complimentary DNA sequences are radiolabeled on agarose gel for visualization
in this method. This method is used to identify the origins of a particular plant
species.
This method is not much favored for DNA fingerprinting, as it has many drawbacks.
The results cannot indicate the chance of match between two organisms.
The other drawback of RFLPs is a costly process which involves lot of labor and
money.
Randomly Amplified Polymorphic DNAs (RAPDs)
This method is most commonly used for primary assay.
This method helps in screening the differences in DNA
sequences of two species of plants.
This method is used to search the sequences required for random
amplification. In this method, using short single primers at low
annealing tempratures, DNA is cut and amplified.
Using electrophoresis and superimposing the gels, a banding
pattern is identified. The gel is cut where the target band is found
and the DNA is isolated and sequenced.
This target is used to assess DNA from other cultivars. This
technique is more cost-effective than RFLPs. The drawback for
this method is that RAPDs lack specificity due to low annealing
temperatures and easier reaction conditions.
Simple Sequence Repeats (SSR)
Simple sequence repeats are microsarellites. They
show high degree of polymorphism.
They are isolated using hybridized probes followed by
their sequences. They are detected by gel
electrophoresis using specific dyes or radiolabelling.
The advantage of SSRs is that the amount of DNA
required is less than RFLPs. The assays involving SSRs
are more robust, making them more efficient than
RAPDs.
The drawback of this method is that seperate SSR
primers are needed for each species.
Amplified Fragment Length Polymorphism (AFLP)
This method is a PCR based derivative of RFLP. Here
sequences are selectively amplified using the
primers. This method is more useful than RFLP or
RAPD as more loci can be evaluated. AFLP helps in
determining a large number of polymorphism. This
method is also cost effective.
ADVANTAGES OF DNA FINGERPRINTING IN PLANTS ARE AS
FOLLOWS:
DNA fingerprinting is used for the identification of genetic diversity
within a breeding population. It is used to identify a gene of interest.
In the United States, it is also used to detect a genetically modified
organisms in agriculture.
RFLP markers are used to detect the genetic distance in wheat.
RAPD markers are used for characterization, estimation of genetic
relatedness and determination of genetic diversity of tea
germplasm. It is also used to find genetic relatedness and difference
in figs.
AFLP markers help in assessing genetic diversity among cultivars
such as wheat. It also helps detect higher level of polymorphism.
DNA fingerprinting of herbal drugs can be useful in authenticating
the various claims of medical uses related to the plants.