1. Blotting techniques
Visualization of specific DNA, RNA or Protein out of contaminants can be done by
blotting techniques like those shown in figure above. These procedure help to
determine the number of copies of genes present in the given tissue or whether there
are any gross alteration in a gene (deletions, insertions, or rearrangements) because
any alteration will change the size which can be resolved by electrophoresis.
Fig. Blotting Technique
Southern Blot:
This is a DNA blot transfer. It combines the use of restriction enzyme, electrophoresis
and DNA probes. DNA isolated from cell line or tissue is digested with one or more
restriction enzymes. This mixture is pipetted into a well in an agarose or polyacrylamide
gel and electrophoresed. DNA being negatively charged migrates towards the anode;
the smaller fragments move rapidly. (The length of fragments can be determined by
comparison of the position of band relative to standard fragment of known size). After
this, the DNA is denatured by exposure to mild alkali and transferred to nitrocellulose
or nylon paper, resulting in an exact replica of the pattern on the gel. The DNA is bound
to the paper by exposure to heat or UV, and the paper is then exposed to the labelled
cDNA probe, which hybridizes the complementary fragments on the filter. After through
washing the paper is exposed to x-ray film, which produces specific bands corresponding
to the DNA fragment that recognized the sequences in the cDNA probe.
2. Blotting techniques
Visualization of specific DNA, RNA or Protein out of contaminants can be done by
blotting techniques like those shown in figure above. These procedure help to
determine the number of copies of genes present in the given tissue or whether there
are any gross alteration in a gene (deletions, insertions, or rearrangements) because
any alteration will change the size which can be resolved by electrophoresis.
Fig. Blotting Technique
Southern Blot:
This is a DNA blot transfer. It combines the use of restriction enzyme, electrophoresis
and DNA probes. DNA isolated from cell line or tissue is digested with one or more
restriction enzymes. This mixture is pipetted into a well in an agarose or
polyacrylamide gel and electrophoresed. DNA being negatively charged migrates
towards the anode; the smaller fragments move rapidly. (The length of fragments
can be determined by comparison of the position of band relative to standard
fragment of known size). After this, the DNA is denatured by exposure to mild alkali
and transferred to nitrocellulose or nylon paper, resulting in an exact replica of the
pattern on the gel. The DNA is bound to the paper by exposure to heat or UV, and
the paper is then exposed to the labelled cDNA probe, which hybridizes the
complementary fragments on the filter. After through washing the paper is exposed
to x-ray film, which produces specific bands corresponding to the DNA fragment that
recognized the sequences in the cDNA probe.
3. Fig. Western blotting
Northern Blot:
This is similar to southern blot. RNA is subjected to electrophoresis before blot
transfer.
Western Blot:
This is for proteins. Proteins are electrophoresed and transferred to special paper
that avidly binds macromolecules and then probed with specific antibody or other
probe molecules.
5. Fig. Southern Blotting
Colony or plaque hybridization:
It is one of the methods used to identify a particular DNA fragment from a plasmid
gene library. Large clones are grown on agar plate as colonies. These colonies are
overlaid with nitrocellulose filter paper where cells will stick. These cells are fixed
by heat or UV, and subsequent NaOH treatment will lyse the cell and denatures DNA
so that it is available to hybridize with probe. A radioactive probe is added to filter
and the hybrid complex is localized by exposing the filter to x-ray film or imaging
screen. By matching the spot on the autoradiograph to a colony, the colonies can be
picked from the plate. Similar process is used to identify fragments in phase library.
PCR secreening of gene libraries
6. Hybrid select technique:
This is for screening of cDNA libraries. Here plasmid colonies harboring cDNA is plated
on medium. DNA is extracted from each colony denatured and immobilized in
membrane. It is exposed to total cellular mRNA and allowed to hybridize with
complementary strand. Bound mRNA is eluted from each membrane and is directed
to in vitro translation. The proteins produced are then characterized and the clone
containing its corresponding cDNA is isolated. They are then further grown for further
analysis.
Screening expression cDNA libraries:
This is also for screening of cDNA libraries. Here the cDNA is inserted along with gene
to produce fusion protein which can be assayed. This recombinant vectors are plated
and allowed to express the protein of interest. The colonies are transferred to
membrane and incubated with antibody specific for that protein which will produce
color. This allows visualization of plaque or colony that contains the cloned cDNA for
that protein which may then be picked from the agar plate and pure preparations
grown for analysis.
- See more at: http://edusanjalbiochemist.blogspot.in/2013/06/blotting-techniques.
html#sthash.yPQ1sFjt.dpuf
7. Fig. Western blotting
Northern Blot:
This is similar to southern blot. RNA is subjected to electrophoresis before blot transfer.
Western Blot:
This is for proteins. Proteins are electrophoresed and transferred to special paper that
avidly binds macromolecules and then probed with specific antibody or other probe
molecules.
9. Fig. Southern Blotting
Colony or plaque hybridization:
It is one of the methods used to identify a particular DNA fragment from a plasmid gene
library. Large clones are grown on agar plate as colonies. These colonies are overlaid
with nitrocellulose filter paper where cells will stick. These cells are fixed by heat or
UV, and subsequent NaOH treatment will lyse the cell and denatures DNA so that it is
available to hybridize with probe. A radioactive probe is added to filter and the hybrid
complex is localized by exposing the filter to x-ray film or imaging screen. By matching
the spot on the autoradiograph to a colony, the colonies can be picked from the plate.
Similar process is used to identify fragments in phase library.
PCR secreening of gene libraries
10. Hybrid select technique:
This is for screening of cDNA libraries. Here plasmid colonies harboring cDNA is plated
on medium. DNA is extracted from each colony denatured and immobilized in
membrane. It is exposed to total cellular mRNA and allowed to hybridize with
complementary strand. Bound mRNA is eluted from each membrane and is directed to
in vitro translation. The proteins produced are then characterized and the clone
containing its corresponding cDNA is isolated. They are then further grown for further
analysis.
Screening expression cDNA libraries:
This is also for screening of cDNA libraries. Here the cDNA is inserted along with gene
to produce fusion protein which can be assayed. This recombinant vectors are plated
and allowed to express the protein of interest. The colonies are transferred to
membrane and incubated with antibody specific for that protein which will produce
color. This allows visualization of plaque or colony that contains the cloned cDNA for
that protein which may then be picked from the agar plate and pure preparations grown
for analysis.
- See more at: http://edusanjalbiochemist.blogspot.in/2013/06/blotting-techniques.
html#sthash.yPQ1sFjt.dpuf
