2. The identification of specific clone from a DNA
library can be carried out by exploiting either
(1)the sequence of the clone or
(2)the structure/function of its expressed product.
Former type can be applied to genomic or cDNA
libraries and nucleic acid hybridization is done
with the help of probes or primers.
Screening the product of a clone is applied only
to expression libraries where the DNA fragment is
expressed to yield proteins and the product is
recognized by antibody /ligand
SEQUENCE DEPENDENT SCREENING
Screening by hybridization
Nucleic acid hybridization is the most commonly
used method and it is rapid
3. Grunstein &hogness (1975) developed a screening procedure to detect
DNA sequences in transformed colonies by hybridization with radioactive
RNA probes.
The variation of this method is devised
By benton and davis and it is called as
Plaque lift method.
This method is widely used in isolating
Recombinant phage particles.
Thus hybridiztion has potential to
isolate any sequence if probe is
available.
A number of alternative labeling
methods are available that avoid
use of radioacticity
Incorporation of digoxigenin or
biotin detected by specific antibody
4. SCREENING BY PCR
The PCR is widely used to isolate specific DNA sequences from uncloned
genomic DNA and now It has been a useful technique for library
screening.
This method is first demonstrated by takumi . And lodish in 1994
Versatile like hybridization.
To isolate a specific clone the PCR is carried out with gene specific
primers that flank a unique sequence in the target.
Pools of clones are maintained in multiwell plates.
Each well is screened by the PCR and positive wells are
identified
The clone in each positive well are then identified are then
diluted into series in a secondary set of plates and screened again
5. The process is repeated until wells carrying
homogeneous clones corresponding the
gene of interest have been identified.
EXPRESSION LIBRARIES SCREENING.
If a DNA library is established using expression
vectors, each individual clone can be
expressed to yield a polypeptide.
This type of screening is important where the
DNA sequence of the target sequence is
unknown.
6. IMMUNOLOGICAL SCREENING
Developed in 1970 when plasmid vectors are used to construct genomic
libraries
Immunological screening involves the use of antibodies
that specifically recognize antigenic determinants
on the polypeptide
This technique can be applied to any protein for which
an antibody is available.
The molecular target for recognition is generally an
Epitope.
It is a short sequence of amino acid that folds in a particular three
dimensional conformation on the surface of the
protein.
7. Broome and Gilbert method was widely used.
Transformed cells were plated in Petri dishes and allowed to form
colonies.
The replica plating is done and colonies were lysed.
A sheet of polyvinyl that had been coated with appropriate antibody
was then applied to surface allowing antigen antibody complex
formation
Then sheet was then removed and exposed to 125I labeled IgG and then
exposed to X-Ray film.
This technique also known as sandwich
technique and the protein must have
atleast two determinants.
8. SOUTHWESTERN AND NORTH WESTERN SCREENING
a plaque lift is carried out to transfer a print of the library onto
nitrocellulose membrane .
Here screening is carried out by incubating radio labeled double
stranded DNA oligonucleotide probe containing recognition sequence
for the DNA-binding protein .
Combines the principles of southern and western blots. It has been
particularly successful in the isolation of clones expressing cDNA
sequences.
A limitation of this technique is that since individual plaques contain only
single cDNA clones, transcription factors that function only in the form of
heterodimers or multimeric complex do not recognize DNA probe.
The affinity of the polypeptide for specific DNA sequence must be high.
To isolate RNA binding proteins single stranded RNA is used and this
method is called as north western screening
9. Alternatively ligands can be used to identify
polypeptides that specifically bind certain molecules.
It is not widely used because of low sensitivity .
