SCREENING OF CLONES

Library screening is the process of identification of the clones carrying the

gene of interest. It relies on a unique property of a clone in a library. The
DNA libraries consist of a collection of probably many thousand clones in the
form of either plaques or colonies on a plate

The process of identifying one particular clone containing the gene of interest
from among the very large number of others in the gene library is called
screening.

The identification of specific clone from a DNA library can be carried out by

using either:

(1) The sequence of the clone or

(2) The structure function of its expressed product

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.
Screening by hybridization
• Nucleic acid hybridization is the most commonly used method of library
screening .
• It relies on the fact that a single-stranded DNA molecule, used as a probe
can hybridize to its complementary sequence and identify the specific
sequences.
• This method is quick, can handle a very large number of clones and used in
the identification of cDNA clones which are not full-length (and therefore
cannot be expressed). The commonly used methods of hybridization are,
a) Colony hybridization
b) Plaque hybridization.
Colony hybridization

It is a rapid method of isolating a colony containing a plasmid harboring a

particular sequence or a gene from a mixed population.

1. The colonies to be screened are first replica-plated on to a nitrocellulose

filter disc that has been placed on the surface of an agar plate prior to
inoculation.
2. Master plate is retained for reference set of colonies.
3. The filter bearing the colonies is removed and treated with alkali so that
the bacterial colonies are lysed and the DNA they contain is denatured.
4. The filter is then treated with proteinase K to remove protein and leave
denatured DNA bound to the nitrocellulose.
5. The DNA is fixed firmly by baking the filter at 80°C.
6. A labeled probe is hybridized to this DNA which is monitored by
autoradiography.
7. A colony whose DNA print gives a positive auto radiographic result on X-
ray film can then be picked from the reference plate.

Colony hybridization can be used to screen plasmid or cosmid based

libraries. Compare the developed autoradiogram with the master plate
to identify the colonies containing a gene of interest. The cells which
contain the desired gene can grow in the liquid medium and can further
process for the isolation of recombinant plasmid DNA.

Therefore, the colony hybridization method is the “Screening technique” which

makes the use of the radioactive probe. The radioactively labelled probe then
screen or isolate the particular gene from the number of live bacterial colonies
from the master plate.

Plaque hybridization
Plaque hybridization, also known as Plaque lift, was developed by Benton and
Davis in 1977 and employs a filter lift method applied to phage plaques. This
procedure is successfully applied to the isolation of recombinant phage by
nucleic acid hybridization and probably is the most widely applied method of
library screening.
The method of screening library by plaque hybridization is described below-
• The nitrocellulose filter is applied to the upper surface of agar plates, making
a direct contact between plaques and filter.
• The plaques contain phage particles, as well as a considerable amount of
unpackaged recombinant DNA which bind to the filter.
• The DNA is denatured, fixed to the filter, hybridized with radioactive probes
and assayed by autoradiography.
Advantages
• This method results in a ‘cleaner’ background and distinct signal (less
background probe hybridization) for λ plaque screening due to less DNA
transfer from the bacterial host to the nitrocellulose membrane while lifting
plaques rather than bacterial colonies.
• Multiple screens can be performed from the same plate as plaques can be
lifted several times.
• Screening can be performed at very high density by screening small plaques.
High-density screening has the advantage that a large number of recombinant
clones can be screened for the presence of sequences homologous to the
probe in a single experiment.
Immunoscreening
 Developed in 1970
 Immunoscreening 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.
 Earlier immunoscreening methods employed radio-labelled primary
antibodies to detect antibody binding to the nitrocellulose sheet.
 It is now superseded by antibody sandwiches resulting in highly
amplified signals.
 The secondary antibody recognizes the constant region of the primary
antibody and is additionally conjugated to an easily assayable enzyme
(eg- horseradish peroxidase or alkaline phosphatase) which can be
assayed using colorimetric change or emission of light using X-ray film.
Procedure
 In this technique, the cells are grown as colonies on master plates and
are then transferred to solid matrix (nitrocellulose sheet)
 These colonies are subjected to lysis releasing the protein which bind to
the matrix.
 These proteins are treated with a primary antibody which specifically
bind to the protein (antigen). The unbound proteins are washed away.
 A secondary antibody is added which specifically binds to the primary
antibody.
 The secondary antibody carries an enzyme label bound to it which
converts colorless substrate to colored products. The colonies with
positive results (i.e. colored spots) are identified and sub-cultured.
Restriction digest for screening of clones:

Another way to distinguish between colonies containing plasmids with inserts

from those that do not is by using a restriction digest.

The key is to be strategic about your enzyme choices. You’ll want to pick
enzymes that will give you different sized bands depending on whether the
plasmid contains your insert of interest or not.

For example, choose enzymes that only cut once on either side of the expected
insert. If the plasmid contains your insert, you will see two bands: one
representing your vector backbone and the other representing the insert.

If the plasmid doesn’t contain the insert, you will just see the vector backbone
band. Or, you can choose an enzyme that cuts once in the plasmid backbone
and another enzyme that cuts within the insert. If the plasmid contains the
insert, you will see two bands but if the plasmid doesn’t, you will just see one
band. These are only a few examples of how you can use restriction digest to
screen your clones, but there are many other ways to choose restriction
enzymes for this approach.

Using restriction enzymes to check the presence and direction of your insert is
a precise and easy method for screening colonies.  First, restriction
mapping should be performed to identify which restriction enzymes can be
used to easily identify the presence of your insert within the plasmid.
After isolating a plasmid DNA from an overnight bacterial culture, digest the
purified plasmid DNA from recombinant clones using restriction enzymes.
Once digested, run the plasmid on an agarose gel to verify that the vector
backbone and insert are of the expected sizes .
Figure 1. Five minute plasmid DNA digestions with M: Thermo Scientific
GeneRuler Express Ladder, 1: Control, undigested plasmid DNA, 2: FastDigest
EcoRI, 3: FastDigest EcoRI and FastDigest KpnI, 4: FastDigest EcoRI, FastDigest
KpnI and FastDigest SmaI.
Alpha complementation/ Blue White Screening
PCR based Screening
 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 was first demonstrated by Takumi and Lodish in 1994.
 The molecular weight of known members of the family can be predicted
and novel mRNAs may give rise to amplification products.
 It is more stringent since three oligonucleotides (the two PCR primers,
and the hybridization probe) are required to give a true positive signal.

 Colony PCR is a method for rapidly screening colonies of yeast or

bacteria that have grown up on selective media following a
transformation step, to verify that the desired genetic construct is
present, or to amplify a portion of the construct.