Different methods by which genes are screened. The identification of specific clone from a library can be carried out.

1. Amit Gothe
2016MSB1337
Screening methods for Cloned
Libraries

2. Introduction
 A DNA library is a collection of DNA fragments that
have been cloned into vectors so that researchers can
identify and isolate the DNA fragments that interest
them for further study.
 There are basically two kinds of libraries: genomic
DNA and cDNA libraries.
 Genomic DNA libraries contain large fragments of DNA
in either bacteriophages or bacterial or P1-derived
artificial chromosomes (BACs and. PACs). cDNA
libraries are made with cloned, reverse-transcribed
mRNA, and therefore lack DNA sequences
corresponding to genomic regions that are not
expressed, such as introns and 5′ and 3′ noncoding
regions.
 cDNA libraries generally contain much smaller

3. Library Screening
 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 Ab/ligand.

4. Sequence dependent Screening
 Screening by Hybridization
 Nucleic acid hybridization is most commonly used
method and it is rapid.
 Developed by Grunstein and Hogness (1975).
 This method is widely used in isolating
Recombinant phage particles.
 Thus, hybridization has potential to isolate any
sequence if probe is available.
 Variation of this method devised by Benton is
called Plaque lift method.

6. PCR 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.

8. South-western 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
labelled 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.
 Alternatively ligands can be used to identify
polypeptides that specifically bind certain
molecules.

9. Thank You!
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