Seminar on

cDNA & Genomic libraries.

Guided by,
Dr.Vedhmurthy
By,
HOD,
Swapnil.K Dept.of Biotechnonolgy,
M.Sc.,Biotechnology, Oxford College of Science,
2nd year. Banglore.
 Contents
• Introduction.
• Genomic libraries.
• Storage of genomic libraries.
• Advantages & disadvantages of genomic libraries.
• Applications of genomic libraries.
• cDNA library.
• Methods for synthesis of cDNA.
• Screening procedures.
– Nucleic acid hybridisation.
– Blue-white screening.
• Applications of cDNA libraries.
• Conclusion.
• References.
 Introduction
• Libraries: A  library is a population of host bacteria,
each of which carries a DNA molecule that was
inserted into a cloning vector, such that the collection
of cloned DNA molecules represents the
entire genome of the source organism.

• They can be
– Genomic libraries.
– cDNA libraries.
• Genomic DNA : For making libraries, genomic DNA, usually
prepared by protease digestion and phase extraction, is
fragmented randomly by physical shearing or restriction
enzyme digestion to give a size range appropriate for the
chosen vector. Often combination of restriction enzymes are
used to partially digest the DNA.
• Vectors : Plasmids, λ phage, cosmid, BAC or yeast artificial
chromosome vectors can be used to construct genomic
libraries, the choice depending on the genome size. The upper
size limit of these vectors is about 10,23,45,350 and 1000 kb
respectively. The genomic DNA fragments are ligated to the
prepared vector molecule using T4 DNA ligase.
 Genomic library.
• A genomic library: is a population of host bacteria, each of
which carries a DNA molecule that was inserted into a cloning
vector, such that the collection of
cloned DNA molecules represents the entire genome of the
source organism.
• Vectors for genomic libraries
• λ-phage - 9-23 kb → convenient and easy to handle
• Cosmids - 30-45kb
• BAC, YAC →artificial chromosomes that handle large inserts.
• Fosmids- 40kb
• The entire human genome is about 3 x 109 bp long while a
plasmid may carry up to 20 kb fragment.  This would require
1.5 x 105 recombinant plasmids. When plating E. coli colonies
on a petri dish, the maximum number of individual colonies is
about 200 colonies per dish.  Thus, at least 700 petri dishes are
required to construct a human genomic library. 
• As many as 5 x 104  phage plagues can be screened on a
typical petri dish.  This requires only 30 petri dishes to
construct a human genomic library. 
• Another advantage of phage vector is that its transformation
efficiency is about 1000 times higher than the plasmid vector.
Storage of Libraries.
 Advantages & Disadvantages of genomic
libraries.
• Entire genome of an organism is used.
• Genomic libraries are not useful while working
with eukaryotes.
• Screening sometimes becomes difficult.
• However, genomic libraries allow us to study
the genome sequence of a particular gene.
Applications of Genomic Library
1. Genomic library construction is the first step
in any DNA sequencing projects.
2. Genomic library helps in identification of
the novel pharmaceutically important genes.
3. Genomic library helps in identification of
new genes which were silent in the host.
4. It helps us in understanding the complexity
of genomes.
cDNA library
Advantages of cDNA libraries
There are no introns, so there is no danger of
pieces of your gene being chopped onto separate
clones; and the library is (hopefully) enriched for
your gene, since instead of one or two copies, as in
the genomic library, you have as many copies as
the cell could produce mRNA's for that gene. So
most molecular biologists, when searching for a
new gene, start by screening a cDNA library from
a tissue or organism that they suspect is actively
using that gene. 
Protocol for construction of
an cDNA library.
Tailing & priming method
The self-priming method
Rnase H method for cDNA synthesis.
 Screening procedures
• Screening : Screening to isolate one particular clone from a
gene library routinely involves using a nucleic acid probe for
hybridization. The probe will bind to its complementary
sequence allowing the required clone to be identified.
• Clony and plaque hybridization: A copy of the position of
colonies or plaques on a petri dish is made on the surface of a
membrane, which is then incubated in a solution of labeled
probe. After hybridization and washing, the location of the
bound label is determined. The group of colonies/plaques to
which the label has bound is diluted and re-plated in
subsequent rounds of screening until an individual clone is
obtained.
LE 20-5

Nucleic Acid Hybridization Colonies

containing
gene of
Master plate interest
Probe Master plate
DNA
Radioactive
Solution single-stranded Gene of
containing DNA interest
probe Film
Filter Single-stranded
DNA from cell
Filter lifted
and flipped over

Hybridization
on filter
A special filter paper The filter is treated to break The filter is laid under After the
is pressed against open the cells and denature photographic film, developed film is
the master plate, their DNA; the resulting allowing any flipped over, the
transferring cells to single-stranded DNA radioactive areas to reference marks
the bottom side of molecules are treated so that expose the film on the film and
the filter. they stick to the filter. (autoradiography). master plate are
aligned to locate
colonies carrying
the gene of
interest.
• Blue white screening.
pUC18 - a common
cloning vector

Essential features
Polylinker
Selectable marker (Ampr)
Screenable Marker (LacZ)
Bacterial Origin of replication (oriR)
 LacZ- a screenable marker
EcoR1
EcoR1 EcoR1

Lac Z gene Interrupted

Lac gene
pUC18 pUC18

“Recombinant
Molecules”

Beta-galactosidase NO Beta-galactosidase
X-gal Gal + X(Blue dye)
blue colonies White colonies
(colorless)

Allows for easy visual “screening” of bacterial colonies that contain

recombinant DNA molecules
Bacterial colonies transformed with pUC18

White colonies blue colonies

(contain recombinant DNA (contain non-recombinant DNA
molecules) molecules)
 Applications of cDNA libraries.
• cDNA libraries creates acess to some
organisms genomes who do not posses a true
DNA.
• cDNA libraries allows us to eliminate the real
junk in an entire DNA.
• Expressions of genes is easy.
 Conclusion.
• The genomic library contains DNA fragments
representing the entire genome of an organism.
• The cDNA library contains only complementary
DNA molecules synthesized from mRNA molecules
in a cell.
• They are important as they provide a gateway for
cloning.
• Even a single gene of interest can be easily isolated
and cloned.
• It allows us to properly notify the expressions of
various genes present in the genome.
 References.
• B.D.Singh, Biotechnology expanding horizons, 2009
edition, Kalyani publishers, 25-36.
• Ernst-L.Winnacker, From genes to genomes, 2003
edition, Panima publishing house, 32-46.
• Jeremy.W.Dale & Malcolm von Schantz, From genes
to genomes concept & applications, 2002 edition,
British library publications, 99-116.
• Julia Lodge , Pete Lund & Steve Mirchin, Gene
cloning principles & applications, Taylor & francis
group, 85-141.
THANK YOU