DNA Library

• 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 (Complementary DNA) libraries.
 (Complementary DNA library)
cDNA Library (Complementary DNA library) Genomic DNA library

What it is?
Collection of clones having complementary DNA
to the mRNA of an entity Collection of clones having the complete genomic
DNA of an entity
What does it contain?

Represents genes expressed in a particular cell at

a given period of time Represents all genes

Size

Smaller compared to genomic DNA library Vast in comparison to cDNA library

Coding and non-coding sequences

Clone contains sequences seen on mRNA only, not Contains sequences for introns and exons.
the complete gene. Genomic clones can have sequences of the
complete gene

Important substance required for their

construction Ligases and restriction endonucleases
cDNA Library (Complementary DNA library) Genomic DNA library

Material required to start

mRNA DNA
Expression in prokaryotic system

Can be directly expressed as it has only Gene expression extracted from the
coding sequences genomic library is challenging in the
prokaryotic systems, as they are devoid of a
splicing mechanism

Role in reverse transcriptase

Occurs in the synthesis of the first cDNA Not involved

strand

Vectors used

Phagemids, Plasmids, Lambda phage for Cosmids, plasmids, Lambda phage, YAC, BAC
harbouring smaller fragments due to the to harbour larger fragments
absence of introns
 How is the Genomic Library created?
There is the same procedure followed to create a genomic library for all the living
beings.

1. Extract and purify DNA.

2. Digest the DNA with a restriction
enzyme. This creates fragments that
are similar in size, each containing
one or more genes.
3. Insert the fragments of DNA into
vectors that were cut with the same
restriction enzyme. Use the enzyme
DNA ligase to seal the DNA
fragments into the vector. This
creates a large pool of recombinant
molecules.
4. These recombinant molecules are
taken up by a host bacterium
by transformation, creating a DNA
library.
 What are the major applications of Genomic Library?

The Genomic Library used in a variety of things.

•After a library is created, the genome of an organism can
be sequenced to elucidate how genes affect an organism or to
compare similar organisms at the genome-level.
•The genome-wide association studies can identify candidate
genes stemming from many functional traits.
•to compare genetic sequences of different individuals to
elucidate similarities and differences within chromosomal
regions
•Genes can be isolated through genomic libraries and used on
human cell lines or animal models to further research.
 How is the cDNA Library created?

• The construction of a cDNA library involves

 Extraction and purification of mRNA
 These mRNAs are used as a template for the synthesis of cDNA by the
process of reverse transcription in the presence of oligo dT primer.
 The oligo dT primer binds with the poly-A tail of mRNA followed by
synthesis of the first strand of cDNA by using reverse
transcriptase enzyme.

Oligo dT are oligonucleotides that contain a segment of repeating

deoxythymidines (dT). The dT anneal to the polyadenosine (polyA) tails of
messenger RNA (mRNA), guiding the synthesis of complementary DNA
(cDNA).
How is the insertion of rDNA in the host cell?
 How is the insertion of rDNA in the host cell?

• In this step, the recombinant DNA is introduced into a

recipient host cell.
• This process is termed as Transformation.
• Transformation is defined as the process of taking up of naked
DNA from the surroundings into the host cell
• Transformation can be brought about by the following
methods.
1. Transformation by Natural competence
2. Transformation by Induced competence
3. Protoplast Transformation and Protoplast Fusion
4. Electroporation
1.Transformation by Natural competence
Some Cells belonging to naturally competent and can take up naked DNA
Pneumococcus, Bacillus and Haemophilus spp. etc.
2. Transformation by Induced competence
Since DNA is a very hydrophilic molecule, it won't normally pass through a
bacterial cell's membrane.
In order to make bacteria take up the plasmid, they must first be made "competent"
to take up DNA.
This involves addition of calcium chloride to the cell suspension which enhances
the binding of plasmid DNA to lipopolysaccharide (LPS).
Positively charged calcium ions attract both the negatively charged DNA and the
negatively charged groups in the LPS inner core.
The plasmid DNA can then pass into the cell upon heat shock, wherein cells are
cooled to a low temperature (4°C) and then heated to a high temperature (42°C)
for a short time. This causes the bacteria to take up the DNA.
3. Protoplast Transformation and Protoplast Fusion
• Initially, protoplasts are generated by enzymatic removal of cell
walls of bacteria which are then transformed using polyethylene
glycol (PEG) along with the DNA.
• PEG is then removed and protoplasts are allowed to regenerate on
an osmotically stabilised medium.
• The process wherein protoplasts from two different cells fuse
together is called protoplast fusion.
• Protoplast transformation/fusion is also used in bacteria such
as Bacillus spp., Lactococcus spp. and Lactobacillus spp. etc. during
their genetic manipulation.
 4. Electroporation
• Bacterial cells mixed with plasmid DNA are subjected to a brief pulse of
high voltage electricity using electroporator
• The membrane of the cells exposed to high intensity electric pulse is
temporarily destabilized in specific regions of cell.
• Cells become permeable and DNA enters the cells.

Gene gun: This technique fires microscopic gold or tungsten particles coated with
foreign DNA at cells using a compressed air gun.(for plant cell)
Microinjection: A cell is held on a glass pipette with diameter much smaller than the
cell, under a microscope and foreign DNA is injected directly into the nucleus using an
incredibly fine micropipette, which punctures the plasma membrane.(for animal cell)
• CONJUGATION : It is a natural microbial recombinant process during
which two live bacteria come together join by cytoplasmic bridges and
transfer single stranded DNA(from donor to recipient).
• The most common model organism used as a
potential host for cloning is E. coli since it is
very convenient to use.
• Besides, a large number of E. coli vectors are
available.
• the recombinant DNA can be easily
introduced into E. coli by transformation and a
high level expression of recombinant proteins
can be obtained