NOTES ON DNA
LIBRARY
KAJALI KARNGAR (BSc, MSc)
Instructor, Biology 305 Section
Department of Biological Sciences
T.J.R. Falkner College of Science & Technology
University of Liberia
INTRODUCTION
Library is a collection of DNA fragments that is stored
and propagated in a population of micro- organisms
through the process of molecular cloning
A genomic library is a collection of recombinant clones
representing the entire genome of an organism
Each clone is like a book in that library containing part
of the information contained in the genome
It contains all DNA sequences such as expressed genes,
non-expressed genes, exons and introns, promoter
and terminator regions and intervening
Uses of DNA/Gene Libraries
To obtain the sequences of genes for analysis,
amplification, cloning, and expression
Once the sequence is known probes, primers,
etc. can be synthesized for further diagnostic
work using, for example, hybridization reactions,
blots and PCR
Knowledge of a gene sequence also offers the
possibility of gene therapy
Also, gene expression can be used to synthesize a
product in particular host cells, e.g. synthesis of
human gene products in prokaryotic cells
Types of DNA/GENE Library:
Two types
GENOMIC LIBRARY
Genomic library contains DNA fragments
representing the entire genome of an
organism
cDNA LIBRARY
cDNA library contains only complementary
DNA molecules synthesized from mRNA
molecules in a cell
Genomic Library
Made from nuclear DNA/whole genome of an
organism or species
DNA is cut into clonable size pieces as randomly
possible using restriction endonuclease
It contains all DNA sequences such as expressed
genes, non-expressed genes, exons and introns,
promoter and terminator regions and intervening
Each clone is like a book in that library containing
part of the information contained in the genome
Construction of Genomic Libraries
1. Isolation of genomic DNA and vector.
2. Cleavage of Genomic DNA and vector by
Restriction Endonucleases.
3. Ligation of fragmented DNA with the
vector.
4. Transformation of r-DNA in the bacterial
cell.
5. Amplification of the r-DNA in bacterial
cells.
Step1. Extraction/isolation of genomic DNA
Step2. Cleaving/cutting the DNA
Mechanisms for cleaving/cutting
Physical method
Enzymatic method
Physical method
• It involves mechanical shearing of genomic DNA
using a narrow-gauge syringe needle or sonication
to break up the DNA into suitable size fragments
that can be cloned
• This method requires large quantities of DNA
Enzymatic method
It involves use of restriction enzyme for the
fragmentation of purified DNA
This method is limited by distribution probability of site
prone to the action of restriction enzymes which will
generate shorter DNA fragments than the desired size
The two factors which govern the selection of the
restriction enzymes are- type of ends (blunt or sticky)
generated by the enzyme action and susceptibility of the
enzyme to chemical modification of bases like
methylation which can inhibit the enzyme activity
The fragments of desired size can be recovered by either
agarose gel electrophoresis or sucrose gradient technique
and ligated to suitable vectors
Step3. Ligate the gene with the vector which
cleaves by same enzyme
Step4. Transformation
Transfer (transform) into bacteria Cells which are
able to undergo this treatment are termed as
competent cells
CaCl 2 causes DNA to precipitate on the outside
walls of bacterial cells.
Step5. Amplification
Example. [Link] cells are grown in an agar medium
containing ampicillin or tetracyclin
Step5. Amplification of Libraries
The primary library created is usually of a low
titer and unstable
The stability and titer can be increased by
amplification (For this, the phages or bacterial
colonies are plated out several times and the
resulting progenies are collected to form an
amplified library)
The amplified library can then be stored almost
indefinitely due to long shelf-life of phages
It usually has a much larger volume than the
primary library, and consequently may be
screened several times
Screening of Genomic Library
Once the genomic library has been created, it is screened to
identify the genes of interest.
One of the most common library screening technique is called
colony hybridization another one is agarose gel
In the process of library construction, phage vectors are used then
the process of identification of genes of interest involved is the
plaques hybridization
Colony hybridization →Colony Hybridization is the screening of
library with a labeled probe (radioactive, etc.) to identify a specific
sequence of DNA, RNA, enzyme, protein, or antibody
Plaque hybridization →The plaques are screened by a technique
,based on the hybridization of oligonucleotide probe to target DNA
DNA is transferred directly from the Petri dish to the filter→ which
is then incubated with labeled probes
• Advantages of genomic libraries
Identification of a clone encoding a particular
gene of interest
It is useful for prokaryotic organisms having
relatively small genomes
Genomic libraries from eukaryotic organisms
are very important to study the genome
sequence of a particular gene, including its
regulatory sequences and its pattern of
introns and exons
Disadvantages of Genomic library
Genome libraries from eukaryotes having
very large genomes contain a lot of DNA
which does not code for proteins and also
contain non-coding DNA such as repetitive
DNA and regulatory regions which makes
them less than ideal
Genomic library from a eukaryotic organism
will not work if the screening method requires
the expression of a 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.
5. Serving as a source of genomic sequence for
generation of transgenic animals through genetic
engineering
6. Study of the function of regulatory sequences in vitro.
7. Study of genetic mutations in cancer tissues
cDNA LIBRARY
Complementary DNA molecules synthesized
from mRNA molecules in a cell
Constitute some portion of the transcriptome
of the organism
cDNA is produced from fully transcribed
mRNA found in the nucleus
Therefore contains only the expressed genes
of an organism→So ,tissue specific cDNA
libraries can be produced
cDNA Library cont.
cDNA is created from a mature mRNA from a
eukaryotic cell with the use of an enzyme
known as reverse transcriptase
In eukaryotes, apoly-(A) tail distinguishes
mRNA from tRNA and rRNA and can therefore
be used as a primer site for reverse
transcription
Steps in making a cDNA Library
• mRNA extraction
• By using oligomeric dT nucleotide coated
resins where only the mRNA having the poly-A
tail will bind.
• Once mRNA is purified, oligo-dT is tagged as a
complementary primer which binds to the
poly-A tail providing a free 3'-OH end that can
be extended by reverse transcriptase to create
the complementary DNA strand
Steps in making a cDNA Library cont.
• Now, the mRNA is removed by using a RNAseH enzyme
leaving a single stranded cDNA (sscDNA)
• This sscDNA is converted into a double stranded DNA
with the help of DNA polymerase
• However, for DNA polymerase to synthesize a
complementary strand a free 3'-OH end is needed
• This is provided by the sscDNA itself by generating a
hairpin loop at the 3' end by coiling on itself.
• Polymerase extends the 3'-OH end and later the loop
at 3' end is opened by the scissoring action of S1
nuclease
Steps in making a cDNA Library cont
• Restriction endonucleases and DNA ligase are
then used to clone the sequences into
bacterial plasmids
• The cloned bacteria are then selected,
commonly through the use of antibiotic
selection.
• Once selected, stocks of the bacteria are
created which can later be grown and
sequenced to compile the cDNA library
cDNA Library uses
cDNA libraries are commonly used when
reproducing eukaryotic genomes, as the amount
of information is reduced to remove the large
numbers of non-coding regions from the library
cDNA libraries are used to express eukaryotic
genes in prokaryotes
cDNA libraries are most useful in reverse genetics
where the additional genomic information is of
less use
Also, it is useful for subsequently isolating the
gene that codes for that mRNA.
Comparison of Genomic and cDNA Libraries