2. Gene Library and Types
A large collection of DNA fragments cloned from a given
organism, tissue, organ, or cell type. It may contain entire
genomic sequences or complementary DNA sequences formed
from messenger RNA .
3. For the construction of Gene libraries we have to know the size of
the gene and the insert size or capacity of the vector. So that we
can determine the size of Gene library and also to ensure the high
probability of the insertion.
Also the molecular tools like Restriction enzymes( Molecular
scissors) and ligases( Molecular paste) were used .
Vectors like Plasmids, Lambda phage, cosmid, BAC or YAC( yeast
artificial chromosome) vectors can be used to construct genomic
libraries, the choice depending on the genome size.
4. Vectors , Insert size, Clone
requirement
Vector type
Maximum
insert size
Cloned DNA (kb)
Approx. No. of clones
required in library
Plasmid ~20 kb >105
lambda phage 20 kb 5 x 105
Cosmid 45 kb 2 x 105
BAC (bacterial artificial
chromosome)
> 500 kb 5 x 104
YAC (yeast artificial
chromosome)
1 Mb 105
5. Genome:
It is the totality of DNA sequences present in an
organism
This includes the DNA of Chloroplast and Mitochondria
The genome size of an individual is expressed in terms
of number of base pairs either in kilobases (1000 bp) or
in megabases (one million bp).
The smallest genome about 130mbp for Arabidopsis
thaliana (thale cress)
For us the genome size- 3300mbp
6. Genomic DNA Libraries
• These libraries are made from genomic DNA
Genomic DNA molecules are very large , so they
must be fragmented into small pieces to insert into
vectors.
• This is done through digestion using one or more
appropriate restriction endonucleases.
• The DNA is then ligated into the vector, which could
be a plasmid, a cosmid or in a Phage
7. Construction of Genomic
Libraries
• The construction of a genomic library begins with cleaving the
genome into small pieces by a restriction endonuclease.
• These genomic fragments are then either cloned into vectors &
introduced into a microbe or packed into phage particles and
assembled that are used to infect the host.
• In either case, many thousands of different clones- each with a
different genomic DNA insert –are created.
• Therefore each clone will act as a “book” in this “library” of DNA
fragments.
• If the genomic library has been inserted into a microbe that
expresses the foreign gene, it may be possible to assay each
clone for a specific protein or phenotype.
9. Screening of the
Genomic Library
The libraries were screened to identify the genes of interest.
For example: finding the gene for alanine production in a bacterium
we have to host genomic sequence of that bacterium in E.coli
Then they were grown in alanine deficit medium to remove the clone
having other genes
They were labeled in the petri plate which indicates that the gene for
alanine production is stored in the bacteria.
This method is called expression screening
Or we can use probes for screening(hybridization technique)
Probes may be a oligonucleotide or short segment of DNA which may
be labeled or taken from related species
These probes are hybridized with the DNA in the Nylon membrane
and visualized in UV or X-ray.
Or we can do the blotting methods for checking the prescence of gene
of interest
10. cDNA Libraries
These libraries are made from cDNA (complimentary
DNA), which are DNA copies from mRNA molecules.
To make cDNA, mRNA is isolated from a tissue or whole
organism, and DNA is copied from the mRNA template
using an enzyme called reverse transcriptase.
This enzyme uses the reverse genetics principle and
found in retrovirus.
The resulting cDNA molecules are then engineered so
that they have restriction enzyme recognition sites at
each end of every molecule, which allows them to be
digested and inserted into a vector
11. mRNA extraction
The mRNA was extracted and purified
by trizol extraction and column purification.
Column purification is done by using oligomeric dT
nucleotide coated resins where only the mRNA having
the poly-A tail will bind.
The rest of the RNAs are eluted out.
The mRNA is eluted by using eluting buffer and some
heat to separate the mRNA strands from oligo-dT.
12. cDNA construction
• Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymine
nucleotides) 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.
• Then the mRNA is removed by using a RNAse 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.
• The polymerase extends the 3'-OH end and later the loop at 3' end is
opened by the scissoring action of S1 nuclease. Restrictio
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.
13. Screening
Once a particular DNA
fragment is identified, it
can be isolated and
amplified to determine its
sequence. If we know the
partial sequence of a gene
and want to determine its
entire sequence, the
probe should contain the
known sequence so that
the detected DNA
fragment may contain the
gene of interest.
14. cDNA library vs.
Genomic Library
• In Eukaryotic genomes, the entire sequence of genes
is often very large because coding sequence (exons)
is interrupted by introns that do not contain coding
DNA sequence.
• The advantage of a cDNA library is that only the
exons, or DNA representing expressed sequence, are
templates for the creation of DNA (via Reverse
Transcriptase) to collect the preferred genes.
• Prokaryotes generally do not contain the apparatus
necessary to splice together exons. Furthermore, the
introns can increase the genomic space taken up by
the gene to a point where prokaryotic genetic
manipulation schemes cannot handle the length of the
DNA.
15. • When generating gene libraries from prokaryotic species,
Genomic DNA libraries are often used. Genes from prokaryotic
cells generally do not contain introns, and the DNA is therefore
much shorter, and the RNA does not require much post-
processing to be expressed correctly.
• Also, prokaryotic mRNA can be difficult to isolate; because of
this a genomic library is more preferable for prokaryotic genomic
studies.
• Therefore both Genomic and cDNA libraries are important in
their own aspects and hold key to the various unanswered
questions in context to the book of life- the Genome.
16. Digestion of the chromosomal DNA
with restriction endonuclease
Production of cDNA
(complimentary DNA)
Genomic Library
Insertion of each DNA fragment into
vector (recombinant DNA)
Transformation of Bacteria
using the recombinant vectors
Cloning of
the bacterial cells
Each clone produced is a “book”
in the “Library” of DNA fragments
cDNA Library
Extraction of mRNAExtraction of chromosomal
DNA
Insertion of each cDNA into vector
(recombinant DNA)
Transformation of bacteria
Cloning of each recombinant
Production of Bacterial cell (clones)
containing the gene of interest
17. 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.
Prokaryotes do not have introns in their DNA and therefore do
not possess any enzymes that can cut it out during
transcription process. cDNA does not have introns and
therefore can be expressed in prokaryotic cells. 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.
