Principle and procedure for making Genomic library and cDNA library.pptx
1. Assignment presentation
Principle and procedure for making Genomic library
and cDNA library
Dr. Shambhoo Prasad
Asso. Prof. Department of PMB &GE
ID. No. A-10046/17/19
2. Gene Library
A gene library is a collection of different DNA sequences from
an organism, which has been cloned into a vector for ease of
purification, storage and analysis.
All gene libraries are collections of DNA fragments that
represent a particular biological system of interest.
There are two types of gene library that can be made
depending upon the source of the DNA used.
3. Genomic Libraries
Genomic libraries are libraries of genomic DNA sequences.
These can be produced using DNA from any organism.
Principle of Genomic Libraries:
A genomic library contains all the sequences present in the
genome of an organism.
It is a collection of cloned, restriction enzyme digested DNA
fragments containing at least one copy of every DNA sequence
in a genome.
The entire genome of an organism is represented as a set of
DNA fragments inserted into a vector molecule.
4. Vectors used for the
Construction of Genomic Library
Fosmid YAC Cosmid
5. Types of Genomic
Nuclear Genomic Library
• This is a genomic library which includes the total DNA content of the
nucleus. While making such a library we specifically extract the
nuclear DNA and use it for the making of the library.
Organelle Genomic Library
• In this case we exclude the nuclear DNA and targets the total DNA of
either mitochondria, chloroplast or both.
6. Procedure in the
Construction of Genomic
8. Genome Library Construction Service via
Bacteriophage lambda is a temperate bacteriophage of E. coli, which can undergo either
lytic or lysogenic lifecycle.
In the lysogenic state, the phage is stably replicated along with the host by integrating its
genome into the bacterial chromosome; while in the lytic state, progeny phages are
rapidly assembled and released by cell lysis, just like T4 and T7.
Lambda phage has an icosahedral head composed of two major coat proteins, gpE (415
copies) and gpD (405-420 copies), and a single tail composed of gpV. Inside the head
there contains its double strand DNA genome (48502 bp) with two cohesive ends.
In contrast to conventional filamentous phage vehicles, lambda phage offers unique
Particularly, lambda phage represents a feasible choice for constructing complex
genome libraries because of its simple structure and flexible DNA.
9. A genomic library is a set of clones that collectively contain every DNA sequence in
the genome of a specific organism. With the help of restriction enzymes, extracted
genome DNA can be prepared as numerous fragments (15-20 kbp) and inserted into
engineered lambda vectors.
After packaging the ligated recombinant DNA into lambda phage particles and
amplification in host bacterial, a comprehensive library of phage clones each carry a
different DNA fragment can be obtained, which collectively constitute the whole
genome sequence of the indicated organism.
Lambda phage genomic library contributes to various applications, especially for
antigen discovery and immune response investigations. By constructing genome
libraries derived from specific pathogens (i.e. virus, bacteria or organ) and screening
against the whole antibody repertoire of infected individuals, efficient identification of
a large panel of antigenic regions can be achieved. Therefore, lambda phage genome
library provides a highlight for improving diagnosis, prophylaxis, and therapy for
many life-threatening diseases.
11. Advantages of Genomic Library
It helps in the determination of the complete genome sequence
of a given organism.
It serves as a source of genomic sequence for generation of
transgenic animals through genetic engineering.
It helps in the study of the function of regulatory sequences in
It helps in the study of genetic mutations in cancer tissues.
Genomic library helps in identification of the novel
pharmaceutical important genes.
It helps us in understanding the complexity of genomes.
12. Disadvantages of Genomic
The main reason behind making a genomic library is to identify a clone from
the library which encodes a particular gene or genes of interest. Genomic
libraries are particularly useful when you are working with prokaryotic
organisms, which have relatively small genomes.
On the face of it, genome libraries might be expected to be less practical when
you are working with eukaryotes, which have very large genomes containing a
lot of DNA which does not code for proteins.
A library representation of a eukaryotic organism would contain a very large
number of clones, many of which would contain non coding DNA such as
repetitive DNA and regulatory regions.
Also, eukaryotic genes often contain introns, which are untranslated regions
interrupting the coding sequence.
13. cDNA LIBRARY
A cDNA library is defined as a collection of cDNA fragments, each of which
has been cloned into a separate vector molecule.
Principle of cDNA Library:
In the case of cDNA libraries we produce DNA copies of the RNA sequences
(usually the mRNA) of an organism and clone them. It is called a cDNA library
because all the DNA in this library is complementary to mRNA and are
produced by the reverse transcription of the latter.
Much of eukaryotic DNA consists of repetitive sequences that are not
transcribed into mRNA and the sequences are not represented in a cDNA
It must be noted that prokaryotes and lower eukaryotes do not contain introns,
and preparation of cDNA is generally unnecessary for these organisms. Hence,
cDNA libraries are produced only from higher eukaryotes.
15. Vectors used in the
Construction of cDNA Library
16. The steps involved in the construction
of a cDNA library are as follows:
1. Extraction of mRNA from the eukaryotic Cell:
2. Construction of cDNA from the Extracted mRNA:
(a) The RNase Method:
(b) The Self Priming method:
(c) Land et al. Strategy:
(d) Homopolymer Tailing:
(e) Rapid Amplification of cDNA Ends (RACE):
3. Cloning the cDNA:
(b) Incorporation of Restriction Sites:
(c) Homopolymer Tailing of cDNA:
17. Procedure in the Construction of
The steps involved in the construction of a cDNA library are as
1. Extraction of mRNA from the eukaryotic Cell:
Firstly, the mRNA is obtained and purified from the rest of the RNAs.
Several methods exist for purifying RNA such as trizol extraction and
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
18. 2. Construction of cDNA from the Extracted mRNA:
There are different strategies for the construction of a cDNA. These are
discussed as follows:
(a) The RNase Method:
The principle of this method is that a complementary DNA strand is synthesized
using reverse transcriptase to make an RNA: DNA duplex. The RNA strand is
then nicked and replaced by DNA. In this method the first step is to anneal a
chemically synthesized oligo dT primer to the 3′ poly A tail of the RNA.
The primer is typically 10-15 residues long, and it primes (by providing a free 3′
end) the synthesis of the first DNA strand in the presence of reverse
transcriptase and deoxyribonucleotides. This leaves an RNA: DNA duplex.
The next step is to replace the RNA strand with a DNA strand. This is done by
using RNase H enzyme which removes the RNA from RNA: DNA duplex. The
DNA strand thus left behind is then considered as the template and the second
DNA strand is synthesized by the action of DNA polymerase II.
20. (b) The Self-Priming method:
This involved the use of an oligo dT primer annealing at the
polyadenylate tail of the mRNA to prime first DNA strand synthesis
against the mRNA.
This cDNA thus formed has the tendency to transiently fold back on
itself, forming a hairpin loop. This results in the self priming of the
After the synthesis of the second DNA strand, this loop must be cleaved
with a single strand specific nuclease, e.g., SI nuclease, to allow
insertion into the cloning vector.
This method has a serious disadvantage. The cleavage with SI nuclease
results in the loss of a certain amount of sequence at the 5′ end of the
22. Advantages of cDNA
A cDNA library has two additional advantages. First, it is
enriched with fragments from actively transcribed genes.
Second, introns do not interrupt the cloned sequences; introns
would pose a problem when the goal is to produce a
eukaryotic protein in bacteria, because most bacteria have no
means of removing the introns.
23. Disadvantages of cDNA Library
The disadvantage of a cDNA library is that it contains only sequences that are
present in mature mRNA.
Intons and any other sequences that are altered after transcription are not
present; sequences, such as promoters and enhancers, that are not transcribed
into RNA also are not present in a cDNA library.
It is also important to note that the cDNA library represents only those gene
sequences expressed in the tissue from which the RNA was isolated.
Furthermore, the frequency of a particular DNA sequence in a cDNA library
depends on the abundance of the corresponding mRNA in the given tissue.
In contrast, almost all genes are present at the same frequency in a genomic
24. Applications of cDNA
Discovery of novel genes.
Cloning of full length cDNA molecules for in vitro
study of gene function.
Study of the repertoire of mRNAs expressed in different
cells or tissues.
Study of alternative splicing in different cells or tissues.
27. Difference Between Genomic Library And cDNA
S. No. Genomic Library cDNA library
1. Genomic Library is prepared directly from the genomic
cDNA Library is prepared by using mRNA as template.
2. Genomic Library represents entire genome of the
cDNA Library represents only those genes of genome which
express under specific conditions.
3. Restriction endonucleases and ligases are important for
Reverse transcriptase enzyme plays an important role in its
4. They carry introns also. They lack introns.
5. They may represent the DNA of both eukaryotic and
They represent the DNA of only eukaryotic organisms.
6. They are not capable of expression in prokaryotes (like
the bacteria) because they carry introns and prokaryotes
do not have machinery to process introns.
They are capable of expression in bacteria because they lack