DNA libraries:

Genomic and cDNA

libraries
Dr. Rezwana Ahmed
Assistant Professor
 Genome
• A genome is all the genetic information of an organism. It
consists of all the nucleotide sequences of DNA.
 DNA Library (Genomic, cDNA)

• DNA Library is simply the collection of DNA fragments cloned

into vectors and stored within host organisms.

• They contain either the entire genome of a particular organism

or the genes that are expressed at a given time.

• The genome is vast and complex. To understand the entire

genome or study specific genes, it is important to study it in
smaller and more manageable fragments.

• DNA libraries make the entire genome accessible in small

fragments. These libraries are used to identify, isolate, and
study particular genes of interest.
Cloning

• So far, we have described

the amplification of a single
DNA fragment.
DNA Library

• In reality, when a genome is cut

by a restriction nuclease, millions
of different DNA fragments are
generated.

• How can the single fragment that

contains the DNA of interest be
isolated from this collection?
DNA Library

• The solution involves introducing all of

the fragments into bacteria and then
selecting those bacterial cells that
have amplified the desired DNA
molecule.

• That means we have to generate a

genomic library.

(Figure 10–10).
DNA Library
• The entire collection of DNA fragments can
be ligated into plasmid vectors, using
conditions that favor the insertion of a single
DNA fragment into each plasmid molecule.
• These recombinant plasmids are then
introduced into E. coli at a concentration that
ensures that no more than one plasmid
molecule is taken up by each bacterium.
• The collection of DNA fragments in this
bacterial culture is known as a DNA library.
• As the DNA fragments were derived directly
from the chromosomal/genomic DNA of the
organism of interest, the resulting collection
is called a genomic library.
• The bacterial cells can be grown
in a petridish, and each clone of
cells contains a plasmid with a
particular DNA fragment.

Now the question is, how do we

identify which bacterial colony
contains a specific DNA fragment?

?
 Finding the specific DNA fragment
• To find a particular gene within this library, one can use a labeled
DNA probe designed to bind specifically to part of the gene’s DNA
sequence.
• Using such a probe, the rare bacterial clones in the DNA library
that contain the gene—or a portion of it—can be identified by
hybridization (binding).

Figure 10–11 A bacterial colony carrying a specific DNA clone can be identified by
hybridization.
 Finding the specific DNA fragment

• A replica of the arrangement of the bacterial colonies (clones) on

the Petri dish is made by pressing a piece of absorbent paper
against the surface of the dish.

• This replica is treated with alkali (to lyse the cells and dissociate
the plasmid DNA into single strands), and the paper is then
hybridized to a highly radioactive DNA probe.

• Those bacterial colonies that have bound the probe are identified
by autoradiography.

• Living bacterial cells containing the plasmid can then be isolated

from the original Petri dish.
 cDNA Libraries Represent the mRNAs Produced by
Particular Cells

• A cDNA library is similar to a genomic library in that it also contains

numerous clones containing many different DNA sequences.
• The only difference is that the DNA sequences in a cDNA library do
not contain any intronic sequences. The DNA that goes into a
cDNA library is not genomic DNA; it is DNA copied from the mRNAs
present in a particular type of cell.
• To prepare a cDNA library, all of the mRNAs are extracted and the
mRNAs are used to produce the cDNA.
• These cDNA molecules are then introduced into bacteria and
amplified, similar to genomic DNA fragments.
• The gene of interest, without its introns, can then be isolated by
using a probe that hybridizes to the DNA sequence.
Figure 10–13
Figure 10–13 Genomic DNA clones and cDNA clones derived from
the same region of the genome are different.
In this example, gene A is infrequently transcribed, whereas gene B
is frequently transcribed, and both genes contain introns (orange).
In the genomic DNA library, both introns and non-transcribed DNA
(gray) are included in the clones, and most clones will contain either
non-coding sequence or only part of the coding sequence of a gene
(red).
In the cDNA clones, the intron sequences have been removed by
RNA splicing during the formation of the mRNA (blue), and a
continuous coding sequence is therefore present in each clone.
Because gene B is transcribed more frequently than gene A in the
cells from which the cDNA library was made, it will be represented
much more often than A in the cDNA library. In contrast, genes A and
B should be represented equally in the genomic library.
 Differences between genomic and cDNA clones

1. Genomic clones from eukaryotes contain coding DNA, noncoding

DNA, repetitive DNA sequences, introns and regulatory DNA.
cDNA clones contain predominantly protein-coding sequences.

2. Genomic clones represent all of the DNA sequences found in an

organism’s genome and, with very rare exceptions, will contain
the same sequences regardless of the cell type from which the
DNA came. cDNA clones contain only the protein-coding
sequences, and only those for genes that have been transcribed
into mRNA in the cells from mRNA were collected. As different
types of cells produce distinct sets of mRNA molecules, each cell
yields a different cDNA library.
 Differences between genomic and
cDNA clones
3. The more frequently transcribed gene in the cell will be
represented much more often the cDNA library compared to
the gene that is transcribed less. However, in a genomic DNA
all the genes should be represented equally.

4. Furthermore, patterns of gene expression change during

development, so cells at different stages in their development
will also yield different cDNA libraries. In contrast, genomic
library generated is always same, irrespective of the cell type or
stage of development.
Characteristics Genomic Library cDNA Library

Genomic libraries contain

cDNA libraries consist of
DNA fragments that
Content only cDNA sequences that
represent the entire
are actively expressed.
genome of an organism.
The DNA fragments are
larger, ranging from cDNA libraries contain
Size of DNA fragments
thousands to millions of smaller DNA fragments.
base pairs.
It contains both coding
It contains only coding
Coding and non-coding and noncoding regions
regions or expressed
regions including introns and
genes.
regulatory sequences.
Characteristics Genomic Library cDNA Library

The source of DNA is the The source of DNA for a

total genomic DNA cDNA library is mRNA
Source of DNA
extracted from cells or which is converted to
tissues. cDNA.
It uses vectors such as
bacteriophages and cDNA libraries generally
bacterial artificial use plasmid vectors, which
Vectors used
chromosomes (BACs), are suitable for the smaller
which can hold large DNA cDNA fragments.
fragments.
Genomic libraries are used cDNA libraries are
for genomic mapping, particularly useful for
Application comparative genomics, studying gene expression
and studying gene patterns and the functions
regulation. of expressed genes.