DNA Libraries: Collections

of Cloned Genes
Dr. Hina Fatima
• During the first several decades of DNA cloning, many cloning
strategies began by preparing a DNA library— a collection of cloned
DNA fragments from a particular organism contained within bacteria
or viruses as the hosts.
• Libraries can be saved for relatively long periods and “screened” to
pick out different genes of interest.
• Two types of libraries were typically used for cloning, genomic DNA
libraries and complementary DNA libraries (cDNA libraries).
• A genomic library is a collection of the total genomic DNA from a
single organism.
• The DNA is stored in a population of identical vectors, each
containing a different insert of DNA.
• In order to construct a genomic library, the organism's DNA
is extracted from cells and then digested with a restriction enzyme to
cut the DNA into fragments of a specific size.
• The fragments are then inserted into the vector using DNA ligase. 
• Next, the vector DNA can be taken up by a host organism - commonly
a population of Escherichia coli or yeast - with each cell containing
only one vector molecule.
• Using a host cell to carry the vector allows for easy amplification and
retrieval of specific clones from the library for analysis.
1. In a genomic library, chromosomal DNA from the tissue of interest
is isolated and then digested with a restriction enzyme.
2. This process produces fragments of DNA that include the organism’s
entire genome.
3. A plasmid, BAC, YAC, or bacteriophage vector is digested with the
same enzyme, and DNA ligase is used to ligate genomic DNA pieces
and vector DNA randomly.
4. In theory, all DNA fragments in the genome will be cloned into a
vector.
5. Recombinant vectors are then used to transform bacteria, and each
bacterial cell clone will contain recombinant vector with a plasmid
containing a genomic DNA fragment.
6. Consider each clone a “book” in this “library” of DNA fragments.
Disadvantages
• One disadvantage of creating this type of library for eukaryotic genes
is that
• non-protein-coding pieces of DNA, called introns, are cloned in
addition to protein-coding sequences (exons).
• Because a majority of DNA in any eukaryotic organism consists of
introns, many of the clones in a genomic library will contain non-
protein-coding pieces of DNA.
• Another limitation of genomic libraries is that many organisms,
including humans, have such large genomes that searching for a gene
of interest would be like searching for a needle in a haystack
Complementary DNA libraries or cDNA
library
• In cDNA library, mRNA from the tissue of interest is isolated and used
for making the library.
• However, mRNA cannot be digested with restriction enzymes, so it
must be converted to a double-stranded DNA molecule.
• An enzyme called reverse transcriptase (RT) is used to catalyze the
synthesis of single-stranded DNA from the mRNA.
• This enzyme is made by viruses called retroviruses—so named
because they are exceptions to the usual flow of genetic information.
• Instead of having a DNA genome that can be used to make RNA,
retroviruses have an RNA genome.
• After infecting host cells, retroviruses use RT to convert their RNA
encoded viral genome into DNA, so that they can replicate.
• Human immunodeficiency virus (HIV) is a retrovirus.
• Because RT synthesizes DNA that is an exact copy of mRNA, it is
called complementary DNA (cDNA).
• The mRNA is degraded by treatment with an alkaline solution or
enzymatically digested; then DNA polymerase is used to synthesize a
second strand to create double-stranded cDNA
• Because cDNA sequences do not necessarily have a convenient
restriction site at each end, short, double stranded DNA sequences
called linker sequences are often enzymatically added to the ends of
the cDNA.
• Linkers contain restriction sites.
• Different linkers for different restriction sites are commercially
available.
• By adding linkers, cDNA can now be ligated into a convenient
restriction site in a vector of choice, often a plasmid.
• The recombinant plasmid is then used to transform bacteria
Advantage
• One primary advantage of cDNA libraries over genomic libraries is
that they are a collection of actively expressed genes in the cells or
tissue from which the mRNA was isolated.
• Also, introns are not cloned in a cDNA library.
• This is one reason why cDNA libraries are typically preferred over
genomic libraries when attempting to clone and express a gene of
interest.
• Another advantage of cDNA libraries is that they can be created and
screened to isolate genes that are primarily expressed only under
certain conditions in a tissue
• For example, during development, cell death, cancer, and other
biological processes.
• One can use these libraries to compare expressed genes from normal
tissues and diseased tissues.
• This approach has been widely used to identify genes involved in
cancer formation, such as those genes that contribute to progression
from a normal cell to a cancer cell and genes involved in cancer cell
metastasis.
• Libraries have become such a routine aspect of molecular biology that
many companies sell libraries prepared from a range of tissues from
different species.
• One disadvantage is that cDNA libraries can be difficult to create and
screen if a source tissue with an abundant amount of mRNA for the
gene is not available.
• But a technique called the polymerase chain reaction (PCR) can
frequently solve this problem