Construction of Genomic and cDNA Library

Genomic library- 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. Genomic libraries
have the potential to contain a clone of every gene found in the cells of any organism. This is because the
starting material in a genomic library is the DNA extracted from the cells of the organism of interest. The steps
performed to make a genomic library are listed below.

1) Isolate a sample of DNA from the organism’s cells

2) Cut the DNA into “gene-size” pieces with a restriction enzyme or enzymes.

3) Cut a cloning plasmid with the same restriction enzymes.

4) Add the cut plasmid and genomic DNA to the same test tube and add DNA ligase. This will make a collection
of recombinant and non recombinant plasmids.

5) Transform the collection of plasmids into a laboratory strain of E. coli that is susceptible to antibiotics.

6) Plate the bacteria on Amp/X-gal plates. Incubate at body temperature overnight.

7) Observe the plates, select white colonies and transfer to new Amp plates.

8) Make a large collection of white colonies to form the genomic library.

Types of Vectors- Genome size varies among different organisms and the cloning vector must be selected
accordingly. For a large genome, a vector with a large capacity should be chosen so that a relatively small
number of clones are sufficient for coverage of the entire genome. However, it is often more difficult to
characterize an insert contained in a higher capacity vector.

Plasmids- A plasmid is a double stranded circular DNA molecule commonly used for molecular cloning.

Plasmids are generally 2 to 4 kilobase-pairs (kb) in length and are capable of carrying inserts up to 15kb.
Plasmids contain an origin of replication allowing them to replicate inside a bacterium independently of the
host chromosome. Plasmids commonly carry a gene for antibiotic resistance that allows for the selection of
bacterial cells containing the plasmid.

Phage lambda (λ)]- Phage λ is a double-stranded DNA virus that infects E. coli. The λ chromosome is 48.5kb
long and can carry inserts up to 25kb. These inserts replace non-essential viral sequences in the λ
chromosome, while the genes required for formation of viral particles and infection remain intact. The insert
DNA is replicated with the viral DNA; thus, together they are packaged into viral particles. These particles are
very efficient at infection and multiplication leading to a higher production of the recombinant λ
chromosomes.

Cosmids- Cosmid vectors are plasmids that contain a small region of bacteriophage λ DNA called the cos
sequence. This sequence allows the cosmid to be packaged into bacteriophage λ particles. These particles-
containing a linearized cosmid- are introduced into the host cell by transduction. Once inside the host, the
cosmids circularize with the aid of the host's DNA ligase and then function as plasmids. Cosmids are capable of
carrying inserts up to 40kb in size.
Bacteriophage P1 vectors- Bacteriophage P1 vectors can hold inserts 70 – 100kb in size. They begin as linear
DNA molecules packaged into bacteriophage P1 particles. These particles are injected into an E. coli strain
expressing Cre recombinase. The linear P1 vector becomes circularized by recombination between two loxP
sites in the vector. P1 vectors generally contain a gene for antibiotic resistance and a positive selection marker
to distinguish clones containing an insert from those that do not.
P1 artificial chromosomes- P1 artificial chromosomes (PACs) have features of both P1 vectors and Bacterial
Artificial Chromosomes (BACs). Similar to P1 vectors, they contain a plasmid and a lytic replicon as described
above. Unlike P1 vectors, they do not need to be packaged into bacteriophage particles for transduction.
Instead they are introduced into E. coli as circular DNA molecules through electroporation just as BACs are.
Bacterial artificial chromosomes- Bacterial artificial chromosomes (BACs) are circular DNA molecules, usually
about 7kb in length, that are capable of holding inserts up to 300kb in size. BAC vectors contain a replicon
derived from E. coli F factor, which ensures they are maintained at one copy per cell.[4] Once an insert is ligated
into a BAC, the BAC is introduced into recombination deficient strains of E. coli by electroporation. Most BAC
vectors contain a gene for antibiotic resistance and also a positive selection marker.

Yeast artificial chromosomes- Yeast artificial chromosomes (YACs) are linear DNA molecules containing the
necessary features of an authentic yeast chromosome, including telomeres, a centromere, and an origin of
replication. Large inserts of DNA can be ligated into the middle of the YAC so that there is an “arm” of the YAC
on either side of the insert. The recombinant YAC is introduced into yeast by transformation; selectable
markers present in the YAC allow for the identification of successful transformants. YACs can hold inserts up to
2000kb, but most YAC libraries contain inserts 250-400kb in size.

cDNA Libraries- A cDNA library is a collection of cloned DNA sequences that are complementary to the
mRNA that was extracted from an organism or tissue (the ‘c’ in cDNA stands for ‘complementary’).  cDNA is
produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes
of an organism. Similarly, tissue-specific cDNA libraries can be produced. In eukaryotic cells the mature mRNA
is already spliced, hence the cDNA produced lacks introns and can be readily expressed in a bacterial cell.
While information in cDNA libraries is a powerful and useful tool since gene products are easily identified, the
libraries lack information about enhancers, introns, and other regulatory elements found in a genomic DNA
library.

1. Isolation of mRNA- First of all, it involves the isolation of total mRNA from a cell type or tissue of
interest. It may be desirable to remove highly abundant tRNAs and rRNAs which might otherwise
constitute the majority of the final library to the detriment of the detection of low abundance RNAs.
We routinely remove tRNAs and other small RNAs<200 nt using a Kit from Creative Biogene and
remove rRNAs using magnetic bead-based depletion kits. The 3’ ends of eukaryotic mRNA are
composed of a string of 50 -250 adenylate residues (poly A Tail) which makes the separation easy from
the much more prevalent rRNAs and tRNAs in a cell extract through a column containing oligo-dTs
tagged onto its matrix.

The number of desired mRNA can be increased by the following methods:

Chromatographic purification of mRNA through the oligo-dT column, which retains mRNA molecules, resulting
in their enrichment.

Spinning down mRNA using density gradient centrifugation.

mRNA preparation from specialized cell types, such as chicken oviduct, erythrocytes, developing seeds,β cells
of pancreas etc.
 2. Synthesis of the first strand of cDNA- mRNA being single-stranded cannot be cloned and is not a
substrate for DNA ligase. It is first converted into DNA prior to insertion into a suitable vector.
1) A short oligo (dT) primer is annealed to the Poly (A) tail on the mRNA.
2) Reverse transcriptase extends the 3´-end of the primer by mRNA molecule as a template producing
a cDNA: mRNA hybrid.
3) The mRNA from the cDNA: mRNA hybrid can be removed by alkaline hydrolysis or RNase H to give a
single stranded (ss)-cDNA molecule.

Note: No primer is required because the 3´end of this ss-cDNA serves as its own primer generating a short
hairpin loop at this end. The free 3´-OH is required for the synthesis of its complementary strand.

3. The second strand of cDNA generation- The ss-cDNA is converted into double stranded (ds) cDNA by
either RTase or E. coli DNA polymerase. (It is essential to use only the minimal number of amplification
cycles needed to obtain sufficient material for sequencing to avoid over-amplification of the libraries,
which is a major source of bias in the results.)
4. Incorporation of cDNA into a vector- The ds-cDNA can be trimmed with S1 nuclease to obtain blunt–
ended ds-cDNA molecule followed by addition of terminal transferase to tail the cDNA with C's and
ligation into a vector. Because the blunt-end ligation is inefficient, short restriction-site linkers are first
ligated to both ends.
5. Cloning of cDNAs- cDNAs are commonly cloned in phage insertion vectors. Bacteriophage vectors
possess the following advantageous over plasmid vectors:
1. Are more desirable when a large number of recombinants are required for cloning low-abundant
mRNAs as recombinant phages are produced by in vitro packaging.
2. Can easily handle and store large numbers of phage clones, as compared to the bacterial colonies
carrying plasmids.

Applications of libraries- Such libraries provide convenient access to a genome for both laboratory and
breeding applications. Genomic libraries can be used as substrates to physically map and sequence entire
genomes, clone agriculturally important genes and to investigate gene expression patterns. Further, genomic
libraries also provide powerful tools and resources for evaluating germplasm conservation stocks and
biological diversity.

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.

8. Create CDNA libraries to determine what genes are being expressed at a particular time.

Applications of CDNA Library

1. Storage of reduced amount of information due to the removal of non-coding regions.
2. Directly expressed in prokaryotic organisms.

3. CDNA libraries are useful in reverse genetics where the additional genomic information is of less use.

4. CDNA library is useful for isolating gene that codes for particular mRNA.

5. CDNA library is a powerful and useful tool in the area of biotechnology.

6. It is helpful in expressing eukaryotic genes in prokaryotes, which helps in the transcription process of
prokaryotes.

7. It is used to isolate DNA sequences to code MRNA.

8. Advantage of CDNA library is to isolate homologous genes.

9. used for the screening genomic libraries to isolate specific CDNA.

10. CDNA of proteins can facilitate to generate antibodies and monoclonal antibodies.

11. the most important application of CDNA library is to study expression of mRNA.