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The cDNA Library

(NOTES)

Complementary DNA (cDNA) is double-stranded DNA synthesized from a single

stranded RNA template (e.g. messenger RNA or microRNA template) in a reaction
catalyzed by the enzyme reverse transcriptase.

MicroRNA (miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in
plants, animals and some viruses, which functions in post-transcriptional regulation of gene
expression.

The cDNA is created from a mature mRNA (originated from a eukaryotic cell) with
the use of reverse transcriptase enzyme. In eukaryotes, a poly-(A) tail, consisting
of a long sequence of adenine nucleotides, facilitates to distinguish
the mRNA from tRNA and rRNA; it can therefore be used as a primer site for
reverse transcription.

Primer is a short strand of DNA or RNA that serves as a starting point for DNA synthesis.

DNA Replication

Primer
Activity of DNA polymerase →

Oligo-dT (5’ dt Primer) is a short chain of

thymine deoxynucleotides; it can be used as a
The primer is required for DNA replication primer during cDNA synthesis, because
because DNA polymerase (DNA replication adenine deoxynucleotides (A A A A A A) of
enzymes) can only add new nucleotides to
poly-(A) tail can base-pair with thymine
an existing strand of DNA (the primer).
Various primers, with known nucleotide deoxynucleotides (T T T T T T) of Oligo-dT.
sequences, are used in biotechnology labs.

The enzyme reverse transcriptase can be used, along with an oligo-dT primer (that is
complementary to the poly-(A) tail of mRNA) to synthesize a complementary DNA
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(cDNA) molecule. The cDNA can then be cloned into a plasmid or it may be amplified by
PCR by ligating (joining) adapters that contain restriction endonuclease cleavage sites
or PCR primer-sequences.

The cDNA library is a combination of cloned fragments of cDNA inserted into a

collection of host cells. When all messenger RNA molecules in one cell or a
population of cells are converted into cDNA clones, it is known as cDNA library.
The tissue-specific cDNA libraries can also be produced, using cDNA clones
prepared from cell-genome of the tissue concerned.

Molecular cloning of eukaryotic genes is often impracticable because they contain

numerous large introns. Eukaryotic genes (with large introns) cannot be cloned in
plasmid because plasmid vectors have a practical size limit of less than 10 kilo-
base pairs (kbp), and PCR is also difficult beyond 10 kb.

The cDNA is produced in the nucleus from fully transcribed mRNA (with no
introns) and therefore contains only the expressed genes of an organism. In fact,
the mRNA, lacking introns, is a compact version of a eukaryotic gene that retains
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all of the protein-coding information and, hence, can be readily expressed in a

bacterial cell.

The cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists
want to express a specific protein in a cell that does not normally produce that
protein, the scientists insert the cDNA in a vector and transfer it as recombinant
DNA molecule into the host cell. The desired gene, carried by the cDNA, then gets
expressed in the recipient prokaryotic cell to produce the specific protein.

Information in cDNA libraries is a powerful and useful tool since gene products
are easily identified, but the libraries lack information about enhancers, introns,
and other regulatory elements that are found in a genomic DNA library.

Synthesis of cDNA molecule

Although there are several methods for cDNA synthesis, cDNA is most often
synthesized from mature mRNA (with no introns) using the enzyme reverse
transcriptase. This enzyme operates on a single strand of mRNA, generating its
complementary DNA based on the pairing of RNA nucleotides (A, U, G and C) to
their DNA complements (T, A, C and G, respectively).
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STEPS:
(1) The mRNA extraction: The mature mRNA strands are extracted from the cell.
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.

Purification of mRNA by affinity chromatography with oligo(dt) – cellulose

(2) Enzyme Reverse transcriptase is added, along with deoxyribonucleotide

triphosphates (A, T, G, C). This synthesizes one complementary strand of DNA
hybridized to the original mRNA strand.

(3) The resulting mRNA-cDNA hybrid is treated with alkali or an enzyme (RNase
H.) to hydrolyze the RNA, leaving the single-stranded cDNA.
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(4) After digestion of the mRNA, a single stranded DNA (ssDNA) is left, and
because single stranded nucleic acids are hydrophobic, the ssDNA tends to loop
around itself. As a result, a hairpin loop is formed in the ssDNA at the 3'-end.

Generation of double-stranded cDNA

(5) DNA polymerase can now synthesize the complementary DNA strand. For this,
the looped-around 3’-end of the first DNA strand can often be used as a primer.
An enzyme called S1 nuclease is then used to cleave the loop.

(6) The double stranded cDNA is lastly incubated with the enzyme terminal
transferase to add single stranded tails to the cDNA strands at 3’-end. Now, the
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double-stranded cDNA molecule is ready for insertion into an appropriate cloning

vector for amplification.

The construction of cDNA library involves following steps:

1. Isolation of mRNA
2. Synthesis of first and second strand of cDNA
3. Incorporation of cDNA into a vector
4. Cloning of cDNAs
Steps 1 and 2 have been explained above. The third and fourth steps are as
follows:

Incorporation of cDNA into a vector and its cloning

Restriction endonucleases and DNA ligase are used to clone the plasmid-confined
cDNA inside the host cell.

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.
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Applications of cDNA

Complementary DNA is often used in gene cloning or as gene probes or in the

creation of a cDNA library. When scientists transfer a gene from one cell into
another cell in order to express the new genetic material as a protein in the
recipient cell, the cDNA will be added to the recipient cell (rather than the entire
gene), because the DNA for an entire gene may include DNA that does not code
for the protein or that interrupts the coding sequence of the protein
(e.g., introns/enhancers).

There are following applications of cDNA library:

1. The cDNA library is a powerful and useful tool in the area of biotechnology.
2. It is helpful in expressing eukaryotic genes in prokaryotes, which helps in
the transcription process of prokaryotes.
3. It is used to isolate DNA sequences to code mRNA.
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4. Advantage of cDNA library is to isolate homologous genes.

5. It is also used for the screening genomic libraries to isolate specific cDNA.
6. The cDNA of proteins can facilitate to generate antibodies and monoclonal
antibodies.
7. The most important application of cDNA library is to study expression of
mRNA.

Difference between genomic and library cDNA library

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Difference between genomic library and cDNA library

S. No. Genomic Library cDNA Library

1. Genomic library includes all possible The cDNA library carries only
fragments of DNA from a given cell or expressed gene sequences.
organism.
2. It is larger in size. It is smaller in size.

3. It represents entire genome of an It represents only the expressed part

organism having both coding and non- of the genome and contain only
coding regions. coding sequences called ESTs
(expressed sequence tags).
4. Expression of genes taken from The cDNA carries only coding
genomic library is difficult in sequences, which can be directly
prokaryotic systems like bacteria due expressed in eukaryotic systems.
to absence of splicing mechanism in
prokaryotes.

5. Vectors used in genomic library include Vectors used in cDNA library include
plasmid, cosmid, lambda phage, BAC plasmid, phagemid, lambda phage,
(Bacterial artificial chromosomes) and etc. to accommodate small DNA
YAC (Yeast artificial chromosome) in fragments as cDNA contains no
order to accommodate large fragments introns.
of DNA.

6. Genomic library contains elaborated The cDNA library contains limited

sequence information, representing sequence information because it is
total genome of the cell or organism prepared from coding sequences
because it is prepared from total only.
genomic DNA.