This is a brief overview of the process of making a complementary DNA (cDNA) library from mRNA (messenger RNA).

1. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
CDNA LIBRARY
“A cDNA library is defined as a collection of cDNA fragments. Complementary DNA
(cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA) template in
a reaction catalyzed by the enzyme reverse transcriptase."
Principle of cDNA Library: To make cDNA libraries, DNA reverse transcripts of the RNA
sequences (usually the mRNA) of an organism are produced and then cloned. It is called a
cDNA library because all the DNA in this library is complementary to mRNA and is produced
by the reverse transcription. Much of eukaryotic DNA consists of repetitive sequences that
are not transcribed into mRNA and those sequences are not represented in a cDNA library.
Prokaryotes and lower eukaryotes do not contain introns (non-coding sequences), rendering
the preparation of cDNA unnecessary. Hence, cDNA libraries are produced only from higher
eukaryotes such as humans.
Vectors of cDNA Library: Vectors are used to mediate the cloning of the cDNA fragments,
two of the most important vectors used for this purpose are: (1) Bacterial Plasmid and (2)
Bacteriophages.
PROCEDURE IN THE CONSTRUCTION OF cDNA LIBRARY:
The steps involved in the construction of a cDNA library are as follows:
1. Extraction of mRNA (from a 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. The rest of the RNAs are discarded.
2. Construction of cDNA from the extracted mRNA: There are different strategies for the
construction of a cDNA, two of them are as follows:
The RNase Method: In this method, 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. The first step is to add a primer to the 3’ end of the RNA. This primer is
extended into a DNA strand by reverse transcriptase. 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
(RNA digesting enzyme) enzyme which removes the RNA from RNA: DNA duplex. A single
DNA strand is left behind which acts as a template. The second DNA strand is synthesized by
the action of DNA polymerase.
The Self-Priming method: A primer is attached at the polyadenylate tail (poly-A tail) of the
mRNA to prime the DNA strand synthesis against the mRNA. The cDNA that forms, as a
result, can fold back on itself, forming a hairpin loop, hence the cDNA self-primes itself for

2. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
the synthesis of the second strand. After the synthesis of the second DNA strand, this loop
must be cleaved with a single-strand-specific nuclease. This method has a flaw: there is a
risk of losing some of the nucleotide sequence of the DNA because of the nuclease.
3. Cloning the cDNA: The cDNA can be cloned by incorporating it into a vector (bacterial
plasmid, bacteriophage, etc.) and allowing the said vector to multiply with the copy of
the cDNA within. Following are some methods of incorporating the DNA fragment into
the vector genome:
Blunt End Ligation: The double-stranded, blunt-ended cDNA molecules can be attached to
the vector molecules by blunt-ended ligation, i.e. the blunt ends of the cDNA are
ligated/joined to the blunt ends of the vector DNA.
Addition of Linkers: The cDNA molecules can also be attached to the vector molecules by the
addition of linkers (small segments of DNA containing many restriction sites). The addition
of linkers is followed by digestion with the relevant enzyme and then ligation into a vector.
Incorporation of Restriction Sites: Another option is to modify the steps of the creation of
DNA strands to include restriction sites in the cDNA. The cDNA can incorporate into the
vector by these restriction sites.
Figure: Procedure for the construction of the cDNA library. First of all, mRNA is isolated from
a eukaryotic cell. Then, the mRNA is used as a template to produce DNA. This DNA is then
incorporated into a vector and cloned. (Source: Wikimedia, CC-BY-SA)
ADVANTAGES OF cDNA LIBRARY:

3. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
A cDNA library has two advantages over other genome libraries:
 It is enriched with fragments from actively transcribed genes (the genes that are
transcribed a lot).
 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.
DISADVANTAGES OF cDNA LIBRARY:
The major disadvantage of a cDNA library is that it contains only sequences that are present
in a mature mRNA. Introns and any other sequences that are altered after transcription are
not present; sequences, such as promoters and enhancers, which 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 ex-
pressed in the tissue from which the RNA was isolated. 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
DNA library.
APPLICATIONS OF cDNA LIBRARY:
Following are the applications of cDNA libraries:
 Discovery of new genes.
 Cloning of full-length cDNA molecules for in vitro study of gene function.
 Study of various mRNAs expressed in different cells or tissues.
 Study of alternative splicing in different cells or tissues.