cDNA Library
Dr. S. Bhangale
What is cDNA?
Complementary DNA (cDNA) is synthesized in
the laboratory from messenger RNA.
cDNA is not genomic DNA, because the
transcript of genomic RNA has been processed
(i.e., it lacks promoters and introns).
The enzyme reverse transcriptase is used to
synthesize double-stranded DNA that is a
complementary copy of the mRNA.
Dr. S. Bhangale
A cDNA library is a combination of cloned
cDNA (Complementary DNA) fragments
inserted into a collection of host cells, which
together constitute some portion of
the transcriptomes (set of all massenger RNA
molecules in one cell) of the organism and
stored as a “library".
cDNA is produced from fully
transcribed mRNA found in the nucleus and
therefore contains only the expressed genes of
an organism.
Dr. S. Bhangale
CONSTRUCTION OF cDNA LIBRARY
The construction of cDNA has following steps:
1. Isolation of mRNA
2. Preparation of complementary DNA fragments
3. Cloning in suitable vector system
4. Transformation in suitable host
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Dr. S. Bhangale
1. Isolation of mRNA
The structure of a typical mRNA has a CAP
structure at 5’, coding sequence and a poly A
tail at its 3’ region.
The Nucleotide A forms 2 hydrogen bonding
with nucleotide T and this pairing is very
specific.
m-RNA population can be isolated from
RNA pool using a poly-T affinity column.
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Dr. S. Bhangale
Dr. S. Bhangale
2. Preparation of complementary DNA (cDNA)
fragments
The synthesis of double stranded cDNA suitable for
insertion into a cloning vector involves three major
steps:
A. First strand DNA synthesis on the mRNA template,
carried out with a reverse transcriptase.
• Reverse transcriptase is also called as RNA dependent DNA
polymerase
• Reverse transcriptase have two activities;
i. DNA polymerase activity: it will transcribe both ssRNA and
ssDNA templates.
ii. RNase H activity: It is a ribonuclease that degrades the RNA
from RNA-DNA hybrids, which are formed during reverse
transcription of RNA template. The enzyme functions as both
an endonuclease and exonuclease. Dr. S. Bhangale
B. Removal of the RNA templates: The
mRNA from the cDNA: mRNA hybrid
can be removed by RNase H or Alkaline
hydrolysis to give a ss-cDNA molecule.
C. Second strand of DNA synthesis: It is
carried out using the first DNA strand as a
template, carried out with a DNA-
dependant DNA polymerase.
Dr. S. Bhangale
No primer is required as the 3´end of this ss-cDNA
serves as its own primer generating a short hairpin
loop at this end.
This free 3´-OH is required for the synthesis of its
complementary strand.
The single stranded (ss) cDNA is then converted into
double stranded (ds) cDNA by either RTase or E.
coli DNA polymerase.
The ds-cDNA can be trimmed with S1 nuclease to
obtain blunt–ended ds-cDNA molecule
Dr. S. Bhangale
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Dr. S. Bhangale
3. Cloning in suitable vector system
Once cDNA molecules have been
synthesized, they must be cloned.
The cloning of cDNA is done by using a
restriction site linker which is a relatively
short double stranded fragment of DNA
which is about 8-10 nucleotide pairs in
length.
The linker contains a restriction site e.g. Bam
HI restriction site.
Dr. S. Bhangale
Both the cDNA molecules and linkers have
blunt ends and they can be ligated together at
a higher concentrations of T4 DNA ligase.
Cohesive ends (sticky ends) are produced in
the cDNA molecule by cleaving the cDNA
with Bam HI.
The resulting DNA is inserted into a cloning
vector that has also being cleaved with Bam
HI.
Dr. S. Bhangale
Common vectors used in library construction
1. Plasmids
Up to 10kb inserts
2. Modified Lambda phage
Up to 20kb inserts/40kbp for cosmids
3. Artificial cloning vectors
BAC- Vectors (bacterial artificial
chromosome)
•Up to 100-150kbp inserts
YAC-Vectors (yeast artificial chromosome)
•Up to 500kbp inserts Dr. S. Bhangale
Dr. S. Bhangale
4. Transformation in suitable host
Post ligation, clones are transformed in a
suitable host to get colonies.
A suitable host can be bacterial strain or
yeast.
Generally, the r-DNA (recombinant DNA)
molecule is produce is transformed into the E.
coli host cell for cloning.
Dr. S. Bhangale
Steps in construction of cDNA library
Dr. S. Bhangale