cDNA preparation and
its uses in gene
analysis
Prepared by:
Shelke P. D.
K.K Wagh College of
Agril Biotechnology.
What is cDNA library?
cDNAlibrary is a combination of cloned cDNA
(complementary DNA) fragments inserted into a
collection of host cells, which together constitute some
portion of the transcriptome of the organism.
cDNA is produced from fully transcribed mRNA
found in the nucleus and therefore contains only the
expressed genes of an organism.
What is cDNA library?
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.
Intron and Exon in Eukaryotic Cells
exon exon exon
promotor intron intron
DNA
3’ 5’
start codon stop codon
Transcription
5’ mRNA
3’
Processing
cap
Splicing poly A
tail
Intron deleted
mature mRNA To cytoplasm
Take place in nucleus
cDNA Library
preparation:
cDNA is created from a mature mRNA from a eukaryotic
cell with the use of an enzyme known as reverse
transcriptase. In eukaryotes, a poly-(A) tail
(consisting of a long sequence of adenine
nucleotides) distinguishes mRNA from tRNA and
rRNA and can therefore be used as a primer site for
reverse transcription.
mRNA
extraction:
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
Column purification is done by using oligomeric dT
nucleotide coated resins where only the mRNA
having the poly-A tail will bind.
The rest of the RNAs are eluted out. The mRNA is
eluted by using eluting buffer and some heat to
separate the mRNA strands from oligo-dT.
mRNA
extraction:
Purification can be performed by binding mRNAs on a
solid matrix to which short strings of thymidylate
residues are attached (oligo dT matrix).
The mRNAs are removed again by washing in a low salt
buffer.
mRNA Extraction
cDNA construction:
Once mRNA is purified, oligo-dT is tagged as a
complementary primer which binds to the poly-A tail
providing a free 3'-OH end that can be extended by
reverse transcriptase to create the complementary
DNA strand.
Now, the mRNA is removed by using an RNAse enzyme
leaving a single stranded cDNA (sscDNA).
This sscDNA is converted into a double stranded DNA
with the help of DNA polymerase. However, for DNA
polymerase to synthesize a complementary strand a
free 3'-OH end is needed.
cDNA construction:
This is provided by the sscDNA itself by generating a hair
pin loop at the 3' end by coiling on it.
The polymerase extends the 3'-OH end and later the loop
at 3' end is opened by the scissoring action of S1 nuclease.
Restriction endonucleases and DNA ligase are then used to
clone the sequences into bacterial plasmids.
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.
cDNA construction
cDNA Is Reverse Transcribed from mRNA
mature mRNA
5’ 3’
poly A tail
Reverse
TTTT
transcription
3’ 5’
RNA hydrolysis
3’ 5’
DNA polymerase
3’ CCC 5’
5’ 3’
GGG
cDNA Library
Genes in expression
mRNA
Reverse transcription
cDNA Complete gene
Smaller
Library
Vector: Plasmid
Uses of cDNA library:
cDNA libraries are commonly used when
reproducing eukaryotic genomes, as the
amount of information is reduced to remove the
large numbers of non-coding regions from the
library.
cDNA libraries are used to express
eukaryotic genes in prokaryotes. Prokaryotes
do not have introns in their
DNA and therefore do not possess any
enzymes that can cut it out during
transcription process. cDNA do not have
introns and therefore can be expressed in
prokaryotic cells.
Uses of cDNA library:
cDNA libraries are most useful in reverse
genetics where the additional genomic
information is of less use.
Also, it is useful for subsequently isolating the
gene that codes for that mRNA.
Discovery of novel genes.
Clonning of full length cDNA molecules for in
vitro study of gene function.
Study of the repertoire if mRNAs expressed in
different cells or tissues.
Study of alternative splicing in different cells or
tissues .
Thank You