A cDNA library is a combination of cloned cDNA 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. 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.
cDNA Library Construction 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 distinguishes mRNA from tRNA and rRNA
and can therefore be used as a primer site for reverse transcription. This has
the problem that not all transcripts, such as those for the histone, encode a
poly-A tail. = 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.
= 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 a RNAse enzyme
leaving a single stranded cDNA. 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. This is
provided by the sscDNA itself by generating a hairpin loop at the 3' end
by coiling on itself. 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 Library uses
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
does not have introns and therefore can be expressed in prokaryotic cells. 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. = cDNA Library vs. Genomic DNA Library =
cDNA library lacks the non-coding and regulatory elements found in genomic
DNA. Genomic DNA libraries provide more detailed information about the organism,
but are more resource-intensive to generate and maintain.
Cloning of cDNA cDNA molecules can be cloned by using
restriction site linkers. Linkers are short, double stranded pieces of DNA
about 8 to 12 nucleotide pairs long that include a restriction endonuclease
cleavage site e.g. BamHI. Both the cDNA and the linker have blunt ends which can
be ligated together using a high concentration of T4 DNA ligase. Then
sticky ends are produced in the cDNA molecule by cleaving the cDNA ends with
the appropriate endonuclease. A cloning vector is then also cleaved with the
appropriate endonuclease. Following "sticky end" ligation of the insert into
the vector the resulting recombinant DNA molecule is transferred into E. coli
host cell for cloning. External links
cDNA library-Properties Functional Annotation of the Mouse
database examples of cDNA synthesis and cloning
Custom cDNA Library Construction