cDNA library

A cDNA library is a collection of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute the entire genome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. 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

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Formation of a cDNA library.

cDNA is created from a mature mRNA from an 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 usig oligomeric dT nuclotide 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 seperate the mRNA strands from oligo-dT.

cDNA construction:

Once mRNA is purified, oligo-dT (a short sequece of deoxy-thymine nucleotides) is tagged as a complementary primer which binds to the poly-A tail providing a free 3'-OH end that can beextended by reverse transcriptase to create the complementary DNA strand. Now, the mRNA is removed by using a 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 need. This is privided by the sscDNA itself by generating a hair pin 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 most useful in reverse genetics where the additional genomic information is of less use.

cDNA Library vs. Genomic DNA Library

As previously mentioned, a cDNA library lacks the non-coding and regulatory elements found in genomic DNA. Genomic DNA libraries provide much more detailed information about the organism, but are much more resource-intensive to generate and maintain.

External links

• cDNA library-Properties
• Functional Annotation of the Mouse database
• examples of cDNA synthesis and cloning