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cDNA Library Construction
cDNA is created from mRNA with the use of an enzyme known as reverse transcriptase. In eukaryotic cells, a poly-(A) tail (consisting of a long sequence of adenine nucleotides) distinguishes mRNA from tRNA and rRNA. Extracted RNA content from a cell can be passed through a chromatography column | on which a strand of complementary nucleotides7#151;such as short uracil [poly(U)] or thymidine extacts (oligo-dT) are attached to a matrix in the column. The mRNA's poly-(A) tail can bind to the oligo-DTs, whilst the rest is washed away. Elution with low-salt buffer releases the mRNA from the oligo-dTs.
Once the purified mRNA is recovered, reverse transcriptase is used to make templates of double-stranded DNA from the mRNA templates. The newly formed DNA templates are then inserted into bacteria plasmids using restriction enzymes (to cut open the plasmid) and ligase (to seal up the ends once the DNA has annealed into the plasmid).
Unfortunately, a very low percentage of plasmids actually have the DNA successfully inserted into the plasmid despite a high percentage cut by restriction enzymes. Many plasmids simply remain cut open or anneal with itself again. Therefore, to isolate those plasmids that do have the DNA successfully inserted, an extra antibiotic resistance gene is usually inserted along with the DNA. The bacteria is then grown in a medium with this antibiotic. All bacteria with plasmids that have successfully taken in the DNA (and hence the antibiotic resistance), will then survive, with all the others dying off.
The surviving colonies are then grown to reproduce the plasmid with the DNA, which is then purified. This is also why these types of libraries are called cDNA or complementary DNA, because it is a complemetary strand of DNA to the mRNA in question.
cDNA Library uses
Such a library has several uses. A cDNA of an eukaryotic organism (for example, a human) can be cloned into a prokaryotic organism (for example, E. coli) and expressed (translated into the appropriate protein) there (with limitations, for example posttranslational modification). A cDNA library is also important for analysis through bioinformatics. The complete cDNA library of an organism gives the total of the proteins it can possibly express. Also, comparison of cDNA sequence between libraries constructed from cells derived from different organisms can provide insight into the genetic and evolutionary relationship between organisms through the similarity of their cDNA.
cDNA Library vs. Genomic DNA Library
Why must cDNAs be used to create gene libraries from eukaryotic species?
In Eukaryotic genomes, the entire sequence of genes is often very large because coding sequence (exons) is interrupted by introns that do not contain coding DNA sequence. The advantage of a cDNA library is that only the exons, or DNA representing expressed sequence, are templates for the creation of DNA (via Reverse Transcriptase) to collect the preferred genes. Prokarya generally do not contain the apparatus necessary to splice together exons. Furthermore, the introns can increase the genomic space taken up by the gene to a point where prokaryotic genetic manipulation schemes cannot handle the length of the DNA.
When generating gene libraries from prokaryotic species, Genomic DNA libraries are often used. Genes from prokaryotic cells generally do not contain introns, and the DNA is therefore much shorter, and the RNA does not require much post-processing to be expressed correctly. Also, prokaryotic mRNA can be difficult to isolate; because of this a genomic library is more preferable for prokaryotic genomic studies.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "CDNA_library". A list of authors is available in Wikipedia.|