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What is a cDNA Library?
Keywords
Complementary DNA, transcriptome, mRNA, eukaryotic cells, bacterial cell, genomic DNA.
Introduction
A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which constitute some portion of the transcriptome of the organism and are 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.
In eukaryotic cells the mature mRNA is already spliced, hence the cDNA produced lacks introns and can be readily expressed in a bacterial cell. 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.
Principle of cDNA Library
To construct cDNA libraries, DNA copies from mRNA sequences of organisms are produced and then they are cloned.
The term cDNA is given as all the DNA in the library are complementary to the mRNAs and are produced by reverse transcription of mRNAs.
Most eukaryotic DNA consists of repeated sequences that are not transcribed into mRNA, and in a cDNA library the sequences are not represented.
It should be remembered that prokaryotes and lower eukaryotes do not contain introns, and cDNA preparation for these species is usually needless.
Therefore, cDNA libraries are only created from higher eukaryotes.
For the construction of cDNA library, both the bacterial and bacteriophage DNA can be used as vectors.
cDNA Library Construction
cDNA is created from a mature mRNA from a eukaryotic cell with the use of 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. This has the problem that not all transcripts, such as those for the histone, encode a poly-A tail.
mRNA extraction
First, mRNA template needs to be isolated for the creation of cDNA libraries. Since mRNA only contains exons, the integrity of the isolated mRNA should be considered so that the protein encoded can still be produced. Isolated mRNA should range from 500 bp to 8 kb. Column purification can be done using oligomeric dT nucleotide coated resins, and features of mRNA such as having a poly-A tail can be exploited where only mRNA sequences containing said feature will bind. The desired mRNA bound to the column is then eluted.
cDNA construction
Once mRNA is purified, an oligo-dT primer is bound to the poly-A tail of the RNA. The primer is required to initiate DNA synthesis by the enzyme reverse transcriptase. This results in the creation of RNA-DNA hybrids where a single strand of complementary DNA is bound to a strand of mRNA. DNA polymerase I is then added, the cleaved RNA acts as a primer. The DNA polymerase I can identify and initiate replacement of RNA nucleotides with those of DNA. Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. The cloned bacteria are then selected, commonly using antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library.
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 keep.
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 libraries are most useful in reverse genetics where the additional genomic information is of less use. cDNA libraries are frequently used in functional cloning to identify genes based on the encoded protein's function.
In eukaryotic DNA, expression libraries are constructed using complementary DNA (cDNA) to help ensure the insert is truly a gene.
Advantages
It is enriched with fragments from genes that have been actively transcribed
Introns do not disrupt the cloned sequences; if the goal is to create a eukaryotic protein in bacteria, introns will pose a problem, since most bacteria have no means of eliminating the introns.
Disadvantages
A cDNA library has the drawback that it only includes sequences that are present in mature mRNA.
There are no introns and any other sequences that are modified during transcription; sequences that are not transcribed into RNA, such as promoters and enhancers, are also not present in a library of cDNA.
It is also important to remember that only certain gene sequences expressed in the tissue from which the RNA has been isolated constitute the cDNA library.
In addition, in a cDNA library, the frequency of a specific DNA sequence depends on the abundance of the corresponding mRNA in the given tissue.
In contrast, in a genomic DNA library, almost all genes are present at the same frequency.
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