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Wednesday, May 1, 2013

cDNA Library: Application & USes



cDNA is also known as the complementary DNA, produced after the reverse transcription of RNA by the enzyme reverse transcriptase. cDNA library is collection of host cells bearing cloned cDNA fragments which constitutes a part of the transcriptome of an organism. cDNA libraries are maintained to have the clone of expressed genes of an organism, as the cDNA's are produced from the fully expressed mRNA's.Mature mRNA's are processed mRNA's ie, they undergo post-transcrptional modifications like splicing, where the non-coding regions like introns are removed so the cDNA will also wont contains the codes for intron region. The advantage of cDNA libraries are the gene product identification is easy.

cDNA Library Construction



construction of cDNA library


Creating a cDNA library


As mentioned earlier, cDNA is produced from mature mRNA by a process called reverse transcription. The poly-(A)- tail of mRNA's are utilized here, the poly A tail which distinguishes mRNA from tRNA and rRNA can be used as primer site for the reverse transcription.

Isolation / Extraction of mRNA
For cDNA library creation, the starting material mRNA needs to be obtained. The mRNA need to be purified from other RNA's from the cell. There are several methods for mRNA isolation. Most widely used method is column purification. The resin will have oligomeric dT nucleotide coating, which specifically bind to the poly A tail of mRNA, all other contaminating RNA's will be washed off there by purified mRNA can be obtained.

Construction of cDNA / complementary DNA
As mentioned earlier, for the synthesis of cDNA from mRNA poly A tail is used as priming site,  a short tag of oligo dT with a free 3'OH group will bind and which will be extended by reverse transcriptase to create cDNA. Next job is to remove the mRNA which is achieved by treating it with RNase enzyme resulting in the single stranded cDNA. The single stranded cDNA need to be converted to double stranded cDNA which is achieved with the help of DNA polymerase. The free 3'OH group for polymerase extension is provided by the single stranded cDNA itself by forming a hairpin loop like structure, which can later be cleaved using 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.

Applications and Uses of cDNA Library 

  • Storage of reduced amount of information due to the removal of non-coding regions.
  • cDNA can be directly expressed in prokaryotic organisms.
  • cDNA libraries are useful in reverse genetics where the additional genomic information is of less use.
  • cDNA library is useful for isolating gene that codes for particular 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.