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Cdna Custom Library Construction For Genome Sequencing Applications
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cDNA custom library construction an overview
Like a library is a place sheltering large collection of books on different subjects for public use, a cDNA custom library construction involves collection of DNA sequences from various organisms, serving different purposes. These DNA sequences are stored as recombinant molecules by ligating them with suitable vectors. The two types of gene libraries are genomic library and cDNA library, categorized based on the source from where they were constructed. A genomic library holds the DNA sequences derived from genomic DNA whereas the cDNA library represents the DNA sequences generated from mRNA. The library that represents the source DNA as such can be considered as an ideal gene library.
How does the cDNA custom library construction take place?
Preparation of the genomic cDNA custom library construction involves isolation of genomic DNA, purification of the genomic DNA and fragmentation of genomic DNA into desired size and then cloning of the fragmented DNA using suitable vector. The eukaryotic cell nuclei are purified by adopting digestion by protease and phenol-chloroform applied phase extraction. The derived genomic DNA is long and needs to be cut into desirable fragment sizes. Fragmentation of DNA is achieved by physical method and enzymatic method. Physical method includes pipe ting the DNA molecule or applying intensified ultrasound waves (sonication). The enzymatic method involves the use of restriction enzyme to fragment the purified DNA. The desirable DNA size suitable for the cloning vector decides the method to be used for fragmenting the DNA and the length of exposure of the DNA molecule.
Steps involved in cDNA custom library construction
cDNA custom library construction and developing involves 4 steps.
1. Initial extraction and purification of mRNA,
2. Production of cDNA,
3. Treating the ends of cDNA and
4. Ligation of the cDNA to the vector.
The polyadenylated nature of the eukaryotic mRNA enables easy isolation of mRNA by using oligo (dT). Magnetic beads with oligo(dT) is added to the cell lysate which enables the binding of the poly A tail of the mRNA to the oligo(dT) and using strong magnetic force the binded mRNA is isolated from the total RNA. The recovered mRNA is checked for its integrity by using gel electrophoresis technique. Also translation of isolated mRNA is carried out as a step to check its integrity.
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