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Genomic and cDNA LibraryBY: Nidhi Uppangala | Category: Genetics | Submitted: 2010-06-13 08:54:30
Construction of DNA library helps the scientists to search for particular gene of interest from the whole genome. Human genome contains around three billion DNA base pairs. But the whole genome does not code for the proteins but the genome contains only 25,000 to 30,000 genes. Genes are DNA sequences which code for the production of mRNA by transcription and these mRNA through translation are converted into proteins. DNA library allows the extraction of particular gene from the genome. DNA library is collection of DNA fragments from an organism. But these DNA fragments are stored in another organism like the E. coli bacteria or yeast.
First we need to understand about the basics which are related to DNA library such as DNA, RNA and cDNA.
DNA (deoxyribonucleic acid) is made up of four bases known as adenine, thymine, cytosine and guanine. The particular base pair with particular bases, like Adenine pairs with Thymine and Guanine pairs with Cytisine. The permutation and combination of these base pairs form the chromosome and genome of an organism. Eukaryotic organisms contain exons and introns in the genome. Only the exons are coding part of the genome or in other words exons code for mRNA and proteins. But the prokaryotic genome contains only coding region or exons in their genome.
DNA with the help of enzymes transcribes to form mRNA in the nucleus of the cell. These mRNA are then transported into cytoplasm part of the cell. There they translate themselves to produce proteins.
cDNA or complimentary DNA are produced by reverse transcription of mRNA or messenger RNA. cDNA contains only the coding part of the genome of an organism.
Construction of Genomic DNA Library:
1. Genomic DNA library contains whole genome of an organism. First the genome of an organism is extracted using some chemicals like enzymes and DNA stabilizing agents and are stored using alcohol. Then the double stranded DNA molecules are cleaved using restriction endonuclease enzymes. As result many DNA fragments are formed.
2. Each DNA fragments are inserted into a vector. In most of the cases plasmids are used as vectors. Plasmids are extra chromosomal DNA, which have the capacity to replicate itself. DNA fragments are inserted into plasmids, using ligase enzymes. Now these plasmids are called as recombinant plasmids.
3. These recombinant plasmids are then trasfected into the bacterial cell, where they replicate themselves to form many thousand copies of the same DNA fragment. Then these are stored in vials and preserved in cryopreservator.
Same procedure or method are used to create DNA library of a human or any other organism like bacteria or fungi or animals like dog or elephant.
Construction of cDNA Library:
cDNA library contains only the coding part of the genome. It does not contain the non coding parts such as promoter and regulator regions of the genome. The mRNA is used instead of whole genome to construct the cDNA library. Messenger RNA is reverse transcribed to complementary DNA using enzyme known as reverse transcriptase. Then other steps are followed same as the genomic DNA library construction.
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