c-DNA library

 What is c-DNA library? Illustrate the various steps in producing c-DNA library. How does it differ from genomic DNA library? 2+8+6

c-DNA library:-

       A DNA library is a collection of different DNA sequences from an organism each of which has been cloned into a vector for ease of purification, shortage, and analysis. The c-DNA library is a collection of c-DNA clones generated in vitro from the m-RNA sequence of a single tissue or cell population by using Reverse transcriptase and DNA polymerase.

Steps of c-DNA library processing:-

       For many plants and animals a complete genome library contain such a vast number of clones that the identification of desired gene is a mammoth task. When it is known that the gene of interests is expressed in a particular tissue or cell type then c-DNA libraries are used.

       The strategy of c-DNA cloning involves the copying of m-RNA transcripts into DNA which are then inserted into bacterial plasmid plasmid and then placed into bacteria by transformation. The major steps in the c-DNA cloning are summarized below.

(i) Isolation or extraction of m-RNA:-

       When it is known that the gene of interest is expressed in a particular tissue or cell, a crude extract of the tissue is prepared and then freed from proteins, polysaccharides and other contaminants. It is known that m-RNA have a poly-A tail in 3' end. Under appropriate condition this tail will bind with a string of thymidine residues immobilized on cellulose and then poly-A fragment can be illuted. Two or three phases of poly-A segment through such a column produces a fraction highly enriched for m-RNA. Before cloning it is necessary to check that the sequence of interest is present. This is done by translation of m-RNA in vitro and identification of appropriate polypeptides in the product obtained.

(ii) Synthesis of 1^st strand of c-DNA:-

       The m-RNA fraction is copied into the 1^st DNA strand by using RNA dependent DNA polymerase (reverse transcriptase). The enzyme can only add residues at 3'OH group of an existing primer. The primer is base paired with template for cloning of c-DNA. The most frequently used primer is Oligo dT which is 12-18 nucleotide long. It binds poly-A tract of 3' end of m-RNA molecules. After completion of copying a DNA-RNA hybrid is formed.

(iii) Synthesis of the second strand of c-DNA:-

       Before 2^nd strand synthesis DNA-RNA hybrid is destroyed by alkali treatment. Therefore the 2^nd strand of c-DNA can be  synthesized by 2 method namely self priming c-DNA and replacement synthesis.

Self priming:-

       m-RNA hybrid so obtained is denatured so that 2^nd strand can be synthesized on the single strand of the c-DNA by Klenow fragment of DNA polymerase I which is self priming.

Replacement synthesis:-

       m-RNA hybrid is also used as template for a nick translation reaction. RNAse H produces nicks and gaps in the m-RNA strand of the hybrid creating a series of RNA primers that are used by DNA polymerase I during synthesis of the 2^nd strand of c-DNA.

(iv) Cloning of c-DNA:-

       The most commonly used procedure for cloning c-DNAs involves the addition of complementary homopolymeric tracts to double stranded c-DNA and to plasmid vector a string of cytosine residues are added to the c-DNA using the enzyme terminal transferase to form Oligo dC tails on the 3' ends. Similarly a plasmid is cut open at a unique restriction endonuclease site and tailed with Oligo dG. The vector and the double stranded c-DNA are then joined by hydrogen bonding between the complementary homopolymers to form open circular hybrid molecule capable of transforming E. coli.

(v) Introduction to host cell:-

       The recombinant plasmids are used to transform bacteria usually E. coli. K₁₂ strain. E. coli cells treated with CaCl₂ willtake up plasmid molecules from the surrounding medium and the host cell will repair any gaps in the recombinant plasmid. If the plasmid has been chosen carefully it is possible to select transfor from non transformed bacteria on the basis of antibiotic resistance. This simple selection tales the investigator which colonies carry a c-DNA copy of some sort.

       c-DNA cloning is widely accepted techniques but it is necessary to ensure that only sence strand of c-DNA is transcribed. If the antisence strand is cloned downstream of a bacterial promoter, then the resulting transcript will not encode the intended protein.