c-DNA library: protocol and utility

                    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, storage, 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: When it is known that the gene of interest is expressed in a particular tissue or cell through the synthesis and processing of active m-RNA, the strategy of c-DNA cloning is adopted.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: 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) Reverse Transcription:The family of mRNAs in solution is treated with oligo d(T) primer, the enzyme Reverse transcriptase and four types of deoxy-ribonucleotides.The temperature is maintained at 55°C suitable for polymerization.

 

The oligo d(T) primer, which is 12-18 nucleotide long gets bound with poly A tail of the eukaiyotic mRNAs. The primer provides a tree 3'-OH group to proceed the reverse transcription.Reverse transcriptase adds complementary deoxyribonucleotides one by one to the 3'-OH group of the primer and forms a single stranded DNA. As a result RNA-DNA hybrid is formed.The single stranded DNA which is complementary to m-RNA is called complementary DNA (c-DNA).

 

(iii) Oligo d(C) tailing: After forming  RNA-DNA hybrid it is treated  with the enzyme terminal transferase and nucleotide CTP.This enzyme adds CTP one by one to  the 3'-OH group of both RNA and DNA strand.As a result a short oligo d(C) tail is formed at the 3' end of both strands.

 

(iv) Alkaline Hydrolysis of mRNA: The reaction mixture is treated with alkaline sucrose solution. The alkaline solution  breaks hydrogen bonds between the RNA and DNA strands and separates the two strands.

 

(v) Addition of Oligo d(G) Primer:Oligo d(G) primer is added to the reaction mixture and the temperature is maintained at  55°C. The oligo d(G) primer gets bound oligo d(C) tail of the cDNA and mRNA by  interchain hydrogen bonding. The primer provides 3'-OH group to proceed polymerization reaction.

(vi) Synthesis of the Second DNA Strand: The second strand of c-DNA can be synthesized by two methods…….

      (a) Self priming: To the reaction mixture, klenow enzyme (DNA polymerase) all the four types of deoxyribonucleotides are added.The enzyme adds complementary nucleotides one by one to the 3'-OH group of the primer. As a result, a double stranded DNA called cDNA clone is formed from each cDNA(i.e first strand). This pocess is called self priming.

 

    (b)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.

 

(vii) 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.

 

(viii) 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.

 

(viii) Screening of recombinant E. coli:The recombinant cells are separated from one another using dilution plating and subsequent culture.The microbial cultures are screened to detect the contigs.Based on the contigs analysis, the clones are arranged in the genome library. Screening of DNA Library .

 

                                     Analysis of various clones to arrange them into contigs is called screening of genomic library. The clones are arranged into contigs based on the principle of overlapping between the adjacent clones. The continuous clones representing the entire genomic DNA of an organism in the correct order are called Contigs. Restriction fragment fingerprinting, chromosome walking, repetitive DNA fingerprinting and STS (Sequence tagged sites) mapping are used to arrange the clones into contigs.

 

Uses of cDNA Library:

l.The cDNA libraries help to understand proteins accumulated in the cell during a particular stage.

 

2.They help to know the reading frames within the genome of the organisms.

 

3.The cDNA libraries constructed from m-RNAs at selective stages of cells help us to know the gene expression at those stages. Such libraries are called selective cDNA libraries. They give details of patterns of expression at different stages of cells.

 

4. The cDNA libraries provide genes for gene manipulation works.

 

5.The cDNA libraries are used to study genomic DNA fragments in the light of details of the cDNA clones.

 

6. The cDNA libraries are used to identify new genes‘ in the genomic DNA fragments.

 

7. A cDNA library acts as storehouse for various genes of an organism.

 

8. The cDNA libraries help for the identification of pharmaceutically valuable genes in the genome.