cDNA LIBRARY @ BIOTECNIKA LABS

Biotecnikaglobal

ONE STOP SITE FOR ALL YOUR BIOTECHNOLOGY NEEDS

You are here: Genetic engineering >Gene Library> cDNA library

cDNA library

It is a population of bacterial transformants or phage lysates in which each mRNA isolated form an organism or tissue is represented as its cDNA insertion in a plasmid or a phage vector.

The frequency of a specific cDNA in such library would ordinarily depend on the frequency of the concerned mRNA in the tissue/organism in question .cDNA is the abbreviated form of complementary DNA. A cDNA is a polydeoxyribonucleotide duplex, the sequence of one strand of which is complementary to a RNA molecule & has been derived primarily by the use of the enzyme Reverse Transcriptase on the RNA as template.

Properties of cDNA

· In some cases it is easier to isolate mRNA molecule than the gene itself, because of selective expression of a particular gene, resulting in the relative abundance of that mRNA, for e.g., Insulin mRNA in the islet cells of Pancreas, ovalbumin mRNA in the hen oviduct on treatment with estrogens. In such cases, cDNA cloning is the approach of choice.

· Only about 1 to 10% of the total DNA in plant or animal cell is expressed or is functional genetically. A cDNA library , which would thus represent only the active genes, would be proportionately much smaller & hence much simpler to handle & construct.

Since the cDNA is derived from mRNA, there would be no intervening sequence in it & hence its expression including translation in bacteria like E.coli will be straight forward

Besides there are RNA viruses, such as influenza virus & retroviruses which do not replicate via a DNA intermediate, only their cDNA can be cloned.

ISOLATION OF mRNA

For isolation of mRNA, total RNA is first extracted from a suitable organism/tissue. The amount of desired mRNA in this sample is then increased by using one of the several procedures, some of which are listed below.

1. Chromatography on poly-u sepharase or oligo-T cellulose which retains mRNA molecules since they have 3’-poly-A tails; this enriches the preparation with mRNA of all kinds.

2. Density of gradient centrifugation- to increase the frequency of desired mRNA molecules.

3. Protein produced by a gene, used to produce antibodies specific to it. Then these are used to precipitate the polysomes engaged in synthesis of concerned polypeptides.Now mRNA is isolate dform the precipitated polysomes & purified.

CONSTRUCTION OF cDNA:-

The synthesis of double stranded DNA using mRNA, suitable for insertion into a cloning vector involves 3 major steps:-

1. First strand synthesis of DNA on the mRNA template carried out with a reverse transcriptase

2. Removal of the mRNA template

3. Second strand synthesis of DNA using the first DNA strand as a template, carried out with a DNA dependant DNA polymerase such as E.coli DNA polymerase I.

Techniques available for cDNA cloning

The first report of cDNA cloning was published in mid 1970’s & was based on homopolymer tailing technique. Of several alternative techniques, The one that became most popular was that of MANIATIS et al (1976)This involves the use of an oligo-dt primer annealing at the polyadenylate tail of mRNA to prime first cDNA synthesis & took advantage of the fact that the first cDNA strans has the tendency to transiently fold back on itself forming a hair loop resulting in self priming of the second strand.

After synthesis of second strand this loop must be cleaved with a single strand specific nuclease i.e., S1 nuclease to allow insertion in the cloning vector.Disadvantage of the hairpin method is that cleavage with S1 nuclease results in the loss of a certain amount of sequence at 5’ end of a clone.

One of the simplest strategies is given by Land et al( 1981). After first strand synthesis which is primed with an oligo dT primer as usual the cDNA is tailed with a strung of cytidine residues using the enzyme terminal transferase. This artificial oligo dC tail is then used as an annealing site for a synthetic oligo dG primer allowing synthesis of second strand.