CLONE BANK (cDNA Library)

Definition:

cDNA clone bank is defined as “bacterial transformants population, each containing a plasmid with single cDNA insert, and with a sufficiently huge number of different transformants such that every mRNA molecule is characterized at least once in the bacterial population.” Thus a compete cDNA clone bank should contain about 5000 – 1000 clones of sequences amenable to detection. 

Construction Procedure:

cDNA cloning involves the preparation of a dsDNA copy (dscDNA) of mRNA and its addition into a plasmid or bacteriophage vector for cloning. Such cloned bacteria are used for assembly of a cDNA clone bank. Two basic methods are widely used to obtain dscDNA, both involve priming first stranded synthesis with reverse transcriptase and oligodT.

8   The first stranded sequence is accomplished by primary denaturation of the mRNA with methyl mercuric hydroxide plus then by the addition of RNase inhibitor in order to limit the degradation of mRNA during the reaction.

8   The second stranded synthesis is accomplished by forming a hairpin loop like structure at 3^’ end of the cDNA sequence which can act as a primer for the second strand synthesis by DNA polymerase I. This arrangement can then be cleaved at the single stranded terminal by S1 nuclease to produce a blunt ended, ds(double stranded) cDNA.

Attachment of cDNA sequence into plasmid vectors for propagation and cloning is accomplished by either complementary oligonucleotide tailing of the cDNA and vector addition of synthetic restriction site containing linkers specific to the cDNA.  

Function: The cDNA clone bank may be used as a probe for purpose of sequence nucleotides of a specific gene by comparing with a cDNA. This is very useful during hybridization and site directed mutagenesis.

GENE BANK:

Definition:

It is a whole collection of fragmented cloned DNA form entire genome of an organism. Gene bank is formed by a ‘shotgun’ experiment where tota cell genome in a cloned in the arrangement of random, unidentified clones. 

Construction Procedure:

·   DNA of the cell should be calibrated using restriction endonucleases to form fractions which are suitable for cloning (about 10-30Kb).

·   These fragments are cloned without selection of particular sequences.

·   Bacteria containing individual rDNA are grown plus a set of such bacterial clones which form whole genome of the organism is the gene bank.   PLASMID