|cDNA Clone Bank or cDNA Library
A typical eukaryotic cell contains between 10,000 to 30,000 different mRNA sequences. Williams (1981) has defined the cDNA clone bank as “a population of bacterial transformants, each containing a plasmid with a single cDNA insert, and with a sufficiently large number of individual transformants such that every mRNA molecule is represented at least once in the bacterial population”. A complete bank is that which contains in the order of 5,000 to 10,000 clones of the sequences amenable to detection.
True copy of an mRNA molecule is known as copy DNA or cDNA. The well characterized cDNA molecule is allowed to bind with a suitable vector which then transforms a bacterial cell in such a way that (Joes not disrupt its normal function. The transformed bacterial cell containing plasmid with DNA copy of an mRNA molecule is known as cDNA clone. lt is difficult to have cDNA from double stranded DNA molecules. Therefore, most of the cDNA clones have been prepared from mRNA sequences of eukaryotic cells. The procedure for obtaining cDNA to built a library (Fig. 4.1) is given under cDNA to be cloned (see Isolation of DNA to be cloned
A clone as needed may be screened, identified, isolated and used as required. Following are the benefits from a cDNA clone bank:
Preparation of cDNA clones is easy in many viruses whose genetic material is RNA, for example influenza virus, reovirus, etc.(ii)
Screening of cDNA is easy as it contains less number of cDNAs prepared from mRNA of the eukaryatic cells that may contain between 10,000 to 30,000 mRNA molecules. Hence, there is possibility of 10-30 thousand of cDNA fragments.(iii)
As the genes in prokaryotes do not contain introns (the intervening non-transcribed regions of DNA sequence which interrupt the transcripts of most of the genes); the cDNA clones possess an uninterrupted copy of mRNA molecules. It may also be attributed to the fact that the prokaryotes produce splicing enzymes for ligation process just to remove the sequences of introns. In contrast, in eukaryotes the introns spilt the transcribed. DNA into many fragments which are known as exons.(iv)
Following the hybridization process the nucleotide sequence of the gene interrupted by introns may be determined from a genomic clone by comparing with its mRNA transcripts therefore, a cloned cDNA is the most suitable probe for hybridization.A
dvancement in the techniques of in vitro
DNA synthesis (Narang et al
., 1979) has made it much less daunting task than it would have been previously. This approach has also been used (Houghton et ah,
1980) to prepare cDNA derived from interferon mRNA. Moreover, mRNA synthesized from the insulin gene is highly abundant in pancreatic tissue just a globin mRNA is highly abundant in erythroid tissue. Thus, mRNA cloning of cDNA can be done and used to screen libraries of recombinant representative of the total genomic DNA of an organism, and from this the chromosomal genes can be separated.