21-10-2014_16-13-52 / рис.6-лекц-9

Genomic and cDNA Libraries

Suppose you have known the partial sequence of a gene (e.g., from the sequence of a homologous gene) and want to determine its entire sequence, then you may use the technique described in this section.

Preparation of a DNA Library

DNA library is is a collection of cloned DNA fragments.  There are two types of DNA library:

The genomic library contains DNA fragments representing the entire genome of an organism.

The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. 

Figure 9-B-1.  Preparation of the genomic library using  phage vectors.  It is basically the cloning of all DNA fragments representing the entire genome.

Genomic LibraryThe genomic library is normally made by  phage vectors, instead of plasmid vectors, for the following reasons: The entire human genome is about 3 x 10⁹ bp long while a plamid or  phage vector may carry up to 20 kb fragment.  This would require 1.5 x 10⁵ recombinant plasmids or  phages.   When plating E. coli colonies on a 3" petri dish, the maximum number to allow isolation of individual colonies is about 200 colonies per dish.  Thus, at least 700 petri dishes are required to construct a human genomic library.  By contrast, as many as 5 x 10⁴  phage plagues can be screened on a typical petri dish.  This requires only 30 petri dishes to construct a human genomic library.  Another advantage of  phage vector is that its transformation efficiency is about 1000 times higher than the plasmid vector.

 cDNA LibraryThe advantage of cDNA library is that it contains only the coding region of a genome.  To prepare a cDNA library, the first step is to isolate the total mRNA from the cell type of interest.  Because eukaryotic mRNAs consist of a poly-A tail, they can easily be separated.  Then the enzyme reverse transcriptase is used to synthesize a DNA strand complementary to each mRNA mlecule.  After the single-stranded DNA molecules are converted into double-stranded DNA molecules by DNA polymerase, they are inserted into vectors and cloned.