BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Gene Cloning. Gene Libraries

A clone refers to a population of identical copies derived from a single common ancestor. Gene clones are widely used to investigate gene Structure.

The Essence of cloning lies in inserting a DNA fragment into a bacterial Cell, where it undergoes Replication. A single cell gives rise to a colony comprising a vast number of identical Cells: for instance, when grown on Agar overnight, an E. coli cell produces 10⁷ cells within a single colony. Thus, bacterial cells facilitate the synthesis of substantial quantities of required gene copies.

Stages of Gene cloning:

- Cleavage of DNA using restriction Enzymes;

- Digestion of the cloning vector with restriction enzymes;

- ligation of the two DNA fragments using T₄ phage DNA ligase;

- transformation of recipient cells;

- DNA Amplification within the transformed cells.

A gene library is a collection of various DNA nucleotide sequences cloned into vector molecules, which collectively represent the entire organismal genome.

To construct a gene library, recombinant DNA is packaged into phages, which are then used to infect Bacteria. The shorter the DNA fragments used for gene libraries and the more complex The Genome under study, the larger the number of clones required to ensure the library is complete. For example, to create a genomic library using DNA fragments 20 kbp in length (with any sequence represented at a 99% probability), it is necessary to obtain 460 clones for bacteria, 600,000 for mammals, and several million for angiosperms.

The size of a library containing any given genome sequence with the desired probability can be calculated using the formula:

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where N is the number of required clones;

P is the probability that the library contains the desired DNA

sequence;

f is the fraction of the genome represented in each clone (calculated as The ratio of the average insert size to the Genome Size).

Gene libraries obtained using bacterial cells are stored at -70°C as Suspensions in a 30% glycerol solution, whereas phage particle suspensions are stored in dimethyl sulfoxide. This prevents the destruction of Bacterial cells and phages during freezing and thawing.