Process of rDNA Technology: Isolation and Cutting of DNA

The overall process of recombinant DNA technology can be thought of as a sequence of discrete, well-defined steps, each of which solves one specific practical problem on the way from 'a gene we want' to 'a host cell that makes the product we want'. The first two of these steps are isolation of the genetic material and cutting of the DNA.

Isolation of the genetic material begins with breaking open the cells or tissue that contain the gene of interest, using a combination of enzymes -- lysozyme for bacterial cells, cellulase for plant cells, chitinase for fungal cells -- to digest the protective cell wall, followed by treatment with other enzymes such as ribonuclease and protease to remove RNA and protein contaminants respectively, since the goal is a sample of pure DNA. The resulting DNA, freed from the cell debris, is present in the solution as extremely long, fine, thread-like strands. Because the DNA is not visible in solution in this form, it is finally purified by precipitation: chilled ethanol is added, and the DNA, being insoluble in ethanol, comes out of solution as visible, fine threads that can be spooled out and collected. This purified DNA is then digested with an appropriate restriction enzyme, cutting it into many fragments, one of which contains the gene of interest.

Cutting of DNA at specific locations is carried out using the restriction enzymes described earlier in this chapter. In practice, this is done through a technique called restriction digestion: the purified DNA sample is incubated with a chosen restriction enzyme under specific conditions of temperature and buffer composition suited to that enzyme, allowing it to find and cut every occurrence of its recognition sequence throughout the DNA sample. Because a typical genomic DNA sample contains its recognition sequence at many different locations, this digestion produces a large number of fragments of different sizes. …