Techniques Used in Genetic Engineering

Genetic engineering requires the DNA of interest, various enzymes, a suitable vector, and a method for introducing the

DNA into the new host. The methods used to genetically engineer prokaryotic cells are generally less complicated than those used to genetically alter eukaryotes.

Genetically Engineering Bacteria

An approach frequently used to clone a specific gene is to clone a set of DNA fragments that together make up the entire genome of the organism being studied into a population of E. coli cells. While each cell in the resulting population contains only one fragment of the genome, the entire genome is represented in the population as a whole. Because each cloned molecule can be viewed as one "book" of the total genetic infor mation, the collection of clones is called a DNA library (figure 9.12). Once a DNA library has been prepared, a colony blot can be used to determine which cells contain the gene of interest. ■ origin of replication, p. 172

Obtaining DNA

The first step of a cloning experiment is to obtain the DNA that will be cloned. To isolate DNA, cells in a broth culture are lysed by adding a detergent. As the cells burst open, the relatively fragile DNA is inevitably sheared into many pieces of varying lengths. Because the original broth culture contained a billion or more cells per ml, the resulting solution of DNA contains a like number of copies of the chromosome. This is important to recognize when trying to envision how a cloning experiment works. While descriptions and diagrams only show one or a few DNA molecules, in reality there are billions. The same holds true for the molecules of DNA depicted in most of the techniques covered in this chapter.

Bacterium X

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