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DNA is digested with a restriction enzyme; fragments with cohesive ends are produced

Cohesive ("sticky") ends

Fragment Annealed to Vector

Two Fragments Annealed

Fragment Annealed to Vector

Two Fragments Annealed

Figure 9.14 Cohesive ends Restriction fragments that were cut with the same restriction enzyme can anneal, regardless of their original source.

Vector digested with same restriction enzyme

9.6 Techniques Used in Genetic Engineering 233

ing recombinant plasmids from those that contain intact vector. This is important because when the vector and insert DNA are both cut with the same restriction enzyme and the fragments are mixed together, not all molecules will join to form the desired recombinant plasmids. If a vector anneals to itself, the intact vector is regenerated and will replicate when introduced into a cell. This second marker is a gene that spans the cloning site (figure 9.15). Insertion of a DNA fragment into the vector disrupts the gene, resulting in a nonfunctional gene product and an altered phenotype. One common vector, pUC18, has a multiple-cloning site in the middle of the lacZ gene. The product of the lacZ gene enables cells to cleave a colorless chemical, x-gal, to form a blue compound. The creation of a vector-insert hybrid disrupts the lacZ gene, resulting in a non-functional gene product. Colonies of cells that harbor intact vector have a functional lacZ gene and are blue, whereas those that contain a recombinant molecule are white (figure 9.16). ■ phenotype, p. 192

Vectors Used to Clone Eukaryotic Genes into

Prokaryotic Cells The type of vector used to clone eukaryotic DNA into a bacterial cell depends largely on the ultimate purpose of the procedure. If the goal of cloning is to produce the protein encoded by the DNA, then a vector designed to optimize transcription and translation of the insert DNA is used. To create a DNA library of a human or other eukaryotic genome, however, a vector that can carry a large insert is generally used. Available vectors include:

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