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Herbert Boyer

Stanley N. Cohen

Paul Berg

Herbert Boyer

Stanley N. Cohen

DNAs are called cloning vectors (a vector is a delivery agent). They are typically plasmids or viral DNAs.

3. Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning vector and DNA to be cloned. Composite DNA molecules comprising covalently linked segments from two or more sources are called recombinant DNAs.

4. Moving recombinant DNA from the test tube to a host cell that will provide the enzymatic machinery for DNA replication.

5. Selecting or identifying host cells that contain recombinant DNA.

The methods used to accomplish these and related tasks are collectively referred to as recombinant DNA technology or, more informally, genetic engineering.

Much of our initial discussion will focus on DNA cloning in the bacterium Escherichia coli, the first organism used for recombinant DNA work and still the most common host cell. E. coli has many advantages: its DNA metabolism (like many other of its biochemical processes) is well understood; many naturally occurring cloning vectors associated with E. coli, such as plasmids and bacteriophages (bacterial viruses; also called phages), are well characterized; and techniques are available for moving DNA expeditiously from one bac terial cell to another. We also address DNA cloning in other organisms, a topic discussed more fully later in the chapter.

Restriction Endonucleases and DNA Ligase Yield Recombinant DNA

Particularly important to recombinant DNA technology is a set of enzymes (Table 9-1) made available through decades of research on nucleic acid metabolism. Two classes of enzymes lie at the heart of the general approach to generating and propagating a recombinant DNA molecule (Fig. 9-1). First, restriction endonu-cleases (also called restriction enzymes) recognize and cleave DNA at specific DNA sequences (recognition sequences or restriction sites) to generate a set of smaller fragments. Second, the DNA fragment to be cloned can be joined to a suitable cloning vector by using DNA ligases to link the DNA molecules together. The recombinant vector is then introduced into a host cell, which amplifies the fragment in the course of many generations of cell division.

Restriction endonucleases are found in a wide range of bacterial species. Werner Arber discovered in the early 1960s that their biological function is to recognize and cleave foreign DNA (the DNA of an infecting virus, for example); such DNA is said to be restricted. In the host cell's DNA, the sequence that would be recognized

TABLE 9-1 Some Enzymes Used in Recombinant DNA Technology

Enzyme(s)

Function

Type II restriction endonucleases

Cleave DNAs at specific base sequences

DNA ligase

Joins two DNA molecules or fragments

DNA polymerase I (E. coli)

Fills gaps in duplexes by stepwise addition of nucleotides to 3' ends

Reverse transcriptase

Makes a DNA copy of an RNA molecule

Polynucleotide kinase

Adds a phosphate to the 5'-OH end of a polynucleotide to label it or permit ligation

Terminal transferase

Adds homopolymer tails to the 3'-OH ends of a linear duplex

Exonuclease III

Removes nucleotide residues from the 3' ends of a DNA strand

Bacteriophage A exonuclease

Removes nucleotides from the 5' ends of a duplex to expose single-stranded 3' ends

Alkaline phosphatase

Removes terminal phosphates from either the 5' or 3' end (or both)

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