Cloning genes is now a technically straightforward process. Usually, cloning uses recombinant DNA techniques, which were developed in the early 1970s by Paul Berg, of Stanford University, and, independently, by Stanley Cohen and Herbert Boyer, of Stanford and the University of California. These researchers devised methods for excising genes from DNA at precise positions, using restriction enzymes and then using the enzyme known as DNA ligase to splice the resulting gene-containing fragment into a plasmid vector.
Plasmids are small, circular DNA molecules that occur naturally in many species of bacteria. The plasmids naturally replicate and are passed on to future generations of bacterial cells. To replicate, all plasmids must contain a sequence, called an origin of replication, which directs the bacterial DNA polymerase to replicate the DNA molecule. In addition, recombinant plasmids contain one or more selectable markers. A selectable marker is a gene that confers on the bacterium harboring the plasmid the ability to survive under conditions in which bacteria lacking the plasmid would otherwise die. Usually, such genes encode enzymes that enable the bacterium to live and grow despite the presence of an antibiotic drug.
The recombinant plasmid is then introduced into a host cell, such as an Escherichia coli bacterium, by a process called transformation, and the cell is allowed to multiply and form a large population of cells. Each of these cells harbors many identical copies of the recombinant plasmid. The cells are then cultured in growth media containing the antibiotic to which the plasmid confers resistance. This ensures that only cells containing the recombinant plasmid will survive and replicate. A researcher then harvests the cells and can extract and purify many copies of the plasmid.
Another method to produce many copies of a DNA molecule, which is even simpler than traditional recombinant cloning methods, is the polymerase chain reaction (PCR). PCR amplifies the DNA in a reaction tube without the need for a plasmid to be grown in bacteria.
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