Scientists often need to obtain large amounts of DNA containing specific genes, or unique regions of genes. Two procedures are critical to rapidly producing large quantities of a target DNA sequence.

Cloning is the process of duplicating genetic information. Cloning occurs in nature and is a common form of reproduction in single-celled organisms and even some plants. In the laboratory, cloning can refer to the replication of any specific DNA sequence, or to the duplication of a whole organism for the purpose of producing a genetically identical copy. For this discussion, we will restrict the definition to single-sequence cloning.

Most laboratory cloning benefits from the assistance of bacteria - an easily maintained, rapidly reproducing organism. The single, circular chromosome of a bacterium (referred to as vector DNA in the laboratory) is isolated and cut with an enzyme called a restriction enzyme to produce either a blunt or staggered end (figure 2-A). The vector DNA is then mixed together with the target sequence (the insert ) that has been cut with the same enzyme (2-B). Under the right temperature, salinity and other conditions, the insert enters the bacterial cell and the two pieces of DNA are joined (2-C). In a matter of hours, thousands of copies are made as the bacteria divide. The insert can then be cut out, or excised, from the host DNA using the same restriction enzyme, and then isolated by electrophoresis.

Figure 2 - Graphic by CSS, Inc.

Genetically modified organisms (GMO's) are a derivative of single-sequence cloning as described above. In the case of GMO's, the target gene is transferred to a plant or animal so that they can produce chemicals or proteins that are useful for agricultural production or human medicine. For example, goats and sheep are being engineered to produce bioactive molecules in their milk for medical treatments such as human serum albuman; tomatoes have been developed containing a gene insert to delay ripening and extend shelf life, and researchers are exploring the possibility of producing cancer drugs from modified tobacco plants.

PCR was developed in the mid 1980's and revolutionized the study of genetics because it allows accurate production of millions of copies of a target sequence in a matter of hours. In addition, a single target sequence within a mixture of millions of other sequences can be faithfully amplified (replicated). The method mimics the same process that cells use to copy DNA.

The essential components of the reaction are a pair of short priming sequences (10-20 bases) complementary to the ends of the target sequence, a special DNA polymerase from the bacterium Thermus aquaticus (Taq polymerse), and a solution of the four DNA bases. These reactants are all placed in the same small tube containing a few copies of the target sequence. The remarkable amplification of the target DNA is accomplished in repeated cycles of three steps:

• Step 1: The solution is heated to 95°C to unzip (dissociate) the two chains of the double-stranded target DNA (3-A);
• Step 2: The solution is then cooled to about 55°C to allow the primers to bind (anneal) to the ends of the DNA strands (3-B);
• Step 3: The solution is reheated to about 75°C, the optimal temperature for the Taq polymerase to synthesize complementary copies of each strand. One PCR cycle takes less than two minutes to complete (3-C).

Figure 3 - Graphic by CSS, Inc.

Since each cycle doubles the number of copies of the target sequence, 50 cycles creates hundreds of thousands of copies in just a few hours. Discovery of the Taq polymerase, which lives in the hot springs of Yellowstone National Park and can survive heating to 95°C, was a key to the immense success of PCR because it meant that the enzyme could be added just once to the reaction tube. Other polymerases were destroyed at 95°C, so new enzyme had to be added at each cycle. Eliminating the need to open the tubes between each cycle to add new polymerase reduces the chance of contamination and increases the reliability and repeatability of the procedure.