Figure 1. Cleavage of double-stranded DNA by the restriction endonuclease EcoR1. It cuts one strand between G–A (direction 5′ to 3′) and the other strand, shifted by four bases, between A–G (direction 5′ to 3′). Sticky ends are produced that “find each other” under appropriate conditions. (Diagram: Anne Kemmling, Goettingen, Germany.)

Figure 2. Cloning of a gene. DNA with the desired gene as well as a plasmid are cut with EcoR1. The fragment with the desired gene and the “opened”, linearized plasmid, are incubated together under conditions under which the two fragments associate because of their sticky ends. The two gaps between the fragments are closed by addition of the enzyme ligase. The resulting plasmid containing the desired gene is introduced into a bacterial host by transformation. With proliferation of the bacteria, the plasmid is also proliferated. The desired gene can then be isolated from the cells. Under certain conditions, the gene can be expressed so that the encoded protein is produced. (Diagram: Anne Kemmling, Goettingen, Germany, on the basis of Figure 2 in Das Spiel by M. Eigen and R. Winkler, Piper Publishers, Munich and Zurich, 1981.)

Figure 3. Demonstration of a PCR product by agarose gel electrophoresis. Left and right lane (lanes 1 and 7): the so-called DNA ladder, DNA fragments from 10 000 base pairs down to several hundred base pairs; lane 2, chromosomal DNA that hardly migrates; lane 3 to 6, the PCR products after 17, 20, 23, and 25 cycles, respectively. A 3500 base-pair-long region on the chromosome was amplified using specific primers. Separation (melting) of the two chromosomal strands was achieved by incubation at 96 °C for two minutes. After cooling to 56 °C, primers bind and DNA synthesis takes place. After 4 minutes the sample is heated again, then recooled, and so on. Separation of the DNA fragments in agarose gel was done at 85 V for 90 minutes. Arrows indicate the application level of the samples at the beginning of the run. (Experiment: Frauke-Dorothee Meyer, Goettingen, Germany.)

Figure 4. Principle of PCR. Cycle 1: melting of the double-stranded DNA is followed by primer binding and DNA synthesis; cycle 2: after melting and primer binding, DNA is synthesized. After cycle 3, two double-stranded products have the correct length, and eight after the fourth cycle. After 20 or so cycles, the fragments with an overhang are practically negligible. (Diagram: Anne Kemmling, Goettingen, Germany.)