The genetic material of any organism is the substance that carries the information that determines its life cycle and its characteristics. There is a procedure by which this genetic material is used in living processes; this is the central dogma of genetics. Before the development of modern genetics, it was commonly believed that the substance responsible for heredity was a protein. Once DNA was recognized as the genetic material, the central dogma was established. This states that the information contained in DNA is translated into protein through the processes of transcription and translation. The protein is then used in all life processes, from cell division to electron transport in photosynthesis. For this to occur, DNA is copied (transcribed) into mRNA, and the mRNA is used as template for production of the protein in a process called translation. The message coded by the mRNA sequence gene is translated into a sequence of amino acids, the basic components of protein. Cells cannot produce a protein by simply aligning amino acids; they need to use an RNA template. Additionally, the use of an intermediate mRNA template in protein synthesis reduces the risk of damage to the DNA that can occur from repeated use. Additionally, the central dogma postulates that the intermediate mRNA molecule, a direct copy of DNA, can be used repeatedly in protein synthesis.

Therefore the main points of the central dogma are as follows:

DNA is a sequence of nucleotides that code for genes. A gene is the smallest physical and functional unit of heredity, and it codes for a specific biological structure or function. Exons are parts of the DNA within a gene that form the RNA transcript. The outline in Figure 2-2 illustrates a typical gene of eukaryotic organisms: animals and plants, showing the promoter, a DNA sequence preceding a gene that contains regulatory sequences controlling the rate of RNA transcription. Promoters control when and in which cells a given gene will be expressed. To the right of the promoter is the coding sequence with the exons and introns, which are transcribed; in other words, they are copied into an mRNA molecule during the expression of the gene. The introns fill in the spaces between the exons in the gene locus. The introns are transcribed, but are spliced out during mRNA processing, just before translation (production of the protein). The exons contain the coding sequence for protein to be synthesized.

Figure 2-2. Simplified structure of a typical gene from eukaryotic organisms.

Although molecular genetics has generated much more detailed information about the sequence and function of each part of the gene locus, the understanding that genes possess several other parts besides the coding region is enough for the scope of this book.

Viruses are microorganisms in the gray area of what is living and nonliving. Viruses are made of a protein envelope, which surrounds the genetic material (DNA or RNA). Although viruses have their own genetic material like many other living organisms, they do not possess the capacity to reproduce by themselves. For that, they need to use the machinery of living cells to produce a new virus.

The viruses that live in bacteria are called bacteriophage. They inject their DNA into bacterium, leaving their protein envelope outside. Inside of the bacterium, the virus is a filament of nucleic acid that contains coded information for the synthesis of new virus particles, which can be released with the bacteria lyses. The genetic engineering came about from the observation of how viruses use cells of other organisms or bacteria to express their own genes. In that sense, the viruses can be considered genetic engineers. One of the first experiments of genetic engineering was carried out using a bacteriophage as a true Trojan horse, to introduce DNA from other organisms into the bacterium.

One of the requirements for genetic engineering experiments is the production of DNA fragments that contain the desired information. At the beginning of the molecular biology era, DNA used to be cleaved with vibration by ultrasound waves. One of the difficulties that scientists faced in those experiments was the random fashion in which the DNA fragmented. In 1970, however, Dr. W. Arber discovered that bacteria themselves possess a mechanism to specifically cut DNA at certain sequences. Bacteria produce proteins called restriction enzymes that cleave the DNA at specific recognition sites. It was only after the discovery of the restriction enzymes that genetic engineering became a reality. The restriction enzymes were developed as a defense mechanism of bacteria against viruses. Viruses inject DNA into bacteria and use their bacteria as a mechanism for reproduction. The bacteria, however, develop a mechanism that fragments the exogenous DNA using restriction enzymes. The restriction enzymes recognize the foreign DNA by means of certain specific nucleotide sequences. Different enzymes recognize and cut the DNA at different sites. Using this knowledge, restriction enzymes became essential tools for the genetic engineer to cut DNA into fragments and build new genes. Hundreds of different restriction enzymes exist, many of which are frequently used in biotechnology. Table 2-1 shows some restriction enzymes and their recognition sites for cleavage.

Before beginning any genetic engineering project, it is necessary to obtain a reasonable amount of relatively pure DNA, which is then cut and ligated to build the new gene. Today, several companies make DNA extraction and purification kits, making the genetic engineering process much simpler. The basic procedure requires the releasing of the DNA from the cells and purification of the DNA to be used in the experiments.

In a typical extraction and purification procedure for plasmid DNA, bacterial cells with the desired plasmid are lysed (broken up) under alkaline conditions and the crude lysate (remains of the cells) is purified using either filters or centrifugation. The lysate is then loaded onto an apparatus where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, then plasmid DNA is released in high-salt buffer. The DNA can then be concentrated and desalted for genetic engineering uses.

General Steps

1. Grow the bacteria in liquid culture.

2. Centrifuge the bacterial suspension to concentrate the bacteria.

3. Discard the supernatant (the liquid part that remains above the pellet).

4. Resuspend the bacteria pellet in a solution with RNAse (enzyme that degrades RNA).

5. Add a buffer to promote an alkaline lyse of the bacteria.

6. Neutralize and adjust the saline conditions of the suspension with the buffer.

7. Centrifuge to separate proteins and other impurities.

8. Adsorb the plasmid DNA onto a membrane by filtration.

9. Rinse the membrane with a solution containing ethanol.

10. Elute plasmid DNA from the membrane with EDTA, a chemical substance that preserves the integrity of DNA.

Figure 2-3 displays the latter steps of this procedure.

Figure 2-3. Purification of plasmid DNA after the precipitation of the bacteria.

Once the DNA has been obtained, it is necessary to cut the DNA into pieces to be used for the engineered gene. Restriction enzymes are used for cutting the DNA at specific sites. As seen in Table 2-1, most restriction enzymes cut the DNA into diametric fragments, as opposed to symmetric fragments. That cut leaves the DNA double helix with a small sequence of nonpairing bases that overhang on the end. These regions of DNA are generally used for ligation, or joining with other DNA fragments. DNA fragments cleaved with a single restriction enzyme or with complementary enzymes can be ligated to each other because the overhanging regions are complementary and will bind together. The ligation of fragments is facilitated with addition of the enzyme DNA ligases. The true art of genetic engineering is putting together the parts of puzzle, where each DNA fragment must be placed in right order and orientation so the gene is functional. As scientists know the sequences of genes encoding important traits or proteins, the information is used to engineer genes that can be used in a variety of applications.

Genetic engineers are able to manipulate tiny pieces of DNA with enzymes to create new genes and DNA sequences used in biotechnology. Relatively simple tools in a small laboratory are needed for these engineers to practice their craft. The products that result from these methods can then be used in many applications of biotechnology.