第17章バイオテクノロジーとゲノミクス

Biotechnology is the use of biological agents for technological advancement. Biotechnology was used for breeding livestock and crops long before people understood the scientific basis of these techniques. Since the discovery of the structure of DNA in 1953, the biotechnology field has grown rapidly through both academic research and private companies. The primary applications of this technology are in medicine (vaccine and antibiotic production) and agriculture (crop genetic modification in order to increase yields). Biotechnology also has many industrial applications, such as fermentation, treating oil spills, and producing biofuels (Figure 17.2).

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. Exposure to high temperatures (DNA denaturation) can separate the two strands and cooling can reanneal them. The DNA polymerase enzyme can replicate the DNA. Unlike DNA, which is located in the eukaryotic cells' nucleus, RNA molecules leave the nucleus. The most common type of RNA that researchers analyze is the messenger RNA (mRNA) because it represents the protein-coding genes that are actively expressed. However, RNA molecules present some other challenges to analysis, as they are often less stable than DNA.

To study or manipulate nucleic acids, one must first isolate or extract the DNA or RNA from the cells. Researchers use various techniques to extract different types of DNA (Figure 17.3). Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as unwanted molecule degradation and separation from the DNA sample). A lysis buffer (a solution which is mostly a detergent) breaks cells. Note that lysis means "to split". These enzymes break apart lipid molecules in the cell membranes and nuclear membranes. Enzymes such as proteases and ribonucleases (RNases) break down proteins and RNA, respectively. Using alcohol precipitates the DNA. Human genomic DNA is usually visible as a gelatinous, white mass. One can store the DNA samples frozen at –80°C for several years.

Scientists perform RNA analysis to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves using various buffers and enzymes to inactivate macromolecules and preserve the RNA.

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, an electric field can mobilize them. Gel electrophoresis is a technique that scientists use to separate molecules on the basis of size, using this charge. One can separate the nucleic acids as whole chromosomes or fragments. The nucleic acids load into a slot near the semisolid, porous gel matrix's negative electrode, and pulled toward the positive electrode at the gel's opposite end. Smaller molecules move through the gel's pores faster than larger molecules. This difference in the migration rate separates the fragments on the basis of size. There are molecular weight standard samples that researchers can run alongside the molecules to provide a size comparison. We can observe nucleic acids in a gel matrix using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the gel's top (the negative electrode end) on the basis of their size (Figure 17.4). A mixture of genomic DNA fragments of varying sizes appear as a long smear; whereas, uncut genomic DNA is usually too large to run through the gel and forms a single large band at the gel's top.