Molecular Biology and Biotechnology: A Guide for Students, Third Edition

A. DNA Structure and Function

• Chapter 6 : Constructing a Paper DNA Model

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    DNA is the genetic material of every living thing on earth. The information encoded in DNA determines the forms and functions of the cells of which each organism is composed and, ultimately, of the entire organism. Proteins are important because they are the molecules that carry out cellular activities, synthesize nonprotein cellular components, and form many cellular structures. The structure of DNA has to allow it to do two things. First, it has to be able to contain instructions for assembling proteins. Second, it has to be easily and accurately duplicated, so that when a cell divides, it can pass on a correct copy of its genetic information to each daughter cell. The DNA molecule is a double helix, which you can imagine as a ladder that has been twisted into a spiral. Watson and Crick, who discovered the structure of the DNA molecule, used cut-and-paste paper models to help them.

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• Chapter 7 : DNA Replication

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    The accurate copying of DNA is one of the most important jobs an organism must do during its life. For such an important task, cells employ not one but a whole team of enzymes. However, the star of the team is the enzyme DNA polymerase. This protein builds the new daughter strand from nucleotides in the cell and checks the new base pairs for accuracy. The other protein team members help DNA polymerase do its job. All bacteria and higher organisms and many viruses have their own DNA polymerase proteins, which are encoded by their DNA. All of these DNA polymerases work in similar ways and even resemble one another. Scientists, therefore, often study the complicated process of DNA replication in simple systems, such as bacterial and animal viruses, and apply what they have learned as they look at higher organisms. From these simple systems, many general facts about DNA polymerases have emerged.

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• Chapter 8 : From Genes to Proteins

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    There are many types of proteins, and each of these types performs an important kind of job in your body. For example, structural proteins form the “bricks and mortar” of your tissues. Two of them, actin and myosin, enable your muscles to contract. Another structural protein, keratin, is the basic component of hair. Carrier proteins transport important nutrients, hormones, and other critical substances around your body. One of these proteins is hemoglobin, which carries oxygen through your blood to your tissues. Enzymes digest your food, synthesize fats so your body can store energy, and carry out the work of making new cells. They make molecules and perform activities necessary for life. Scientists immediately saw the potential of RNA interference (RNAi) for controlling the expression of genes, just as they had recognized the potential of antisense regulation. RNAi has generally proven very fruitful in laboratory experiments, allowing scientists to mimic the effects of gene mutations by shutting off the expression of specific genes. Scientists can introduce the 21- to 23-base-pair dsRNA molecules into cells directly, or they can introduce an artificial DNA gene with a promoter and a self-complementary coding sequence, like the natural miRNAs. Although there were no RNAi drugs on the market at the time of writing this chapter, potential uses of RNAi to fight AIDS, certain inherited diseases, and cancer are being explored.

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• Chapter 9 : Sizes of the Escherichia coli and Human Genomes

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    DNA is stored in cells in the form of chromosomes and plasmids. One of the wonders of biology is that cells are able to store and access the great lengths of DNA needed to encode their hereditary information. The "average" protein-encoding bacterial gene is considered to be 1,200 bp in length. It is capable of high degrees of folding and coiling, an essential feature for packing it into the cell. The Escherichia coli chromosome is circular and is attached to the cell membrane. The real E. coli chromosome occupies about 10% of the cell volume. The human genome can be related to a length of railroad track. The railroad ties represent the base pairs, and the rails represent the sugar-phosphate backbone of the DNA molecule.

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• Chapter 10 : Extraction of Bacterial DNA

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    This chapter discusses the procedure for extraction of bacterial DNA. The preparation of DNA from any cell type involves the same general steps: (i) breaking open the cell (and the nuclear membrane, if applicable), (ii) removing proteins and other cell debris from the nucleic acid, and (iii) final purification. Cell membranes are made of proteins and fats. Just as detergent dissolves fats in a frying pan, a little detergent dissolves cell membranes. After cell lysis, the next step in DNA preparation usually involves purification by removing proteins from the nucleic acid. Furthermore, phenol and water, like oil and water, do not mix but instead form separate layers. Adding phenol to an aqueous (water-based) DNA-protein mixture, like a cell lysate, and mixing them well, makes the protein to dissolve in the phenol. To remove the protein, the phenol layer is removed. Following the removal of the protein, the DNA is usually subjected to additional purification.

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B. Manipulation and Analysis of DNA

• Chapter 11 : DNA Scissors

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    Genetic engineering is possible because of special enzymes that cut DNA. Restriction enzymes are proteins produced by bacteria to prevent or restrict invasion by foreign DNA. They act as DNA scissors, cutting the foreign DNA into pieces so that it cannot function. EcoRI makes one cut between the G and A in each of the DNA strands. The EcoRI cut sites are not directly across from each other on the DNA molecule. When scientists study a DNA molecule, one of the first things they do is to figure out where many restriction sites are. They then create a restriction map, showing the location of cleavage sites for many different enzymes. These maps are used like road maps to the DNA molecule. A restriction map of a plasmid is discussed in this chapter.

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• Chapter 12 : DNA Goes to the Races

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    In the laboratory, scientists separate DNA fragments by a process called gel electrophoresis so that they can look at the results of restriction digests. Under normal circumstances, the phosphate groups in the backbone of DNA are negatively charged. In electrical society, opposites do attract, so DNA molecules are very much attracted to anything that is positively charged. The electric field makes the DNA molecules move, but to cause them to separate and be easy to look at later on, the whole process is carried out in a gel. Since the plan for agarose gels is usually to add DNA to them, scientists place a device called a comb in the liquid agarose after it has been poured into the desired dish and let the agarose harden around the comb. When the comb is removed from the hardened agarose gel, a row of holes in the gel remains. For electrophoresis, the entire gel is placed in a tank of buffer. An electric current is applied across the tank so that it flows through the salt water and the gel. All of the DNA in the gel migrates through the gel toward the positive pole, but the gel material makes it more difficult for larger DNA molecules to move than smaller ones. After a time, the electric current is turned off and the entire gel is placed into a DNA staining solution. After being stained, the DNA can be seen.

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• Chapter 13 : Restriction Analysis of Lambda DNA

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    This chapter discusses the restriction analysis of lambda DNA. In the chapter, a series of steps are carried out that molecular biologists and biotechnologists use very frequently in their work. Restriction fragments of DNA are separated by electrophoresis through an agarose gel, the fragments are stained so that you can see them, and the products of the digest are analyzed. Predigested DNA samples are used or the digestions are set up and carried out. The process from restriction digestion to analysis of gel patterns is called “restriction analysis.” The procedure has been broken into several parts. The first part is setting up the restriction digests. The remaining steps are of preparing and loading the agarose gels, staining the gels, and analyzing the data. The DNA analyzed is from bacteriophage lambda. Lambda is a virus that infects Escherichia coli and destroys it. Lambda DNA with the enzymes EcoRI, HindIII, and possibly BamHI is cut. These enzymes cut the DNA into a number of different-sized pieces called restriction fragments. The mixture of fragments is loaded into a well in the gel, and then electric current is applied. As DNA is negatively charged, the fragments will migrate toward the positive electrode in the gel box. The shorter the fragment, the faster it can progress through the agarose. The DNA is stained so that the fragments can be seen and results can be analyzed by comparing them to a restriction map of bacteriophage lambda.

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• Chapter 14 : Recombinant Paper Plasmids

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    Some of the most important techniques used in biotechnology today involve making recombinant DNA molecules. Recombinant DNA molecules are pieces of DNA that have been reassembled from pieces taken from more than one source of DNA. Plasmids are copied by the cell’s DNA replication enzymes because they contain a special sequence of DNA bases called an origin of replication. Plasmids often contain genes for resistance to antibiotics. Plasmids carrying genes for ampicillin and kanamycin resistance are assembled and the two plasmids are recombined. The plasmid with ampicillin resistance is called as pAMP, the plasmid with kanamycin resistance as pKAN, and the recombinant plasmid as pAMP/KAN. Scientists place real recombinant plasmids back into bacteria, where they are replicated. The multiplying bacteria, carrying the recombinant plasmid, generate millions of copies of the recombinant DNA molecule and the proteins it encodes. New copies of the plasmid are synthesized by the cell’s DNA replication enzymes and passed to daughter cells as the bacteria multiply.

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• Chapter 15 : Restriction Analysis Challenge

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    Restriction analysis is especially important in checking the structures of recombinant DNA molecules and in analyzing an unknown DNA. This chapter shows some examples with some typical restriction analysis problems encountered by scientists in the laboratory. Observing the sizes of fragments produced, scientists determine restriction site locations that would give them the patterns they see. A linear (not circular) piece of DNA is digested with the restriction endonuclease EcoRI and gives fragments of the following sizes: 3,000, 3,600, and 3,400 bp. When digested with BamHI, the DNA molecule gives fragments of the following sizes: 4,500, 3,000, and 2,500 bp. A double digest with EcoRI and BamHI gives fragments of the following sizes: 2,500, 500, 3,600, 3,000, and 400 bp. The distances between the restriction sites in base pairs are indicated, and the sites EcoRI or BamHI are appropriately labeled.

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• Chapter 16 : Detection of Specific DNA Sequences: Part I. Fishing for DNA

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    Scientists often need to fish for particular pieces of DNA, such as the cystic fibrosis gene. When a scientist fishes for a particular piece of DNA, a special DNA “hook” called a probe is used. One type of analysis done with fluorescent probes is called fluorescent (or fluorescence) in situ hybridization (FISH). A FISH analysis consists of applying fluorescently labeled probes to an intact sample, such as a chromosome, a cell, or a thin slice of tissue, rather than to DNA that has been extracted from the sample. The hybridization analysis learnt in Fishing for DNA can indicate whether a given DNA sequence is present in a sample. In 1975, a scientist named Southern figured out a way to transfer DNA fragments from a gel directly to a membrane, so that the exact pattern of the fragments in the gel was preserved. After the fragments were stuck on the membrane, they could be tested for hybridization to a probe.

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• Chapter 18 : DNA Sequencing: The Terminators

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    Determining the base sequence of a piece of DNA is a critical step in applications of biotechnology. In DNA replication, a nucleotide complementary to the template base is brought into position. The DNA polymerase then adds it to the growing DNA strand by forming a bond between the 5' phosphate group of the new nucleotide and the 3' OH group of the previous nucleotide. The chain terminators used in DNA sequencing are the dideoxynucleotides. The newly synthesized molecules are separated by size and visualized by exposing the gel to photographic film. The A reaction lane shows bands that correspond in length to the site of each T in the template. The G reaction lane shows bands whose lengths correspond to the site of template C’s, and so on. The sequence of the new DNA strand is “read” from the sequencing gel by starting at the bottom (the shortest new molecule) and reading upward. As in PCR, the mixture is put through temperature cycles of denaturation, hybridization, and DNA synthesis. Unlike a PCR, only one primer is present, so DNA synthesis occurs using only one strand of the parental DNA as a template. The AIDS virus encodes a special enzyme, reverse transcriptase, that synthesizes DNA using the viral RNA as a template. HIV infects cells as an RNA genome packaged with the enzymes reverse transcriptase and integrase inside a protein envelope. The herpesviruses are DNA viruses with relatively large genomes. Herpesvirus diseases can now be treated with the chain terminator acyclovir.

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C. Transfer of Genetic Information

• Chapter 19 : Transformation of Escherichia coli

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    Transformation is the uptake and expression of foreign DNA by bacterial cells. Escherichia coli is one of the many bacterial strains that do not undergo transformation naturally. Transformation is thought to be an important means by which streptococci undergo genetic change, and they are undergoing genetic changes that are important to humans—they are becoming increasingly resistant to antibiotics. The cell’s DNA replication enzymes duplicate the plasmid DNA just as they duplicate the regular chromosome, so plasmid DNA molecules are inherited by both daughter cells when the bacterium divides. Scientists use plasmids as convenient vehicles for introducing new genes into cells. It is easy to isolate plasmid DNA. A vector is any DNA molecule used to deliver new genes to cells. Even the most carefully conducted transformations of E. coli are very inefficient. Scientists usually use plasmids that carry marker genes when they do recombinant DNA work. Marker genes are genes that produce an easily detected phenotype, such as resistance to an antibiotic or a color change when exposed to certain conditions.

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• Chapter 20 : Conjugative Transfer of Antibiotic Resistance in Escherichia coli

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    This chapter talks about the procedure for conjugative transfer of antibiotic resistance in Escherichia coli. Public health officials are quite concerned about the rise in antibiotic resistance among disease-causing organisms. For example, Campylobacter is a bacterium that causes stomach cramps, diarrhea, and fever. In 1992, 1.3% of cases of Campylobacter infection studied in Minnesota were caused by bacteria resistant to a fairly new class of antibiotics called quinolones (ciprofloxacin [Cipro] is an example of a quinolone). Virtually every clinically important antibiotic resistance gene is carried on a plasmid. Conjugation allows the spread of plasmids, not only between different individuals of the same bacterial species, but also between species and even between genera. Conjugation has been observed to occur in the soil; on plant surfaces; in lakes, rivers, oceans, sediments, and sewage treatment plants; and inside plants, insects, chickens, mice, and humans. It is believed that conjugation is the most important route of transmission of antibiotic resistance in most disease-causing bacteria. The spread of antibiotic resistance is included at the end of the chapter.

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• Chapter 21 : Transduction of an Antibiotic Resistance Gene

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    Transduction is a natural method of gene transfer that occurs in bacteria. The key player in transduction is a bacterial virus, or bacteriophage (phage for short). There are many different bacteriophages that infect many different bacteria. T4 infects Escherichia coli by attaching to its outer membrane and injecting its DNA into the bacterial cell. Once inside the cell, the phage DNA takes over. The E. coli cell becomes a factory for producing many copies of the T4 genome and for producing large amounts of viral proteins. Some of these proteins help replicate the T4 DNA; others are assembled into new T4 heads and tails. After many copies of the T4 genome have been made and many new heads and tails are floating around in the cytoplasm, still other T4 proteins begin to put together new virus particles. These proteins fill the empty phage heads with T4 DNA and then attach the tails. After many new viruses are assembled, the E. coli cell bursts, releasing the virus progeny. The gene for the botulism toxin is not really a C. botulinum gene! Instead, it is carried on a bacteriophage that infects C. botulinum and is thought to have been transduced from another type of bacterium. Other examples of human diseases in which transduction plays a role are Staphylococcus aureus food poisoning, diphtheria, and cholera.

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• Chapter 22 : Agrobacterium tumefaciens: Nature's Plant Genetic Engineer

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    Agrobacterium tumefaciens infects certain types of plants (most dicots [plants with two seed leaves], but not mono-cots) at wound sites. Once in the wound, the bacterium injects a segment of its plasmid, called tumor inducing (Ti), into the adjacent living plant cells. This piece of DNA, called transferred DNA (T-DNA), is only one region of the plasmid. The tumor cells synthesize new chemicals that provide nourishment that is critical to the bacterium but useless to the plant. In the late 1970s, plant scientists realized they might be able to take advantage of A. tumefaciens' natural genetic engineering abilities. They developed an important method for plant genetic engineering based on this organism and its Ti plasmid. In this method, the T-DNA genes that induce tumor formation and nourish the bacterium are removed from the Ti plasmid and replaced with any gene of interest. The new plasmid is returned to A. tumefaciens, which is grown in culture so that many bacteria carrying the engineered plasmid are produced. The plants to be engineered are then infected with the bacterium carrying the "designer" Ti plasmid. A. tumefaciens injects the engineered T-DNA into the plant. Instead of receiving genes for tumor formation, the plant gets the genes inserted into the Ti plasmid by the scientist.

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D. Molecular Biology and Genetics

• Chapter 23 : An Adventure in Dog Hair, Part I

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    This chapter talks about an adventure in dog hair color. It uses the scientific terminology to describe the pigments and the cells that produce them. Pigmentation in dogs and other mammals is caused by the relative amounts and types of two classes of pigment: eumelanin and phaeomelanin. The eumelanins are the black and brown pigments, while the phaeomelanins are red and yellow. Both eumelanins and phaeomelanins are synthesized in pigment-producing cells called melanocytes. A schematic of the synthesis pathways is shown in the chapter. First, the enzyme tyrosinase converts the amino acid tyrosine into a chemical called dopa-quinone. The enzyme called tyrosinase-related protein 2 (TYRP2) is present, it converts the dopaquinone into a version of eumelanin that has a brown color—Cocoa’s pigment. If the enzyme called tyrosinase-related protein 1 (TYRP1) is present, it converts the brown version of eumelanin into the final, black-colored pigment. It talks about two useful terms: genotype and phenotype.

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• Chapter 24 : An Adventure in Dog Hair, Part II: Yellow Labs

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    This chapter highlights the need for the adventure in dog hair, in order to understand how the yellow color is produced. The pigments are synthesized in cells called melanocytes. The phaeomelanins are the family of red and yellow pigments. The exact color of the phaeomelanin depends on the enzymes available for its synthesis, as is the case with the color of the eumelanin (black or brown). In Labrador retrievers, the phaeomelanin is yellow. Yellow Labs are yellow because the receptor for melanocortin 1 (MC1) does not work, so even though the dogs' bodies produce MC1 and have functional genes for TYRP2 and TYRP1, the signal to produce the enzymes is never transmitted to the melanocyte. The gene that causes yellow coat color in Labrador retrievers is actually a nonfunctional allele of the gene for MC1R. Black and brown Labs have at least one functional allele, so their melanocytes receive the hormone signal to make TYRP2 and TYRP1. In the case of yellow Labs, the expression of the black or brown genotype is altered by the alleles of a completely separate gene. Geneticists call this phenomenon epistasis.

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• Chapter 25 : Human Molecular Genetics

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    Human genetics works the same way as dog genetics in terms of chromosomes, genes, enzymes, and other proteins. There are two basic approaches to learning about human genetics. In the classic approach, geneticists do what can only be described as detective work. First, a condition that seems to be inherited must be recognized. This chapter discusses many of the genes that work in dogs and experimental animals, like mice and fruit flies, because people have bred them for generations and created pure breeds that differ very little in genetic makeup from one individual to another. Proto-oncogenes are normal, essential parts of our genetic material that belong to the group of genes in charge of causing and regulating cell growth and division. It is actually changes in these genes that can lead to the development of cancer. Oncogenes are abnormal forms of proto-oncogenes. The most common oncogene in human cancers is ras. Genetic analysis of families with hereditary cancer and individual cancer patients is helping us identify additional genetic loci associated with cancer development. So far, the genetic picture of most common cancers looks very complicated, and no clear interpretation is available. It is clear that cancer is actually many diseases. It is also clear that different sets of mutations can cause similar cancers.

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• Chapter 26 : Genetics in Action

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    This chapter talks about an article that was originally published in the Smithsonian magazine in February 2006. It tells the story of the work of a medical geneticist, D. Holmes Morton, among the Amish and Mennonites in Pennsylvania. Using his knowledge of medicine, some rare conditions he had studied, and basic genetics, Morton has solved several medical mysteries. The article tells his story, the stories of some of the mysteries he solved, and that of the clinic he founded and describes doing genetics among the Anabaptists of Pennsylvania. The article refers to two great historical figures in medicine: the scientist Louis Pasteur and the humanitarian Albert Schweitzer. About two years after Sara Glick's death, Morton, Strauss, clinic lab director Erik Puffenberger, who holds a doctorate in genetics, and researcher Vicky Carlton from the University of California at San Francisco located the precise genetic site of the bile-salt transporter disorder, and devised a test that could tell doctors whether an infant might have it. But Morton's approach to identifying and treating a disease is more than mere genetics. On being notified of the prize, Morton began to read about Schweitzer and found that the great German physician also came to medicine late, after a distinguished career in music and theology—and that he had established his famed hospital in Gabon at age 38, the same age Morton was when he began the clinic in Strasburg.

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E. Genomics

• Chapter 27 : Comparing Genomes

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    This chapter provides information about how genomes change, what kinds of questions can be addressed by comparing genomes, and general approaches to making those comparisons. The raw material for evolution, or genome change, is mutation. It is useful to think about genome changes in terms of time scales. For the genomes of two closely related individuals to be different, the changes must have occurred very fast. At this time scale, the process of recombination is extremely important. In comparisons of animal genomes, scientists have found that even between two species that are not particularly closely related, such as mice and humans, rearrangement events can be traced by identifying segments of chromosomes in which the order of genes is the same in the two organisms. To illustrate how genome comparisons can be useful, several examples from studies of dogs are rounded up. The most obvious way to compare genomes would be to sequence the genomes in question and use computers to help you understand the differences. However, sequencing the genome of an organism such as the dog is an enormous undertaking requiring years to finish. For genome comparisons, several highly variable regions are characterized, and a DNA “profile” is generated. Loci that are highly variable among different individuals are the ones that are useful for DNA typing, while those that are more constant are more useful for comparing breeds or species. A locus used for DNA comparisons is often called a DNA marker.

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• Chapter 28 : Forensic DNA Typing

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    One of the early goals of researchers was to relate known genetic differences in humans, such as hemoglobin differences related to various anemias, to base sequence changes in DNA. The first DNA-typing techniques were based on the minisatellites and used restriction fragment length polymorphism as the typing method. In criminal cases, DNA typing is used to exclude the possibility that a given suspect left DNA-containing evidence at a crime scene. Some crimes have been solved by analysis of DNA that did not come from humans. DNA testing can also be used to establish paternity or maternity. Since an offspring inherits half its chromosomes from its mother and half from its father, its DNA profile should show contributions from both. To establish paternity, profiles of the child, mother, and putative father are generated. The child’s DNA profile is compared to the mother’s, and the bands that match are subtracted. The remainder of the bands in the child’s profile should match bands in the father’s. DNA testing can also establish whether human remains are male or female. Normally, scientists can make that determination based on measurements of the skeleton (if other evidence is not available), but when the skeleton is badly fragmented (or parts of it are missing), it may not be possible. In humans, gender is determined by the so-called sex chromosome.

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• Chapter 29 : Mapping a Disease Gene

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    This chapter explores how DNA markers can be used to map the locations of genes. Maps showing the positions of genes or other inheritable chromosome features, such as short tandem repeats (STRs), as deduced from genetic experiments, are called genetic maps. Genetic mapping is still used, but now it can be combined with physical analyses of DNA to yield much more detailed and accurate information about genes. Genetic mapping was invented long before researchers had the ability to analyze DNA (and before they knew that genes were made of DNA). It is based on the observation of inheritance patterns. Genetic mapping hinges on the measurement of recombination frequency. One recombination event between the nail-patella syndrome (NPS) and ABO loci in 10 paternal haplotypes was observed. The chapter summarizes the frequencies with which alleles of each pair of loci are inherited together. While some scientists were developing better sequencing methods, others focused on the goal of developing detailed maps. To achieve this goal, they needed better mapping techniques and better methods for cloning large amounts of DNA that could be assembled into overlapping fragments. A new discipline within molecular biology, proteomics, focuses on the structures and interactions of proteins.

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• Chapter 30 : Microarray Analysis of Genome Expression

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    As scientists learned more about DNA and gene function, they developed tools based on their knowledge that let them ask even more questions. The structure of DNA was worked out in the 1950s, and during that decade, enough experimental evidence was accumulated to convince even the skeptics that despite its chemical simplicity, it was indeed the hereditary material. The ability to clone DNA let scientists study individual genes and make probes. Armed with these technologies, they focused on understanding basic processes, such as the control of the expression of individual genes and the replication of DNA. One of the new tools that allow scientists to type many genes at once or to measure the expression of every gene in a cell in a single experiment is the microarray. The major difference with microarrays is that thousands, even hundreds of thousands, of different probes are used in a single experiment. Most researchers buy microarrays from companies that design and manufacture them. One of the first broad applications of genotyping will likely concern genes involved in the metabolism of medicines. Physicians have long known that different patients respond differently to the same medicines. Pharmacogenomics is an area of intense scientific research and commercial interest.

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F. Bioinformatics and Evolutionary Analysis of Proteins

• Chapter 31 : Testing for Amylase Activity

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    All organisms that use starch for energy make enzymes that convert it back into glucose molecules. One of these enzymes is amylase, which breaks the chemical linkages between glucose molecules by inserting a water molecule into the bond, a process known as hydrolysis. Humans secrete amylase in their saliva, so conversion of starch to glucose begins as soon as starch enters the mouth. Besides being important for energy production in plants and animals, amylase is an important industrial enzyme. In fact, one of the most common industrial uses of enzymes today involves amylase. Amylase activity can be easily detected by following the hydrolysis of starch. The presence of starch can be detected with iodine. Because of the bond angles between glucose residues in starch, starch molecules assume a helical form. Iodine molecules nestle inside the coils of the helix, forming a complex that has a dark-blue color.

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• Chapter 32 : Electrophoresis of Amylase Samples

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    This chapter talks about the separation of the proteins in amylase samples, such as saliva, by electrophoresis and the identification of the amylase bands. Electrophoresis of DNA is a little different from electrophoresis of proteins. The differences are due to the fact that proteins are quite different from DNA. DNA has a uniform backbone that is negatively charged at the pH of electrophoresis buffers. Therefore, the major difference between DNA fragments is usually their size. During standard electrophoresis, they all migrate toward the positive pole at rates that depend on their sizes. The net effect is that the proteins all migrate toward the positive pole during electrophoresis (as do DNA molecules). After electrophoresis, the gel is cut in half so that two halves are obtained, each with a complete sample set. After electrophoresis, half the gel is stained for total protein, and the other half is assayed for amylase activity. The gel is removed from the electrophoresis chamber, and cut in half vertically with a razor blade, ruler edge, or scissor blade. Many of the complex samples, such as bean extract or human saliva, show multiple bands. To determine which of these bands is amylase, the unstained half of the gel for areas of visible clearing is carefully examined.

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• Chapter 33 : Constructing an Amylase Evolutionary Tree

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    The theory of evolution holds that existing life forms evolved from earlier life forms through genetic change and natural selection. For a genetic change to affect the form or function of an organism, it has to affect a protein: the shape and function of the protein, the amount of it present, the cells in which it is expressed, the stage in development at which it is expressed, and so on. Thus, proteins are a very reasonable level at which to consider evolution. In general, protein evolution tracks organismal evolution. Protein evolution is not always a simple matter of an enzyme accumulating amino acid differences. This chapter states a simple example using three organisms and an imaginary protein called evolutionase. The evolutionase amino acid sequences from organisms 1 and 2 differ by 30 amino acids. The evolutionase sequences from organisms 2 and 3 differ by 29 amino acids. The evolutionase sequences from organisms 1 and 3 differ by 13 amino acids. In the activity stated in this chapter, an amylase evolutionary tree is constructed.

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• Chapter 34 : Exploring Bioinformatics

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    The availability of molecular information and relatively easy means of analyzing it have led to the coining of a new word: bioinformatics. The activities in this chapter provide the user with an introduction to it. This chapter is meant to open a door for the user, and talks about performing a basic local alignment search tool (BLAST) search to identify amylase source organisms. In this activity, the user uses the search tool BLAST to identify the source organisms of the amylase sequences. The databases are constantly growing through the addition of new information. The BLAST searches described in this chapter uses only portions of the amylase sequences. The chapter talks about exploring protein bioinformatics resources in more depth, using human pancreatic amylase as the example protein. It also talks about exploring online biomedical literature references and its procedure.

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Introduction to Societal Issues

• Chapter 35 : Facing the Forces: Science, Technology, and Society

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    People use all technologies for the same purpose: to change the environment so that the natural world suits us better. Technologies not only alter the environment; they change people. Beneficial technologies that meet legitimate societal needs can also have negative societal impacts, some of which are also unpredictable. The concept of technology encompasses the practices and products humans develop to modify and control nature for sustenance and comfort. As science-based understanding of the natural world broadened and deepened, science and technology began to converge, and progress in one began to drive advances in the other. Some people argue that the economic and social benefits that science-based technology development has provided to some of us are not worth the costs, particularly to the environment, paid by all of us. Not only do science and technology affect society, but societies also affect science and technology development. Knowledge really is power, so understanding something about two of the primary forces that make today's world go round-science and technology makes people less vulnerable to manipulation by others. Finally, and probably most surprising to those who think science has all of the answers, one has to be comfortable with ambiguity, uncertainty, and being wrong-a lot. Dismissing decisions about science and technology as someone else's concern makes it easy for a handful of people to control the most powerful forces shaping the modern world.

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• Chapter 36 : Analyzing the Issues of Biotechnology: Cloning

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    Biotechnology began generating public discussion and debate about issues raised by its possible applications at the earliest stages of research, 10 years before any products had been developed. The debate about the potential societal impacts of modern biotechnology has continued unabated throughout its development. This chapter provides an approach and process for analyzing biotechnology-related issues so that one can contribute constructively to the public discourse on biotechnology and make informed decisions that are in ones best interest. The chapter states some critical steps in objectively evaluating societal issues associated with applications of biotechnology. It discusses the formal risk assessment process in detail. Finally, in discussing the risks of any technology, one has to keep in mind that technologies may be used wisely or unwisely, for good or for bad. The issue of animal cloning presents an opportunity to use the strategy to analyze an emotionally charged societal issue raised by a scientific breakthrough related to biotechnology development. The discussion is not meant to be a comprehensive treatment of the issues involved with animal cloning but is included to give one an example of some of the requisite steps in a rational treatment of the issue. If one is interested in extending this discussion to the question of cloning humans, it is essential to make a clear distinction between cloning cells taken from an embryo that is only a few days old and cloning to make babies (reproductive cloning).

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• Chapter 37 : The Risks of Technology: Perception, Reality, or Both?

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    Social scientists have identified a number of psychological elements that skew perceptions of risk, causing people to inflate small risks or trivialize significant risks. Fact-based risk analysis provides much needed perspective on questions of real and perceived risks. The first step in a risk assessment is identifying the hazard, which is anything that could go wrong or might lead to injury or harm. Effective safeguards can minimize the hazard level, the degree of exposure, or both. Assessing risks is a simple task compared with assessing benefits. Benefits are often very difficult to identify in the abstract. The uncertainty of exposure is one of the reasons risks are discussed in terms of probabilities instead of an absolute assessment of the seriousness of the hazard. Risk management is the set of activities that can be undertaken to control a hazard, minimize exposure, or both. This chapter presents two conceptual formulas that illustrate the first steps in using rational risk analysis to assess and manage risks. It talks about the Bacillus thuringiensis (Bt) corn ban and the Cornell study to illustrate the value of rational risk analysis. The chapter also presents student activity for assessing risks and benefits as well as regulatory decision making.

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• Chapter 38 : Making Decisions When There Is No Right Answer

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    Bioethics, or biomedical ethics, has become an immensely important topic. Few major hospitals are without an ethics committee to assist patients or health care providers when they need help. Colleges offer courses of study in medical humanities or bioethics. High school students, too, must be aware of the questions bioethicists study. Paralleling the medical developments described were striking advances in the scientific understanding of cellular and molecular biology. Knowledge of molecular genetics has only recently been applied to specific diseases or patients, but the ethical questions that plague society about any medical technology apply to genetic technologies and other applications of medical biotechnology. To make ethical decisions, society must agree on some basic guidelines about what constitutes moral conduct. In the field of biomedical ethics, certain guides are well established. This chapter presents specific case studies to analyze and a decision-making model to be used in these analyses. Part of the decision-making process is to gather any additional background facts needed to evaluate the situation and to find out what sort of ethical standards that apply to a particular case have been established. The chapter provides a decision as to the best course of action and justifies it in terms of basic ethical principles. The case studies provided raise bioethical dilemmas. Ethical dilemmas revolve around trying to find the best solution when no solution is completely good. A dilemma exists when no choice is ideal but all options have benefits and risks that must be carefully assessed.

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• Chapter 39 : Analyzing Ethical Dilemmas in Genetic Testing

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    Genetic testing is usually thought of in the context of determining whether someone has a genetic disorder or is a carrier of a recessive disease gene, such as a gene mutation that leads to cystic fibrosis (CF). As society enters the new age of molecular genetics, it will be possible to test for more and more of the 3,000 to 4,000 genetic disorders physicians have identified in the past century. This chapter begins with background information and moves on to discussing the societal impacts of genetic testing. It presents two case studies, one of a 6-year-old girl whose mother carried the CF gene. The other study is of a 32-year-old lady whose mother was diagnosed with Huntington’s disease (HD). Questions and assignments are provided for students in the chapter. A section is devoted to further information on genetic testing, which includes government policies and scientific research. It discusses the role, if any, of politicians in determining which paths of scientific inquiry are pursued and therefore which medical breakthroughs are most likely to occur. The focus is on embryonic stem (ES) cell research to describe some of the ways government bodies influence, which scientific questions are pursued and, therefore, which potential technological applications become commercial realities.

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• Chapter 40 : Analyzing Ethical Dilemmas in Gene Therapy

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    In the first approved human gene therapy experiment, W. French Anderson attempted to use genes to treat the genetic disease called severe combined immunodeficiency disease. The need for a standard review procedure for proposed human gene therapy experiments was made clear by an incident in 1979-1980, when a University of California-Los Angeles researcher tried a human gene therapy experiment on two patients without getting approval from the appropriate review committee at his institution. Leroy Walters, of Georgetown University's Kennedy Institute of Bioethics, divided gene therapy into four possible categories. The categories are somatic-cell gene therapy for the cure or prevention of disease, germ line gene therapy for the cure or prevention of disease, somatic-cell enhancement, and germ line enhancement. One of the ongoing issues concerning gene therapy is whether any or all of the four types of manipulation are ethically acceptable. At present, only somatic-cell gene therapy for the cure or prevention of serious disease is considered ethically appropriate, even by researchers like Anderson. In considering any application of gene therapy, basic respect for human dignity is, as always, the underlying moral principle. Like any other experimental medical treatment, gene therapy should be used to benefit the patient. Three case studies have been discussed at the end of this chapter.

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