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

A. DNA Structure and Function

• Chapter 6 : DNA Structure

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    This chapter discusses the classroom activity regarding the visualization of the DNA structure. Having students assemble a model of DNA is probably the most useful way of communicating information about DNA structure to the large population of students who learn by doing. This has been successfully used in classroom settings ranging from junior high school/middle school to college. Advanced procedures, such as DNA sequencing and the polymerase chain reaction, depend on these differences, and students need to understand these aspects of DNA structure before they can understand the procedures. In a DNA molecule, two complementary DNA polymers are connected by the hydrogen bonds between the base pairs. The sugar-phosphate backbones of the polymers are oriented in opposite directions: one is 5' to 3', and the other is 3' to 5'. The two polymers (usually called strands) are twisted around each other to form the famous double helix. The double-helix structure of DNA is ideally suited to the cellular environment of the molecule. Hydrogen bonds "edges" of the base pairs are "visible" to cellular proteins between the spiraling strands of the sugar-phosphate backbone. DNA replication can be taught with this model by having students make extra nucleotides, simulate unwinding of the helix, and build new strands.

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• Chapter 7 : DNA Replication

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    DNA replication is a topic usually presented in 9th-grade biology. The essential fact of DNA replication is that the base-pairing rules make it very easy to generate two identical new helices from one helix. The first part is appropriate for young students; more advanced students will perform both parts of the lesson. The first activity described in this chapter is a simple (and necessarily inaccurate) paper simulation of DNA replication. The second activity is a student reading about DNA polymerase, the central DNA replication enzyme. The background information in the introduction that follows contains far more detail about DNA replication. The two aspects of DNA synthesis that your advanced students need to know are that synthesis is unidirectional and that it absolutely requires a primer. Not surprisingly, the characteristics of DNA polymerase determine the overall features of DNA replication inside the cell and in the test tube. The feature of DNA replication means that DNA synthesis is unidirectional, from 5' to 3'. Uni-directionality presents a problem for chromosome replication that is discussed in this chapter. Chromosomal DNA replication is usually initiated at specific sites along the DNA called replication origins. Instead, several different strategies circumvent the problem created by the specificity of DNA replication enzymes.

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• Chapter 8 : Expression of Genetic Information

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    This chapter presents lessons where students act out transcription and translation, either at their desks with paper models or by assuming roles and moving around the room. This activity also makes a good lead-in to a discussion of the various means of gene regulation. The chapter also includes an advanced Student Activity about RNA interference (RNAi) and antisense RNA. Background for it follows the material on gene regulation. DNA determines the characteristics of an organism by specifying the amino acid sequences (and therefore the structures and functions) of its proteins. In order to direct the synthesis of a protein, the DNA must contain not only codons for each of the amino acids in that protein, but also regulatory sequences that tell the cell’s proteinsynthesizing machinery where to start and stop. Although prokaryotes and eukaryotes use the same genetic code, they have evolved different regulatory signals. The genetic traffic signals involved in converting DNA sequences into proteins (in prokaryotes) are summarized. It appears that there are at least three mechanisms: one for transfer RNA (tRNA), one for mRNA, and one for rRNA. Most of the discussion is related to a form of the activity in which students act out transcription and translation by moving around the classroom. To move the coding information to the ribosome for translation, the cell makes an mRNA copy. The chapter also presents answers to student questions and other ideas for teaching transcription and translation.

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• Chapter 9 : Modeling Genome Size

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    The activity described in this chapter uses models to show the relative sizes of an Escherichia coli cell, the E. coli chromosome, a typical plasmid, and a gene. The models demonstrate graphically how much longer the E. coli chromosome is than the cell. Calculations included in the student questions use the analogies of letters in a book and miles of railroad track to suggest the size of the human genome. DNA is stored in cells in the form of chromosomes and plasmids. The amount of DNA required to store the information necessary for making even a simple organism, such as a bacterial cell, is very large. 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. It is capable of high degrees of folding and coiling, an essential feature for packing it into the cell. Although the degree of folding required to fit the DNA of E. coli into the bacterium is impressive, the folding necessary for packaging DNA into a human cell is even more remarkable. The DNA models consist of appropriate lengths of thread, yarn, or string. At the end of the activity students will able to list some differences between the E. coli and human genomes.

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• Chapter 10 : DNA Extraction

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    In the activity described in this chapter, students can see a mass of stringy DNA fibers precipitate from bacterial cells or, alternatively, yeast, plant, or animal tissue. The bacterial-DNA extraction is very easy to perform. The procedure provided in this chapter is for home-grown cells; the kits of materials available from scientific supply houses come with their own instructions. Additional procedures for extraction of DNA from yeast, plant, and animal tissue are also provided in this chapter. 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. There are several different ways of accomplishing each of these steps, and the method chosen generally depends on how pure the final DNA sample must be and the relative convenience of available options. In the activity described, no attempt is made to purify the DNA, since all that is required is to see it. Students will lyse Escherichia coli with detergent and layer a small amount of alcohol on top of the cell lysate. DNA is insoluble in either alcohol and will form a white, weblike mass (precipitate) at the interface of the alcohol and water layers. This DNA is very impure; the mass contains cellular proteins and other debris, but the stringy fibers are DNA. This easy and fun procedure lets students see DNA with their own eyes and shows its fibrous nature.

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B. Manipulation and Analysis of DNA

• Chapter 11 : Restriction Enzymes

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    Students are introduced to restriction enzymes and simulate the activity of restriction enzymes with scissors. They are also introduced to restriction maps and asked to make simple predictions based on a map. Restriction enzymes were originally discovered through their ability to break down foreign DNA. Restriction enzymes can distinguish between the DNA normally present in the cell and foreign DNA, such as infecting bacteriophage DNA. They defend the cell from invasion by cutting foreign DNA into pieces and thereby rendering it nonfunctional. Restriction enzymes appear to be made exclusively by prokaryotes. The action of restriction enzymes is introduced and modeled in the activity DNA Scissors. The idea of rejoining restriction fragments and the need for complementarity in the single-stranded "tails" are introduced in the activity DNA Scissors. Restriction enzymes and DNA ligase play starring roles in DNA cloning. The discovery of restriction enzymes gave scientists a way to cut DNA into defined pieces. Every time a given piece of DNA was cut with a given enzyme, the same fragments were produced. These defined pieces could be put back together in new ways. A new phrase was coined to describe a DNA molecule that had been assembled from different starting molecules: recombinant DNA. After restriction digestion, the fragments of DNA are often separated by gel electrophoresis. The chapter also presents answers to exercise questions.

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• Chapter 12 : Gel Electrophoresis

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    DNA Goes to the Races is a reading and paper activity that introduces electrophoresis. Students should already be familiar with the activity of restriction enzymes through the activity DNA Scissors. This lesson provides enough background for students to continue with activities (hybridization analysis, DNA sequencing, DNA fingerprinting) that require them to know something about the process. It is also an excellent introduction if you plan to conduct electrophoresis in your classroom. The activity is self-explanatory. Students can read the handout and do the exercises. The students should be shown as much real material (or pictures) as one can: gel boxes, power supplies, an old gel, or a photograph of one. Relate the items to their activity and reading.

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• Chapter 13 : Restriction Analysis

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    In the activity described in this chapter, students perform electrophoresis using precut samples of lambda DNA, or they first carry out the restriction digests themselves and then perform electrophoresis with their samples. DNA Scissors include using restriction maps to predict the sizes of DNA fragments after digestion. The restriction map of bacteriophage lambda should be shown to them; so that they can predict the sizes of the fragments they will see in their gels. The standard method for separating DNA fragments is electrophoresis through agarose gels. Agarose is a polysaccharide, like agar or pectin that dissolves in boiling water and then gels as it cools. In the introductory material, this chapter provides information about different methods of staining DNA and recording data. The procedure outlined in the student activity was designed for the safest DNA stains, which are also the least sensitive. Methylene blue is an alternative stain for DNA gels recommended for classroom use by the National Association of Biology Teachers. The other gel material used in electrophoresis of DNA is polyacrylamide. Polyacrylamide forms a tighter mesh than does agarose, so polyacrylamide gels can separate smaller molecules. Proteins are normally separated by polyacrylamide gel electrophoresis, because proteins are much smaller molecules than the DNA fragments commonly separated on agarose. Protein electrophoresis offers several advantages over DNA electrophoresis. Students typically want to analyze DNA from familiar organisms, often seeking to identify organisms from restriction fragment patterns.

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• Chapter 14 : Recombinant DNA

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    The student activity recombinant paper plasmids introduces some basic recombinant DNA techniques to students. Paper models are used to demonstrate how plasmid DNA is cut with restriction enzymes and recombined to create a recombinant DNA molecule. “Recombinant” DNA is simply a DNA molecule that has been assembled from pieces taken from more than one source of DNA. Making recombinant DNA became possible with the discovery of restriction enzymes and DNA ligase. Restriction enzymes are used to cut DNA into reproducible fragments, and ligase is used to form new phosphodiester bonds between them. When phosphodiester bonds are formed between DNA pieces from different sources, a recombinant DNA molecule is created. The most important application of recombinant DNA technology is gene cloning. Plasmids are (relatively) small circular DNA molecules found in many bacteria and some yeasts. After transformation by plasmids containing antibiotic resistance genes, bacteria that acquired the plasmid (transformants) can be detected by plating the bacteria on media containing the antibiotic(s). To determine whether a given transformant contains the desired recombinant plasmid, the scientist often must prepare plasmid DNA from that transformant. In this activity chapter, students assemble plasmids carrying genes for ampicillin and kanamycin resistance.

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• Chapter 15 : Restriction Analysis Challenge Worksheets

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    The restriction analysis challenge worksheets contain three problems that illustrate actual uses of restriction analysis in the laboratory. The problems, particularly the second and third, require multistep analysis. They are most appropriate for advanced classes or for students who enjoy analytical puzzles. Students must be familiar with restriction enzymes, construction of recombinant DNA molecules with DNA ligase, and gel electrophoresis. The required background is found in the activities DNA Scissors, Recombinant Paper Plasmids, and DNA Goes to the Races. These worksheets require students to apply their knowledge of the construction of recombinant DNA molecules and restriction analysis to three problems like those encountered every day in research laboratories. The first two problems involve analysis of the molecules produced during a ligation, and the third requires students to generate a restriction map from restriction analysis data. In challenge I, the described ligation will produce the regenerated vector (where the ligase has simply resealed the EcoRI site in the plasmid vector), as well as the desired recombinant molecule. Challenge II is an amplification of the ideas introduced in challenge I. Here, the vector is ligated in the presence of two different inserts: the one that is to be removed and the one that is desired. Challenge III should stand on its own. There is no magic method to solve the third problem; students simply have to work with it. The chapter also provides details on restriction mapping laboratory activities, and answers to challenges.

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• Chapter 16 : Detection of Specific DNA Sequences: Hybridization Analysis

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    The activities in this lesson plan use paper models to illustrate basic concepts of hybridization analysis. Hybridization is a technique that takes advantage of the specificity of DNA base pairing for the detection of specific DNA sequences in a mixed sample. It is one of the fundamental methods for analysis of DNA. The first activity (Fishing for DNA) requires students to be familiar with the structure of DNA and the base-pairing rules. The second and third activities assume that students are familiar with restriction enzymes and the process of electrophoresis. If one plans to use the subsequent lessons on DNA sequencing, the polymerase chain reaction, or the analysis of human DNA, it is important to introduce one`s class to hybridization. Restriction digestion, electrophoresis, and staining allow scientists to cut DNA molecules into reproducible fragments and to look at the sizes of those fragments. In brief, hybridization analysis involves separating the strands of (denaturing) the DNA molecules to be analyzed and then mixing those separated strands with many copies of a single-stranded DNA or RNA molecule. This single-stranded DNA or RNA molecule has the complement of the base sequence of interest and is labeled for detection (often with radioactive isotopes). It is called a probe.

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• Chapter 17 : The Polymerase Chain Reaction

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    The polymerase chain reaction (PCR) has become a key tool in molecular biology research and in biotechnology applications. In the student activity, students use paper models to simulate the steps of the PCR. The model exercise demonstrates how DNA polymerase can be used to make multiple copies of a specific DNA fragment and shows how the technique can be used to detect a specific DNA molecule (such as the chromosome of a disease-causing microorganism) in a sample. Automated DNA sequencing is also based on PCR technology. PCR is a simple technique that combines in vitro DNA synthesis by DNA polymerase and hybridization. To start the reaction, the double-stranded sample DNA in the mixture is denatured into single strands by heating it. The mixture is then cooled so that hybridization of complementary strands can occur. In the next step, the DNA polymerase enzyme synthesizes a second DNA strand on each of the two original strands, using the free nucleotides in the solution. The annealed primer serves as a starting point. New DNA is synthesized from the 3' end of each primer, extending in only one direction. In early PCR methods, new DNA polymerase had to be added after each denaturation step, because the high heat necessary for denaturation destroyed the enzyme. This paper model very accurately demonstrates the steps of PCR and shows how a specific DNA segment can be amplified from a single copy. The second part of the activity illustrates how PCR is used as a diagnostic tool.

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• Chapter 18 : DNA Sequencing

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    In this lesson, paper-and-paper clip simulations are used to illustrate the most common methods of DNA sequencing. Two approaches are shown, both based on the same biochemistry. The difference is that one employs a heat-resistant DNA polymerase in a modification of the polymerase chain reaction (PCR) (“cycle sequencing”), while the other uses regular DNA polymerase in a single round of reactions (“original approach”). The authors have added the cycle-sequencing simulation because it is currently the most widely employed sequencing method. The methods most commonly used for sequencing DNA on a small scale employ compounds called chain terminators, chemicals that specifically stop the elongation of a new DNA strand by DNA polymerase. Several chain terminators are also used as antiviral drugs. The reading at the end of this chapter explains how terminators work to fight human immunodeficiency virus and herpesvirus. Cycle sequencing uses the dideoxy method of sequence determination. Here, the DNA polymerase is the Taq polymerase used in PCR, and the sequencing reactions are based on thermal cycles, as is PCR. A major difference between cycle sequencing and PCR is that only one primer is present in cycle sequencing instead of the pair used for DNA amplification. In the 1980s and early 1990s, DNA sequencing was usually performed by individual researchers in their own laboratories.

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C. Transfer of Genetic Information

• Chapter 19 : Reading: Gene Transfer, Escherichia coli, and Disease

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    Transformation occurs when cells take up free DNA molecules from the environment and express encoded information. This phenomenon is of great importance to experimental molecular biology, because it provides a means of inserting new genes into cells. Bacterial cells in a state that allows them to be transformed are said to be competent. Internal proteins then compare the base sequence of the new DNA to the genome of the organism. Natural transformation is believed to be an important mechanism of genetic exchange for a number of species important to humans, notably, Streptococcus pneumoniae, a causative agent of pneumonia. Cells that are not naturally competent can often be artificially induced to take up DNA. In most cases, new linear DNA fragments are destroyed before they even have a chance to recombine into the Escherichia coli genome. Plasmids seem to be extra pieces of DNA; they do not contain any genes that are essential to the life of the organism. The ampicillin resistance gene encodes an enzyme called beta-lactamase that breaks down the ampicillin molecule, allowing cells to multiply in ampicillin-containing media. The kanamycin resistance enzymes stay in the periplasmic space (the area between the inner and outer membranes of E. coli) and modify the drug there so that it cannot cross the inner membrane.

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• Chapter 20 : Conjugation

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    In 1946, Joshua Lederburg and Edward Tatum discovered that genes could be exchanged between Escherichia coli cells in a process that required direct contact between the cells and a special fertility (F) factor in the donor cell. This process was named conjugation, and it is also referred to as bacterial sexuality because of the direct donation of genetic material. The basic form of the F factor is the F plasmid, a very large plasmid that contains several genes required for its conjugational transfer. Any cell that contains the F plasmid can synthesize all the proteins needed for conjugation (from the F genes) and so is "fertile." Fertile cells synthesize a special structure called a pilus, a tubelike appendage that protrudes from the outer membrane. The F plasmid occasionally recombines into the E. coli chromosome. It is believed that conjugation is the most important route of transmission of antibiotic resistance in most disease-causing bacteria. In this activity, students will observe the conjugative transmission of ampicillin resistance to a cell that is already resistant to streptomycin. The antibiotic streptomycin acts by binding to a ribosomal protein and preventing protein synthesis.

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• Chapter 21 : Transduction

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    In the process of transduction, a bacterial virus (bacteriophage) carries bacterial genes from one cell to another. Many different bacteriophages are capable of transduction; the details of transduction by any one of them depend on its life cycle. Cellular enzymes are diverted to make many new copies of the viral genetic material and many viral proteins. The viral DNA lies dormant in the host chromosome until a signal directs it to begin an active (often lytic) infection cycle. There are variations on the lytic and latent infection themes. Virus particles that contain bacterial DNA instead of viral DNA are completely capable of attaching to a new host cell and injecting DNA (those functions are carried out by the protein capsid and are independent of its contents). The usually fatal food poisoning botulism is a phage-borne disease. Staphylococcus aureus food poisoning is associated with a bacterial virus. The most common toxin involved in this disease is encoded by a gene on a lysogenic phage. The disease diphtheria is caused by the bacterium Corynebacterium diphtheriae. Cholera is also associated with transduction. The most severe symptoms of cholera are caused by a single protein, the cholera toxin. The chapter talks about amber mutations and amber suppressors.

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• Chapter 22 : Gene Transfer in Plants by Agrobacterium tumefaciens

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    In this student activity, students inoculate plants with the bacterium Agrobacterium tumefaciens and observe the subsequent plant tumor formation. The common soil bacterium Agrobacterium tumefaciens causes crown gall disease in many dicotyledonous plants. Virulent strains of A. tumefaciens contain genes that cause the plant cells to divide. The plasmid used by A. tumefaciens is the tumor-inducing, or Ti, plasmid. Chemicals secreted from freshly wounded plant tissue attract A. tumefaciens to the wound site. The injected bacterial DNA diverts the plant cell's machinery to tasks that support the growth and reproduction of A. tumefaciens. The strain-specific opine also promotes the conjugational transfer of tumor-inducing plasmids from that virulent strain to avirulent (plasmid-free) strains of Agrobacterium. The gall induced by A. tumefaciens is located at soil level where the roots join the stem (the crown). The vir genes that control gene transfer ability remain intact. Once individual plant cells have received new genetic information, the problem becomes how to regenerate whole plants from these cells. A. tumefaciens is an effective vector for tobacco, petunias, tomatoes, and other dicots—plants with two seed leaves. Microinjection is a new twist on an old idea. Biologists first used fine glass microtools in the late 1800s to dissect animal tissues. Using electroporation, scientists shock protoplasts with electricity until they become receptive to foreign DNA.

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D. Molecular Biology and Genetics

• Chapter 23 : Mendelian Genetics at the Molecular Level: Dominance and Recessivity

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    This chapter contains a reading and thinking activity that connects the observed genetics of dominant and recessive traits to molecular biology, using coat color in Labrador retrievers as an example. The chapter focuses on the molecular biology of black/brown pigmentation in Labrador retrievers, both because it is simple and because most students will be familiar with the animals. The molecular biology of black or brown (called chocolate in the breed) pigmentation in Labrador retrievers is based on the presence or absence of a single enzyme. The pathway to black and brown pigments (the eumelanins) can be viewed as beginning with the amino acid tyrosine. In melanocytes (pigment-producing cells), tyrosine is first converted to the chemical dopaquinone, which is then converted by a second enzyme, tyrosinase-related protein 2 (TYRP2), to a brown form of eumelanin pigment. The genetic basis for brown coat color is a nonfunctional allele for TYRP1. Chocolate is recessive to black because a single functional copy of the gene for TYRP1 leads to the production of plenty of black pigment.

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• Chapter 24 : Mendelian Genetics at the Molecular Level: An Example of Epistasis

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    The black/brown coloration of Labrador retrievers is determined by whether their melanocytes can synthesize TYRP1 and thus convert brown eumelanin to black eumelanin. The yellow coat color is based on the ability of follicle melanocytes to receive a hormone signal that induces production of TYRP2 and TYRP1. These enzymes, which convert dopaquinone to the brown and black pigments, respectively, are not constitutively produced in melanocytes. Instead, the melanocytes receive a signal from the hormone melanocortin 1 (MC1) (also referred to as melanocyte-stimulating hormone), which causes the cell to produce the enzymes. The hormone signal is transmitted when the hormone binds to a membrane receptor protein, melanocortin 1 receptor (MC1R). The melanocytes of yellow Labs cannot receive the hormone signal to produce TYRP2 and TYRP1. Researchers took skin biopsies and used PCR to determine the protein-coding sequence of the TYRP1 gene. They found two common variants that could account for brown coat color—a premature stop codon in exon 5 and the deletion of a proline, also in exon 5—in addition to one less common variant.

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• Chapter 25 : Human Molecular Genetics

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    This activity extends the discussion of the molecular basis of genetics to human beings. The background information for students focuses on specific single-gene disorders and on the difficulties of studying genetics in humans. The activity discusses the molecular genetics of cancer. The characteristics of genetic diseases vary widely, depending on the type of change and the gene in which it occurs. Genetic diseases can be divided into three categories: chromosomal defects, single-gene disorders, and multigenic traits. Chromosomal defects include missing or extra chromosomes and rearrangement or deletion of parts of chromosomes. Chromosomal disorders include gain or loss of an entire chromosome (aneuploidy), loss of part of one or more chromosomes (deletion), transfer of one segment of a chromosome to another chromosome (translocation), and reversal of a segment of a chromosome (inversion). The best-known chromosomal disease is probably Down syndrome, caused by an extra copy of chromosome 21. A chromosomal disorder involving the sex chromosomes is Klinefelter’s syndrome. Translocations and deletions can give valuable clues to the locations and functions of particular genes. Multigene disorders including familial hypercholesterolemia, Huntington’s disease (HD) and cystic fibrosis are discussed in the chapter. Cancer is a multigene disease; evidence shows that disruption of the normal functions of several genes is usually required before cancer develops. Cystic fibrosis is the most common inherited disease of European Americans. It is a recessive disorder.

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• Chapter 26 : Medical Sleuth: A Story of Genetics in Action

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    In this chapter, the authors have reprinted an article by Tom Schachtman that was published in the Smithsonian magazine in February 2006. It tells the story of D. Holmes Morton's medical genetics work among the Amish and Mennonites in Pennsylvania. It touches on all aspects of genetics, from classical pedigree studies to identification of disease genes to microchips. The authors provide this article as a compelling humanitarian story that can be used as a basis for integrating genetics and molecular biology information, as well as for putting medical genetics into historical context. They have supplied some questions for research and discussion that will require information not found in the article. They provide answers to the scientific questions and pointers for the history questions. They do not intend the answers to the questions to be complete—students can find much more information on every topic. The chapter includes answers to questions for research and discussion.

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E. Genomics

• Chapter 27 : Comparing Genomes

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    This chapter provides student activity that introduces the concept of using markers, such as repeated sequences, to compare genomes and discusses the various levels at which genomes can be compared: between genera, species, breeds within a species, or individuals. It contains a paper that provides the foundation for understanding how restriction analysis and polymerase chain reaction (PCR) can be used for genome comparisons and discusses the use of mitochondrial DNA in genetic studies. The information in the student introduction provides a brief overview of how genomes change and what sorts of questions can be addressed with genome comparisons, explains two approaches to DNA typing, and illustrates their use by focusing on the analysis of dog genomes. The activity describes the process for identifying a microsatellite marker. The technique of whole-genome hybridization and the comparison of the appearance of chromosomes in karyotypes were the only methods available for comparing genomes until the 1980s. Some molecular scientists now argue that genetic distance should also be grounds for defining species. The activity uses the dog genome as an example of ways in which genome comparisons and genome typing can be used to answer a variety of questions. It illustrates how PCR or Southern hybridization can be used to distinguish microsatellite alleles.

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• Chapter 28 : Forensic DNA Typing

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    This chapter contains three easy activities that illustrate applications of DNA typing plus a reading about applications of DNA analysis to human remains found at an archaeological site. The original reference for the archaeology case is listed. In Exercise 1, students assign babies to the correct pair of parents based on DNA profiles. In Exercise 2, students analyze DNA typing data to determine if I. M. Megabucks, a recently deceased megabillionaire, is actually the father of any of three children alleged to be his heirs. This activity can be done, with some discussion, by students who have completed Comparing Genomes. Students should also have completed DNA Scissors (the introduction to restriction enzymes) and DNA Goes to the Races (the introduction to electrophoresis) before doing this activity. DNA-based identification methods focus on highly variable regions of the human genome. To conduct DNA typing, the students must have a DNA containing sample. In paternity cases, blood is drawn from the child, its mother, and the alleged father, and DNA is extracted from the white cells. The first DNA-typing methods used Southern hybridization; now, PCR-based approaches are increasingly popular. It is not possible to say that no small ethnic group anywhere has a special, common DNA-typing profile not often seen in any other population—not unless everyone everywhere has been typed. In about one out of three cases, the perpetrator was unknown to the victim.

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• Chapter 29 : Genome Mapping

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    This chapter illustrates how polymorphic loci are used in genetic mapping, using a human disease gene as an example. The Student Activity contains extensive introductory material, including an example of using genetic mapping to determine the relative locations of three chromosomal markers (the ABO blood group locus, the nail-patella syndrome locus, and a hypothetical polymorphic short tandem repeat [STR] locus). The chapter provides students with an idea of how scientists figure out where genes are and how physical markers like STRs are used in mapping process. It also includes a reading about the Human Genome Project. One of the terms introduced in the chapter is haplotype, a contraction of haploid genotype. The term haplotype refers to the DNA content of one contiguous molecule, such as a mitochondrial genome, a single chromosome, or part of a chromosome, even down to very small regions. Occasionally, haplotype is used to refer to half of a genome, in the sense that a gamete contains a haplotype, and it is often used to refer to a specific mitochondrial genotype. A microsatellite haplotype would be the exact configuration of repeated sequences along a single chromosome.

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• Chapter 30 : Microarrays and Genome Analysis

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    This chapter deals with an activity that is an interactive computer simulation of a microarray analysis of gene expression in normal and cancer cells. Hybridization analysis allows scientists to ask questions about specific nucleic acid sequences. A microarray resembles a tiny checkerboard on a glass slide about the size of a coverslip. Microarray manufacturers will design a number of different probes to different portions of the same gene. In fluorescence in situ hybridization or Southern hybridization, the probe DNA is labeled. With microarrays, the probes sit on the gene chip and labeled sample is applied to them. When DNA samples are analyzed by microarrays, the goal is to get information about the genotype. The sample is hybridized to probes that represent many different alleles of genes, and the genotype is revealed by the hybridization patterns. Computerized scanners read the chips after hybridization and interpret the patterns of hybridization into information about specific genes or sequences. Genotyping by microarray allows an investigator to analyze which allele among many possibilities is present in an individual’s genome at many loci simultaneously. One application for which this can be very helpful is genetic mapping.

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Bioinformatics and Evolutionary Analysis of Proteins

• Chapter 31 : Amylase, an Evolutionarily Conserved Enzyme

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    Plants use energy from sunlight to power the synthesis of glucose, which they can use immediately or store as starch, a polymer of glucose. When plants need the energy stored in starch (for example, when a seed germinates), plant enzymes hydrolyze the starch into sugars. Animals use starch for energy, too, but instead of making it, they get it by eating plants. Two enzymes that hydrolyze starch are amylase and amyloglucosidase. The introduction to the student activity includes a description of the brewing process, which involves amylase action. The student activity also notes that humans have a salivary amylase and a pancreatic amylase, which are encoded by different, though similar, genes. In this activity, students are asked to test dog saliva for amylase. In the last chapter of this section, students learn how to use the online bioinformatics resources, which include programs for searching the recent biomedical literature.

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• Chapter 32 : Protein Electrophoresis

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    In this activity, students will perform agarose gel electrophoresis with amylase samples. The goal of this student activity is to separate the proteins in amylase-containing samples by electrophoresis so that students can see different protein bands and to identify amylase bands through their starch-hydrolyzing activity. Electrophoresis of proteins is different from electrophoresis of DNA in a few important respects. A mixture of proteins applied to an electrophoretic gel will likely contain some of each. Proteins assume different shapes, so two proteins of the same molecular weight and charge might migrate differently in a gel. To make proteins migrate more uniformly, they are often treated with the detergent sodium dodecyl sulfate (SDS). Proteins in SDS buffer migrate toward the positive electrode in an electric field, and the distances they migrate are largely proportional to their molecular weights. The gels in this activity are made with fine-sieving agarose, which improves the resolution of protein bands. Starch is also added to the gel. As the amylase activity is reconstituted, the enzyme degrades the starch in its vicinity, creating an area of clearing in the gel. Students can compare the stained half of the gel to the half with amylase activity.

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• Chapter 33 : Analyzing Evolutionary Changes

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    In this activity, students count the differences in amylase amino acid sequences from seven different organisms and use the data to construct a simple evolutionary tree. Evolution is a unifying theme in biology. Students often learn about the evolution of plants and animals from single-celled progenitors, with discussions of adaptations and niches. This chapter looks at evolution of the protein amylase at the molecular level. Proteins from closely related organisms are more similar to one another than are proteins from less closely related organisms. A mutation in one of the proteins could result in large morphological changes. A specific type of change associated with speciation is a change in the timing of body maturity and reproduction. The accumulation of genome sequences is making it easier for researchers to trace evolutionary changes. Evolution requires genetic change, such as mutations or chromosomal rearrangements, and the fixation of the new genotype in a population. If a slightly deleterious change in a protein that for a different reason had a survival advantage arose in an organism, that deleterious change could become fixed. Conserved protein sequences also support the hypothesis that the various proteins are descendants of an ancestral form. If necessary, the student can review the relationship between DNA sequence and protein sequence, emphasizing how changes in DNA can cause changes in proteins. The point of this activity is not the method for producing an evolutionary tree. Rather, it is the idea of protein evolution.

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• Chapter 34 : Bioinformatics

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    The student activity was designed to introduce students to the online bioinformatics resources and to reinforce the fact that proteins from many different organisms are quite similar and have evolved from common ancestral proteins. The National Center for Biotechnology Information (NCBI) is a national resource for molecular biology information. NCBI creates public databases and software for analyzing genomic data, conducts research in computational biology, and disseminates biomedical information. NCBI creates public databases and software for analyzing genomic data, conducts research in computational biology, and disseminates biomedical information. This chapter gives the information available through NCBI. In the activity, students compare their results with the evolutionary tree they constructed. They use data from their searches to explore other features of the databases and will conduct some literature searches.

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• Chapter 35 : Science, Technology, and Society

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    One cannot overestimate the importance of conducting classroom activities that encourage students to think about the complex interrelationships among science, technology, and society. While very few students will pursue biology as a career, all of them will be citizens in a society shaped by science and technology. If they respect the power of these fields to influence society for better or worse, then perhaps they will become concerned citizens, involved in ensuring that science and technology are directed toward maximizing societal benefits and minimizing costs. In addition to providing students with skills for analyzing societal issues and participating productively in discussions about technology development and use, the authors also hope that these activities provide one with a platform for impressing upon students the essential role each citizen must play in a democracy. Science and technology have altered the characteristics of societies throughout history. Some of the forces they have exerted have been revolutionary, and some have been minor. Different aspects of life—social, cultural, ecological, and economic— have been affected, and different groups have been affected in different ways. Often, one group has benefited at another’s expense. The agents guiding technology development along certain paths but not others include economics, ethical values, government policies, market opportunities, consumer preferences, and, in democracies, public opinion. As so many different forces can alter the course of science and technology development, different segments of society with divergent interests have the power to influence the trajectory of scientific research and technological innovation.

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• Chapter 36 : A Framework for Rational Analysis of Issues

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    In this chapter, the author provides an approach and process for analyzing some of the issues associated with biotechnology that are often "debated" through the media. A productive approach to analyzing any issue associated with biotechnology begins with the simple task of placing modern biotechnology within the context of other technologies. Observers are left with the impression that these technologies appeared suddenly, with no historical precedents, and that the issues raised by biotechnology are unique to biotechnology. Knowing that today’s biotechnologies are the next step in a continuum of technologies has important, real-world implications for charting its course. The costs of no technology are difficult to assess, because those costs are usually unknown to people who have lived in a world in which technology has solved certain problems. Gene flow from a transgenic crop to a wild plant depends on the potential for (i) cross-pollination between the transgenic crop (pollen donor) and the wild plant and (ii) successful hybridization. Crops and wild plants can potentially cross-pollinate only if the wild plants occurring near the crop field are closely related to the crop. In fact, botanists have been studying it for at least a century. A corn plant can either self-pollinate or cross-pollinate, and pollen dispersal is driven by wind speed and direction. Canola is a special type of oil that is produced by any of three Brassica species, B. napus, B. rapa, and B. juncea, which are known as the oilseed brassicas.

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• Chapter 37 : Risks and Rationality in Today's Culture of Fear

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    This chapter presents an activity that describes some pitfalls of making choices based on perceived risks rather than actual risks, compares emotion-based risk perception to science-based risk assessment, and discusses the role that government regulatory agencies play in minimizing the potential risks associated with new products, including those developed using biotechnology. A rational explanation is that new risks do arise. A number of emerging infectious diseases, such as human immunodeficiency virus-AIDS and West Nile virus infections, have begun to cause human health problems only recently. For example, viruses that are transmitted in sexual intercourse were a causative agent of cervical cancer centuries before scientists knew viruses existed. The uncertainty of exposure is one of the reasons that risks are discussed in terms of probabilities instead of an absolute assessment of the seriousness of the hazard. To assess the risks of Bt corn pollen to monarchs, regulators must determine: (1) If Bt corn pollen is hazardous to monarch larvae (This step is unnecessary in this case, because it has been widely known for well over 50 years that the Bt protein can kill lepidopteran caterpillars that consume it.); (2) The dose required for harm or injury; (3) The likelihood that an individual larva will be exposed to that dose. In summary, Bt corn pollen can be hazardous to monarch larvae, but the hazard level varies with the dose, the Bt corn variety, and larval age.

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• Chapter 38 : Bioethical Issues: A Decision-Making Model

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    While medical bioethical issues are not derived solely from advances in biotechnology, the new knowledge and powers the biotechnologies provide will force people and governments to wrestle with ethical issues they have never had to address. Teachers have found the bioethics decision-making model, described in this chapter, to be helpful in channeling productive thought and debate. The basic steps, objectives, preparation and resources of this model are explained in detail. Two example cases follow with sample workups and decisions to demonstrate the process. In the field of biomedical ethics, certain guides are well established. The moral-action guides or principles can be divided into four major principles and several secondary ones. Following the prescribed, step-by-step procedure laid out in the model greatly reduces the chance of getting off track. After reading this chapter and completing the exercises, students will have learned the basic principles that guide ethical decision making, become familiar with internationally accepted codes of conduct, learned and practiced a process for making decisions on bioethical issues, learned that some issues present dilemmas to which there is no one right answer. The chapter presents six possible decisions, but the possibility exists for more.

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• Chapter 39 : Bioethics Case Study: Genetic Testing

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    In this chapter, students are asked to use the decision-making model to analyze ethical dilemmas related to newer capacities to test for genetic defects. Before the students tackle ethical dilemmas posed by genetic testing, you should provide information that allows them to place ethical issues associated with genetic testing in context. A first step to informed discussion of genetic testing is establishing a semantic distinction between genetic testing, gene (or genetic marker) identification, and genetic screening. For purposes of this chapter's discussion, genetic testing is referred to a diagnostic test, usually ordered by a physician, to test for a specific genetic disorder because the physician thinks there is a good chance the person might have the defective gene. With the older testing technologies, in order to diagnose a genetic disease, physicians had to have a measurable or observable phenotype that was consistently associated with the genetic defect. Now, however, with only partial DNA sequences of disease genes or nearby marker sequences, physicians can diagnose genetic diseases long before symptoms appear. A table illustrates legally mandated neonatal screening for genetic disorders. The chapter presents two case studies for application of the model. To follow the methodology outlined in the model, students must have a basic knowledge of cystic fibrosis (CF), including its genetic basis, symptoms, and treatments and the prognosis for patients who suffer from the disease.

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• Chapter 40 : Bioethics Case Study: Gene Therapy

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    Leroy Walters, of Georgetown University’s Kennedy Institute of Bioethics, divided gene therapy into four possible categories. (1) Somatic-cell gene therapy for the cure or prevention of disease. Example: insertion of a DNA sequence into a person’s cells to allow production of an enzyme like adenosine deaminase. (2) Germ line gene therapy for cure or prevention of disease. Example: insertion of an adenosine deaminase sequence into early embryo or reproductive cells, which would affect not only the individual but all of his or her offspring. (3) Somatic-cell enhancement. Example: insertion of a DNA sequence to improve memory, increase height, or increase intelligence, which would affect only that individual. (4) Germ line enhancement. Example: insertion of a DNA sequence for enhancement into a blastocyst, sperm, or egg, which would affect future generations. Germ line therapy (altering disease genes so that the individual not only will be healthy but will pass on the healthy genes to his or her offspring) is considered desirable by some, but the techniques used to alter animal embryos have far too high a failure rate to consider their application to humans at this time. In the future, however, physicians will be able to detect many more defective genes than those that cause illnesses that are incurable and untreatable. Some genetic defects that are life threatening will be controllable with therapeutics (e.g., hemophilia). Other genetic defects will simply indicate a propensity to develop a disease.

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