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CHAPTER 9   Biotechnology and DNA Technology 2 Introduction to Biotechnology foreign DNA into a cell. (See more on vectors on page 246.)...

CHAPTER 9   Biotechnology and DNA Technology 2 Introduction to Biotechnology foreign DNA into a cell. (See more on vectors on page 246.) The gene of interest is inserted into the vector DNA in vitro. In LEARNING OBJECTIVES Figure 9.1, the vector is a plasmid. The DNA molecule chosen 9-1 Compare and contrast biotechnology, genetic modification, as a vector must be self-replicating, such as a plasmid or a viral and recombinant DNA technology. genome. This recombinant vector DNA is taken up by a cell 9-2 Identify the roles of a clone and a vector in making such as a bacterium, where it can multiply. The cell contain- recombinant DNA. ing the recombinant vector is then grown in culture to form a Biotechnology is the use of microorganisms, cells, or cell com- clone of many genetically identical cells, each of which carries ponents to make a product. Microbes have been used in the copies of the vector, and therefore many copies of the gene of commercial production of foods, vaccines, antibiotics, and interest. This is why DNA vectors are often called gene-cloning vitamins for years. Bacteria are also used in mining to extract vectors, or simply cloning vectors. (In addition to referring to a valuable elements from ore (see Figure 28.12, page 822). Addi- culture of identical cells, the word clone is also routinely used tionally, animal cells have been used to produce viral vaccines as a verb, to describe the entire process, as in “to clone a gene.”) since the 1950s. Until the 1980s, products made by living cells The final step varies according to whether the gene itself or were all made by naturally occurring cells; the role of scientists the product of the gene is of interest. From the cell clone, the was to find the appropriate cell and develop a method for their researcher may isolate (“harvest”) large quantities of the gene large-scale cultivation. of interest, which may then be used for a variety of purposes. Now, microorganisms and plants are being used as “facto- The gene may even be inserted into another vector for intro- ries” to produce chemicals that the organisms don’t naturally duction into another kind of cell (such as a plant or animal make. This is accomplished by inserting, deleting, or modify- cell). Alternatively, if the gene of interest is expressed (tran- ing genes with recombinant scribed and translated) in the cell clone, its protein product can DNA (rDNA) technology, be harvested and used for a variety of purposes. ASM: Cell genomes can be manipulated which is sometimes called to alter cell function. The advantages of using rDNA for obtaining such pro- genetic engineering. The devel- teins is illustrated by one of its early successes, the produc- opment of rDNA technology is expanding the practical applica- tion of human growth hormone (hGH) in E. coli bacteria. tions of biotechnology almost beyond imagination. Some individuals don’t produce adequate amounts of hGH, so their growth is stunted. In the past, hGH had to be obtained Recombinant DNA Technology from human pituitary glands at autopsy. (Growth hormone Recombination of DNA occurs naturally in microbes (see Chapter 8). In the 1970s and 1980s, scientists developed artifi- cial techniques for making rDNA. CLINICAL CASE No Ordinary Checkup A gene from one organism can be inserted into the DNA of a bacterium or a yeast. In many cases, the recipient can then be made to express the gene, which may code for a commercially D r. B. is closing his dental practice after 20 years. Four years ago, he went to his family doctor because of debilitating exhaustion. He thought he had a flu virus that he useful product. Thus, bacteria with genes for human insulin are could not shake, and he was also having night sweats. His now being used to produce insulin for treating diabetes, and a vac- doctor ordered a myriad of blood tests, but only one came cine for hepatitis B is being made by yeast carrying a gene for part back positive. Dr. B. had HIV. Although he immediately began of the hepatitis virus (the yeast produces a viral coat protein). Sci- an HIV treatment regimen, one year later he was diagnosed entists hope that such an approach may prove useful in produc- with AIDS. Now, two years later, Dr. B. is very ill and can no ing vaccines against other infectious agents, thus eliminating the longer work. need to use whole organisms, as in conventional vaccines. Dr. B. lets his employees know the situation and suggests The rDNA techniques can also be used to make thousands that they all get tested for HIV. All of Dr. B.’s employees, of copies of the same DNA molecule—to amplify DNA—thus including the hygienists, test negative. Dr. B. also writes an open letter to his patients informing them of his decision to generating sufficient DNA for various kinds of experimenta- close his practice and why he is doing so. This letter prompts tion and analysis. This technique has practical application for 400 former patients to be tested for HIV, seven of whom test identifying microbes, such as viruses, that can’t be cultured. positive for antibodies against HIV. What type of test can determine whether these patients An Overview of Recombinant DNA Procedures contracted HIV from Dr. B.? Read on to find out. An overview of some of the procedures typically used for mak- ing rDNA, along with some promising applications, is shown 243 249 252 254 257 in Figure 9.1. A vector is a DNA molecule that transports  F IGU R E 9.1 A Typical Genetic Modification Procedure bacterium 1 Vector, such as a plasmid, is isolated. 2 DNA containing the gene of interest from a different species is cleaved by an enzyme into fragments. Plasmid Bacterial chromosome recombinant DNA containing the gene of interest DNA (plasmid) 3 The desired gene is selected and inserted into a plasmid. 4 The plasmid is taken up by a cell, such as a bacterium. transformed bacterium 5 Cells with the gene of interest are cloned with either of two goals in mind. 6a Create and harvest OR 6b Create and harvest copies of a gene. protein products of a gene. Plasmid RNA Protein product Gene encoding protein for Gene encoding degradative Amylase, cellulase, and other Human growth hormone pest resistance is inserted enzyme to clean up toxic enzymes prepare fabrics for treats stunted growth. into plant cells. waste is inserted into clothing manufacture. bacterial cells. KEY CONCEPTS Genes from one organism’s cells can be inserted and expressed in another organism’s cells. Genetically modified cells can be used to create a wide variety of useful products and applications. 244  CHAPTER 9   Biotechnology and DNA Technology 2 from other animals is not effective in humans.) This practice cultures to radiation, the highest-yielding variant among the was not only expensive but also dangerous because on several survivors was selected for another exposure to a mutagen. occasions neurological diseases were transmitted with the hor- Using mutations, biologists increased the amount of penicillin mone. Human growth hormone produced by genetically modi- the fungus produced by over 1000 times. fied E. coli is a pure and cost-effective product. Recombinant Screening each mutant for penicillin production is a DNA techniques also result in faster production of the hor- tedious process. Site-directed mutagenesis is more targeted mone than traditional methods might allow. and can be used to make a specific change in a gene. Suppose you determine that changing one amino acid will make a laun- CHECK YOUR UNDERSTANDING dry enzyme work better in cold water. Using the genetic code ✓ 9-1 Differentiate biotechnology and rDNA technology. (see Figure 8.8, page 214), you could, using the techniques described next, produce the sequence of DNA that encodes that ✓ 9-2 In one sentence, describe how a vector and clone are used. amino acid and insert it into that enzyme’s gene. The science of molecular genetics has advanced to such a degree that many routine cloning procedures are performed Tools of Biotechnology using prepackaged materials and procedures that are very much like cookbook recipes. Scientists have a grab bag of LEARNING OBJECTIVES methods from which to choose, depending on the ultimate 9-3 Compare selection and mutation. application of their experiments. Next we describe some of the most important tools and techniques, and later we will con- 9-4 Define restriction enzymes, and outline how they are used to make rDNA. sider some applications. 9-5 List the four properties of vectors. Restriction Enzymes 9-6 Describe the use of plasmid and viral vectors. Recombinant DNA technology has its technical roots in the 9-7 Outline the steps in PCR, and provide an example of its use. discovery of restriction enzymes, a special class of DNA- Research scientists and technicians isolate bacteria and fungi cutting enzymes that exist in many bacteria. First isolated from natural environments such as soil and water to find, in 1970, restriction enzymes in nature had actually been or select, the organisms that produce a desired product. The observed earlier, when certain bacteriophages were found to selected organism can be mutated to make more product or to have a restricted host range. If these phages were used to infect make a better product. bacteria other than their usual hosts, restriction enzymes in the new host destroyed almost all the phage DNA. Restriction Selection enzymes protect a bacterial cell by hydrolyzing phage DNA. In nature, organisms with characteristics that enhance survival The bacterial DNA is protected from digestion because the cell are more likely to survive and reproduce than are variants that methylates (adds methyl groups to) some of the cytosines in its lack the desirable traits. This is called natural selection. Humans DNA. The purified forms of these bacterial enzymes are used use artificial selection to select desirable breeds of animals or in today’s laboratories. strains of plants to cultivate. As microbiologists learned how What is important for rDNA techniques is that a restric- to isolate and grow microorganisms in pure culture, they were tion enzyme recognizes and cuts, or digests, only one par- able to select the ones that could accomplish a desired objec- ticular sequence of nucleotide bases in DNA, and it cuts this tive, such as brewing beer more efficiently or producing a new sequence the same way each time. Typical restriction enzymes antibiotic. Over 2000 strains of antibiotic-producing bacteria used in cloning experiments recognize four-, six-, or eight-base have been discovered by testing soil bacteria and selecting the sequences. Hundreds of restriction enzymes are known, each strains that produce an antibiotic. producing DNA fragments with characteristic ends. A few restric- tion enzymes are listed in Table 9.1. You can see they are named Mutation for their bacterial source. Some of these enzymes (e.g., HaeIII) cut both strands of DNA in the same place, producing blunt Mutations are responsible for much of the diversity of life (see ends, and others make staggered cuts in the two strands—cuts Chapter 8). A bacterium with a mutation that confers resistance that are not directly opposite each other (Figure 9.2). These stag- to an antibiotic will survive and reproduce in the presence of gered ends, or sticky ends, are most useful in rDNA because they that antibiotic. Biologists working with antibiotic-producing can be used to join two different pieces of DNA that were cut by microbes discovered that they could create new strains by the same restriction enzyme. The sticky ends “stick” to stretches exposing microbes to mutagens. After random mutations were of single-stranded DNA by complementary base pairing. created in penicillin-producing Penicillium by exposing fungal 246 PART ONE Fundamentals of Microbiology Recognition sites DNA Cut Cut 1 A restriction enzyme cuts (red arrows) GAAT TC GAAT TC double-stranded DNA at its particular recognition sites, shown in blue. CT T AAG CT T AAG Cut Cut AATTC G GAATTC 2 These cuts produce a DNA fragment with two sticky ends. G CTTAA CTTAA G AATTC G DNA from another source, Sticky end perhaps a plasmid, cut with the same restriction enzyme G CTTAA AATTC GA A T T C G 3 When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing. G C T T A AG CTTAA AATT G C TA A G CT 4 The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. A A T T C G Other combinations of fragments can also occur. TAA G CT G A TC AT C T T A A G 5 The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing rDNA G A TTC A a molecule of rDNA. C T T A A G Figure 9.2 A restriction enzyme’s role in making rDNA. Q Why are restriction enzymes used to make rDNA? sources have been produced by the action of the same restric- TABLE 9.1 S  elected Restriction Enzymes Used in rDNA Technology tion enzyme, the two pieces will have identical sets of sticky ends and can be spliced (recombined) in vitro. The sticky Enzyme Bacterial Source Recognition Sequence ends join spontaneously by hydrogen bonding (base pairing). BamHI Bacillus GGATCC The enzyme DNA ligase is used to covalently link the backbones amyloliquefaciens C CTAGG of the DNA pieces, Play Recombinant DNA Technology EcoRI Escherichia GAATTC producing an rDNA @MasteringMicrobiology coli C TTAAG molecule. HaeIII Haemophilus GGCC aegyptius C CGG Vectors HindIII Haemophilus AAGCTT Many different types of DNA molecules can serve as vectors, influenzae T TCGAA provided they have certain properties. The most important property is self-replication; once in a cell, a vector must be capable of replicating. Any DNA that is inserted in the vector Notice in Figure 9.2 that the darker base sequences on the will be replicated in the process. Thus, vectors serve as vehicles two strands are the same but run in opposite directions. Stag- for the replication of desired DNA sequences. gered cuts leave stretches of single-stranded DNA at the ends Vectors also need to be large enough to be manipulated out- of the DNA fragments. If two fragments of DNA from different side the cell during rDNA procedures. Smaller vectors are more  CHAPTER 9   Biotechnology and DNA Technology 2 being used to insert corrective genes into human cells that have defective genes. Gene therapy is discussed on page 255. lacZ HindIII CHECK YOUR UNDERSTANDING BamHI amp EcoRI ✓ 9-3 How are selection and mutation used in biotechnology? pUC19 ✓ 9-4 What is the value of restriction enzymes in rDNA technology? ✓ 9-5 What criteria must a vector meet? ✓ 9-6 Why is a vector used in rDNA technology? ori Polymerase Chain Reaction Figure 9.3 A plasmid used for cloning. A plasmid vector used The polymerase chain reaction (PCR) is a technique by for cloning in the bacterium E. coli is pUC19. An origin of replication which small samples of DNA can be quickly amplified, that is, (ori) allows the plasmid to be self-replicating. Two genes, one encoding resistance to the antibiotic ampicillin (amp) and one encoding the increased to quantities that are large enough for analysis. enzyme β-galactosidase (lacZ), serve as marker genes. Foreign DNA can Starting with just one gene-sized piece of DNA, PCR can be be inserted at the restriction enzyme sites. used to make billions of copies in only a few hours. The PCR process is shown in Figure 9.4. Q What is a vector in rDNA technology? Each strand of the target DNA will serve as a template for DNA synthesis. Added to this DNA are a supply of the four easily manipulated than larger DNA molecules, which tend to nucleotides (for assembly into new DNA) and the enzyme for be more fragile. Preservation is another important property of catalyzing the synthesis, DNA polymerase (see Chapter 8, page vectors. The DNA molecule’s circular form protects the vector’s 209). Short pieces of nucleic acid called primers are also added DNA from being destroyed by its recipient. Notice in Figure 9.3 to help start the reaction. The primers are complementary to that the DNA of a plasmid is circular. Another preservation the ends of the target DNA and will hybridize to the fragments mechanism occurs when a virus’s DNA inserts itself quickly to be amplified. Then, the polymerase synthesizes new com- into the chromosome of the host. plementary strands. After each cycle of synthesis, the DNA is When it is necessary to retrieve cells that contain the vector, heated to convert all the new DNA into single strands. Each a marker gene in the vector often helps make selection easy. newly synthesized DNA strand serves in turn as a template for Common selectable marker genes are for antibiotic resistance more new DNA. or for an enzyme that carries out an easily identified reaction. As a result, the process proceeds exponentially. All of the nec- Plasmids are one of the primary vectors in use, particularly essary reagents are added to a tube, which is placed in a thermal variants of R factor plasmids. Plasmid DNA can be cut with the cycler. The thermal cycler can be set for the desired temperatures, same restriction enzymes as the DNA that will be cloned, so times, and number of cycles. Use of an automated thermal cycler that all pieces of the DNA will have the same sticky ends. When is made possible by the use of DNA polymerase taken from a the pieces are mixed, the DNA to be cloned will be inserted thermophilic bacterium such as Thermus aquaticus; the enzyme into the plasmid (Figure 9.2). Note that other fragment com- from such organisms can survive the heating phase without binations can occur as well, including the plasmid reforming a being destroyed. Thirty cycles, completed in just a few hours, will circle with no DNA inserted. increase the amount of target DNA by more than a billion times. Some plasmids are capable of existing in several different The amplified DNA can be seen by gel electrophoresis. In species. They are called shuttle vectors and can be used to move real-time PCR, or quantitative PCR (qPCR), the newly made DNA cloned DNA sequences among organisms, such as among bac- is tagged with a fluorescent dye, so that the levels of fluores- terial, yeast, and mammalian cells, or among bacterial, fungal, cence can be measured after every PCR cycle (that’s the real and plant cells. Shuttle vectors can be very useful in the process time aspect). Another PCR procedure called reverse-transcription of genetically modifying multicellular organisms—for example, (RT-PCR) uses viral RNA or a cell’s mRNA as the template. The when herbicide resistance genes are inserted into plants. enzyme, reverse transcriptase, makes DNA from the RNA tem- A different kind of vector is viral DNA. This type of vec- plate, and the DNA is then amplified. tor can usually accept much larger pieces of foreign DNA than Note that PCR can only be used to amplify relatively small, plasmids can. After the DNA has been inserted into the viral specific sequences of DNA as determined by the choice of vector, it can be cloned in the virus’s host cells. The choice of a primers. It cannot be used to amplify an entire genome. suitable vector depends on many factors, including the organ- PCR can be applied to any situation that requires the ampli- ism that will receive the new gene and the size of the DNA to fication of DNA. Especially noteworthy are diagnostic tests that be cloned. Retroviruses, adenoviruses, and herpesviruses are use PCR to detect the presence of infectious agents in situations in 248 PART ONE Fundamentals of Microbiology PREPARATION Target DNA 5¿ 3¿ 3¿ 5¿ 1 Add primers, nucleotides, and DNA polymerase. Primer Nucleotides DNA polymerase FIRST CYCLE 5¿ 3¿ 3¿ 5¿ 2 Incubate at 94°C for 1 minute; this temperature will separate the strands. 5¿ 3¿ 3¿ 5¿ 3 Incubate at 60°C for 1 minute; this allows primers 5¿ 3¿ to attach to single-stranded DNA. 3¿ 5¿ 5¿ 3¿ 4 Incubate at 72°C for 1 minute; DNA polymerase 3¿ 5¿ copies the target DNA at this temperature. 5¿ 3¿ 3¿ 5¿ Copy of target DNA Copy of target DNA SECOND CYCLE 5¿ 3¿ 5¿ 3¿ 5 Repeat the cycle of heating and cooling to make two more copies of target DNA. 3¿ 5¿ 3¿ 5¿ Copies of target DNA Copies of target DNA Figure 9.4 The polymerase chain reaction. Deoxynucleotides (dNTPs) base-pair with the target DNA: adenine pairs with thymine, and cytosine pairs with guanine. Q How does reverse-transcription PCR differ from this figure? which they would otherwise be undetectable. A qPCR test provides rapid identification of drug-resistant Mycobacterium tuberculosis. Techniques of Genetic Modification Otherwise, this bacterium can LEARNING OBJECTIVES take up to 6 weeks to culture, Play PCR: Overview, Components, Process 9-8 Describe five ways of getting DNA into a cell. leaving patients untreated for @MasteringMicrobiology 9-9 Describe how a genomic library is made. a significant period of time. 9-10 Differentiate cDNA from synthetic DNA. 9-11 E  xplain how each of the following is used to locate a clone: CHECK YOUR UNDERSTANDING antibiotic-resistance genes, DNA probes, gene products. ✓ 9-7 For what is each of the following used in PCR: primer, 9-12 L ist one advantage of modifying each of the following: E. coli, DNA polymerase, 94°C? Saccharomyces cerevisiae, mammalian cells, plant cells.  CHAPTER 9   Biotechnology and DNA Technology 2 glycol increases the frequency of fusion (Figure 9.5). In the CLINICAL CASE new hybrid cell, the DNA derived from the two “parent” cells may undergo natural recombination. This method is R everse-transcription PCR using a primer for an HIV gene can be used to amplify DNA for analysis. The Centers for Disease Control and Prevention (CDC) interviews the seven especially valuable in the genetic manipulation of plant and algal cells. former patients to determine whether their histories show A remarkable way of introducing foreign DNA into plant any additional risk factors for contracting HIV. Five out of the cells is to literally shoot it directly through the thick cellu- seven have no identified risk factors for HIV other than having lose walls using a gene gun (Figure 9.6). Microscopic particles had invasive procedures performed on them by Dr. B. The of tungsten or gold are coated with DNA and propelled by a CDC then performs reverse- burst of helium through the plant cell walls. Some of the cells Patients transcription PCR on DNA from express the introduced DNA as though it were their own. Dentist A B C D E F G white blood cells in Dr. B.’s DNA can be introduced directly into an animal cell by peripheral blood and the seven microinjection. This technique requires the use of a glass HIV-positive patients (see the micropipette with a diameter that is much smaller than the figure). cell. The micropipette punctures the plasma membrane, and What can be concluded from the PCR amplification in the DNA can be injected through it (Figure 9.7). figure? 243 249 252 254 257 Chromosome Plasma membrane Inserting Foreign DNA into Cells Cell wall Recombinant DNA procedures require that DNA molecules be manipulated outside the cell and then returned to living cells. Bacterial cells 1 Bacterial cell walls There are several ways to introduce DNA into cells. The choice are enzymatically digested, producing of method is usually determined by the type of vector and host protoplasts. cell being used. In nature, plasmids are usually transferred between closely related microbes by cell-to-cell contact, such as in conjuga- Protoplasts tion. To modify a cell, a plasmid must be inserted into a cell by transformation, a procedure during which cells can take 2 In solution, protoplasts are up DNA from the surrounding environment (see Chapter 8, treated with polyethylene glycol. page 232). Many cell types, including E. coli, yeast, and mam- malian cells, do not naturally transform; however, simple 3 Protoplasts fuse. chemical treatments can make all of these cell types compe- tent, or able to take up external DNA. For E. coli, the procedure 4 Segments of the two for making cells competent is to soak them in a solution of chromosomes recombine. calcium chloride for a brief period. Following this treatment, the now-competent cells are mixed with the cloned DNA and Recombinant given a mild heat shock. Some of these cells will then take up cell the DNA. There are other ways to transfer DNA to cells. A process 5 Recombinant cell grows new cell wall. called electroporation uses an electrical current to form microscopic pores in the membranes of cells; the DNA then enters the cells through the pores. Electroporation is generally applicable to all cells; those with cell walls often must be con- verted to protoplasts first. Protoplasts are produced by enzy- matically removing the cell wall, thereby allowing more direct Figure 9.5 Protoplast fusion. Removal of the cell wall leaves only access to the plasma membrane. the delicate plasma membranes, which will fuse together, allowing the The process of protoplast fusion also takes advantage exchange of DNA. of the properties of protoplasts. Protoplasts in solution fuse Q What is a protoplast? at a low but significant rate; the addition of polyethylene 250 PART ONE Fundamentals of Microbiology Obtaining DNA We have seen how genes can be cloned into vectors by using restriction enzymes and how genes can be transformed or transferred into a variety of cell types. But how do biologists obtain the genes they are interested in? There are two main sources of genes: (1) genomic libraries containing either natu- ral copies of genes or cDNA copies of genes made from mRNA, and (2) synthetic DNA. Genomic Libraries Isolating specific genes as individual pieces of DNA is seldom practical. Therefore, researchers interested in genes from a particular organism start by extracting the organism’s DNA, which can be obtained from cells of any organism, whether plant, animal, or microbe, by lysing the cells and precipitating the DNA. This process results in a DNA mass that includes the Figure 9.6 A gene gun, which can be used to insert organism’s entire genome. After the DNA is digested by restric- DNA-coated “bullets” into a cell. tion enzymes, the restriction fragments are then spliced into Q Name four other methods of inserting DNA into a cell. plasmid or phage vectors, and the recombinant vectors are introduced into bacterial cells. The goal is to make a collection of clones large enough to ensure that at least one clone exists Thus, there is a great variety of restriction enzymes, vec- for every gene in the organism. This collection of clones con- tors, and methods of inserting DNA into cells. But foreign taining different DNA fragments is called a genomic library; DNA will survive only if it’s either present on a self-replicating each “book” is a bacterial or phage strain that contains a frag- vector or incorporated into one of the cell’s chromosomes by ment of the genome (Figure 9.8). Such libraries are essential for recombination. Genome to be stored in library is cut up with restriction enzyme Recombinant plasmid OR Host cell Recombinant phage DNA Phage cloning vector LM 80 mm Figure 9.7 The microinjection of foreign DNA into an egg. The egg is first immobilized by applying mild suction to the large, blunt, holding pipette (right). Several hundred copies of the gene of interest Plasmid Library Phage Library are then injected into the nucleus of the cell through the tiny end of the micropipette (left). Figure 9.8 Genomic libraries. Each fragment of DNA, containing about one gene, is carried by a vector, either a plasmid within a bacterial Q Why is microinjection impractical for bacterial and fungal cells? cell or a phage. Q Differentiate a restriction fragment from a gene.  CHAPTER 9   Biotechnology and DNA Technology 2 Exon Intron Exon Intron Exon Nucleus (Figure 9.9). This synthesis is the reverse of the normal DNA-to- RNA transcription process. A DNA copy of mRNA is produced DNA by reverse transcriptase. Following this, the mRNA is enzymati- 1 A gene composed of exons and cally digested away. DNA polymerase then synthesizes a com- introns is transcribed to RNA by plementary strand of DNA, creating a double-stranded piece of RNA polymerase. DNA containing the information from the mRNA. Molecules of RNA transcript cDNA produced from a mixture of all the mRNAs from a tissue 2 Processing enzymes in the nucleus or cell type can then be cloned to form a cDNA library. remove the intron-derived RNA The cDNA method is the most common method of obtain- and splice together the ing eukaryotic genes. A difficulty with this method is that long exon-derived RNA into mRNA. molecules of mRNA may not be completely reverse-transcribed mRNA into DNA; the reverse transcription often aborts, forming only parts of the desired gene. Cytoplasm Synthetic DNA 3 mRNA is isolated from the cell, and reverse Under certain circumstances, genes can be made in vitro with transcriptase is added. the help of DNA synthesis machines (Figure 9.10). A keyboard 4 First strand on the machine is used to enter the desired sequence of nucleo- of DNA is tides, much as letters are entered into a word processor to com- synthesized. pose a sentence. A microprocessor controls the synthesis of the DNA strand 5 The mRNA is digested by being synthesized DNA from stored supplies of nucleotides and the other neces- reverse transcriptase. sary reagents. A short chain of about 200 nucleotides, called an oligonucleotide, can be synthesized by this method. Unless the 6 DNA polymerase is added to gene is very small, at least several chains must be synthesized synthesize second strand separately and linked together to form an entire gene. of DNA. The difficulty of this approach, of course, is that the cDNA of gene without sequence of the gene must be known before it can be synthe- introns Test tube sized. If the gene hasn’t already been isolated, then the only Figure 9.9 Making complementary DNA (cDNA) for a eukaryotic gene. Reverse transcriptase catalyzes the synthesis of double-stranded DNA from an RNA template. Q How does reverse transcriptase differ from DNA polymerase? maintaining and retrieving DNA clones; they can even be pur- chased commercially. Cloning genes from eukaryotic organisms presents a spe- cific problem. Genes of eukaryotic cells generally contain both exons, stretches of DNA that code for protein, and introns, intervening stretches of DNA that do not code for protein. When the RNA transcript of such a gene is converted to mRNA, the introns are removed (see Figure 8.11 on page 219). To clone genes from eukaryotic cells, it’s desirable to use a version of the gene that lacks introns because a gene that includes introns may be too large to work with easily. In addition, if such a gene is put into a bacterial cell, the bacterium usually won’t be able to remove the introns from the RNA transcript. Therefore, it won’t be able to make the correct protein product. However, Figure 9.10 A DNA synthesis machine. Short sequences of DNA an artificial gene that contains only exons can be produced can be synthesized by instruments such as this one. by using an enzyme called reverse transcriptase to synthe- Q What four reagents (in the brown bottles) are necessary to size complementary DNA (cDNA) from an mRNA template synthesize DNA? 252 PART ONE Fundamentals of Microbiology The plasmid vector used contains a gene (amp) encoding CLINICAL CASE resistance to the antibiotic ampicillin. The host bacterium won’t be able to grow on the test medium, which contains ampicillin, T he primer amplifies all eight samples and confirms that Dr. B. and seven of his former patients are all infected with HIV. The CDC then sequences the amplified DNA and unless the vector has transferred the ampicillin-resistance gene. The plasmid vector also contains a second gene, this one for the enzyme β-galactosidase (lacZ). Notice in Figure 9.3 that there compares the sequencing to an HIV isolate from Cleveland (local control) and an isolate from Haiti (outlier). A portion of are several sites in lacZ that can be cut by restriction enzymes. the coding (5' to 3') is shown below. In the blue-white screening procedure shown in Figure 9.11, a library of bacteria is cultured in a medium called X-gal. X-gal Patient A GCTTG GGCTG GCGCT GAAGT GAGA contains two essential components other than those neces- Patient B GCTAT TGCTG GCGCT GAATT GCAC sary to support normal bacterial growth. One is the antibiotic Patient C GCCAT AGCTG GCGCA GAAGT GCAC ampicillin, which prevents the growth of any bacterium that Patient D GCTAT TGGCG TGGCT GACAG AGAA Patient E GCACC TGCTG GCGCT GAAGT GAAA Patient F CAGAT TGTGT TGATT GAACC TCAC b-galactosidase gene (lacZ) Patient G GCTAT TGCTG GCGCT GAAGT GAAA Ampicillin-resistance Dentist GCTAT TGCTG GCGCT GAAGT GCAC gene (amp) Restriction Local control CAGAC TACTG CTAGG AAAAA TATT Plasmid site Outlier GAAGA CGAAA GGACT GCTAT TCAG 1 Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasmid Foreign DNA What is the percent similarity among the viruses? has the genes for lactose hydrolysis (the lacZ gene encodes the enzyme 243 249 252 254 257 b-galactosidase) and ampicillin Restriction resistance. sites way to predict the DNA sequence is by knowing the amino 2 Foreign DNA will insert into Recombinant acid sequence of the gene’s protein product. If this amino the lacZ gene. The bacterium plasmid acid sequence is known, in principle you can work backward receiving the plasmid vector through the genetic code to obtain the DNA sequence. Unfor- will not produce the enzyme b-galactosidase if foreign tunately, the degeneracy of the code prevents definitive deter- DNA has been inserted into mination; thus, if the protein contains a leucine, for example, the plasmid. which of the six codons for leucine is the one in the gene? For these reasons, it’s rare to clone a gene by synthesizing 3 The recombinant plasmid it directly, although some commercial products such as insu- is introduced into a Bacterium lin, interferon, and somatostatin are produced from chemically bacterium, which becomes ampicillin resistant. synthesized genes. Desired restriction sites are added to the synthetic genes so the genes can be inserted into plasmid vec- tors and cloned in E. coli. Synthetic DNA plays a much more 4 All treated bacteria are spread on a nutrient agar plate useful role in selection procedures, as we will see. containing ampicillin and a b-galactosidase substrate and CHECK YOUR UNDERSTANDING incubated. The b-galactosidase substrate is called X-gal. Colonies ✓ 9-8 Contrast the five ways of putting DNA into a cell. with foreign DNA ✓ 9-9 What is the purpose of a genomic library? 5 Only bacteria that picked up the plasmid will grow in the ✓ 9-10 Why isn’t cDNA synthetic? presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and Selecting a Clone an indigo compound. The indigo turns the colonies blue. In cloning, it’s necessary to select the particular cell that con- Bacteria that cannot hydrolyze tains the specific gene of interest. This is difficult to do because X-gal produce white colonies. out of millions of cells, only a very few might contain the Figure 9.11 Blue-white screening, one method of selecting desired gene. Here we’ll examine a typical screening procedure recombinant bacteria. known as blue-white screening, from the color of the bacterial colonies formed at the end of the screening process. Q Why are some colonies blue and others white?  CHAPTER 9   Biotechnology and DNA Technology 2 has not successfully received the ampicillin-resistance gene from the plasmid. The other, called X-gal, is a substrate for Master plate with β-galactosidase. colonies of bacteria Only bacteria that picked up the plasmid will grow, because containing cloned segments of foreign they are now ampicillin resistant. Bacteria that picked up the genes recombinant plasmid—in which the new gene was inserted into the lacZ gene—will not hydrolyze lactose and will produce white colonies. If a bacterium received the original plasmid Nitrocellulose 1 Make replica of master filter plate on nitrocellulose containing the intact lacZ gene, the cells will hydrolyze X-gal filter. to produce a blue-colored compound; the colony will be blue. What remains to be done can still be difficult. The above procedure has isolated white colonies known to contain for- eign DNA, but it is still not known whether it’s the desired frag- ment of foreign DNA. A second procedure is needed to identify 2 Treat filter with detergent (SDS) to these bacteria. If the foreign DNA in the plasmid encodes the lyse bacteria. production of an identifiable product, the bacterial isolate only needs to be grown in culture and tested. However, in some cases the gene itself must be identified in the host bacterium. Strands of Colony hybridization is a common method of identifying 3 Treat filter with sodium bacterial DNA cells that carry a specific cloned gene. DNA probes, short seg- hydroxide (NaOH) to separate DNA into ments of single-stranded DNA that are complementary to the single strands. desired gene, are synthesized. If the DNA probe finds a match, it will adhere to the target gene. The DNA probe is labeled with an enzyme or fluorescent dye so its presence can be detected. A typical colony hybridization experiment is shown in Figure 9.12. Fluorescence- labeled probes An array of DNA probes arranged in a DNA chip can be used to 4 Add labeled probes. identify pathogens (see Figure 10.17, page 287). Making a Gene Product We have just seen how to identify cells carrying a particular gene. Gene products are frequently the reason for genetic mod- Bound Gene of DNA 5 Probe will hybridize ification. Most of the earliest work in genetic modification used probe interest with desired gene from E. coli to synthesize the gene products. E. coli is easily grown, Single- bacterial cells. and researchers are very familiar with the bacterium and its stranded genetics. For example, some inducible promoters, such as that DNA of the lac operon, have been cloned, and cloned genes can be attached to such promoters. The synthesis of great amounts of the cloned gene product can then be directed by the addi- 6 Wash filter to remove unbound probe. tion of an inducer. Such a method has been used to produce gamma interferon in E. coli (Figure 9.13). However, E. coli also has several disadvantages. Like other gram-negative bacteria, it produces endotoxins as part of the outer layer of its cell wall. Colonies containing genes of interest 7 Compare filter with Because endotoxins cause fever and shock in mammals, their replica of master plate accidental presence in products intended for human use would Replica plate to identify colonies be a serious problem. containing gene of interest. Another disadvantage of E. coli is it doesn’t usually secrete protein products. To obtain a product, cells must usually be broken open and the product purified from the resulting Figure 9.12 Colony hybridization: using a DNA probe to “soup” of cell components. Recovering the product from such identify a cloned gene of interest. a mixture is expensive when done on an industrial scale. It’s Q What is a DNA probe? more economical to have an organism secrete the product so it 254 PART ONE Fundamentals of Microbiology against infection. To produce huge amounts of CSF industri- ally, the gene is first inserted into a plasmid. Bacteria are used to make multiple copies of the plasmid (see Figure 9.1), and the resulting recombinant plasmids are then inserted into mam- malian cells that are grown in bottles. Plant cells can also be grown in culture, altered by rDNA techniques, and then used to generate genetically modified plants. Such plants may prove useful as sources of valuable products, such as plant alkaloids (the painkiller codeine, for example), the isoprenoids that are the basis of synthetic rubber, and melanin (the animal skin pigment) for use in sunscreens. Genetically modified plants have many advantages for the pro- duction of human therapeutic agents, including vaccines and antibodies. The advantages include large-scale, low-cost agri- TEM 0.25 mm cultural production and a low risk of product contamination Figure 9.13 E. coli genetically modified to produce gamma by mammalian pathogens or cancer-causing genes. Geneti- interferon, a human protein that promotes an immune cally modifying plants often requires use of a bacterium. We’ll response. The product, visible here as a red mass in a violet ring, can return to the topic of genetically modified plants later in the be released by lysis of the cell. chapter (page 260). Q What is one advantage of using E. coli for genetic engineering? CHECK YOUR UNDERSTANDING One disadvantage? ✓ 9-11 How are recombinant clones identified? ✓ 9-12 What types of cells are used for cloning rDNA? can be recovered continuously from the growth medium. One approach has been to link the product to a natural E. coli pro- tein that the bacterium does secrete. However, gram-positive Applications of DNA Technology bacteria, such as Bacillus subtilis, are more likely to secrete their LEARNING OBJECTIVES products and are often preferred industrially for that reason. Another microbe being used as a vehicle for expressing 9-13 List at least five applications of DNA technology. rDNA is baker’s yeast, Saccharomyces cerevisiae. Its genome is 9-14 Define RNAi. only about four times larger than that of E. coli and is prob- 9-15 Discuss the value of genome projects. ably the best understood eukaryotic genome. Yeasts may carry 9-16 D  efine the following terms: random shotgun sequencing, plasmids, which are easily transferred into yeast cells whose bioinformatics, proteomics. cell walls have been removed. As eukaryotic cells, yeasts may 9-17 D  iagram the Southern blotting procedure, and provide an be more successful in expressing foreign eukaryotic genes than example of its use. bacteria. Furthermore, yeasts are likely to continuously secrete 9-18 D  iagram DNA fingerprinting, and provide an example the product. Because of all these factors, yeasts have become of its use. the eukaryotic workhorse of biotechnology. Mammalian cells in culture, even human cells, can be 9-19 Outline genetic engineering with Agrobacterium. genetically modified much like bacteria to produce various products. Scientists have developed effective methods of grow- ing certain mammalian cells in culture as hosts for growing CLINICAL CASE viruses (see Chapter 13, page 371). Mammalian cells are often the best suited to making protein products for medical use because the cells secrete their products and there’s a low risk T he sequences from Dr. B. and patients A, B, C, E, and G share 87.5% of the nucleotide sequence, which is comparable to reported similarities for known linked of toxins or allergens. Using mammalian cells to make foreign infections. gene products on an industrial scale often requires a prelimi- Identify the amino acids encoded by the viral DNA. Did this nary step of cloning the gene in bacteria. Consider the exam- change the percent similarity? (Hint: Refer to Figure 8.8 on ple of colony-stimulating factor (CSF). A protein produced page 214). naturally in tiny amounts by white blood cells, CSF is valuable because it stimulates the growth of certain cells that protect 243 249 252 254 257  CHAPTER 9   Biotechnology and DNA Technology 2 We have now described the entire sequence of events in cloning DNA vaccines are usually circular plasmids that include a a gene. As indicated earlier, such cloned genes can be applied gene encoding a viral protein that’s under the transcriptional in a variety of ways. One is to produce useful substances more control of a promoter region active in human cells. The plas- efficiently and less expensively. Another is to obtain infor- mids are then cloned in bacteria. A DNA vaccine to protect mation from the cloned DNA that is useful for either basic against Zika virus disease is currently in clinical trials. Vac- research, medicine, or forensics. A third is to use cloned genes cines are discussed in further detail in Chapter 18 (page 503). to alter the characteristics of cells or organisms. Table 9.2 lists some other important rDNA products used in medical therapy. Therapeutic Applications The importance of rDNA technology to medical research An extremely valuable pharmaceutical product is the hormone cannot be emphasized enough. Artificial blood for use in insulin, a small protein produced by the pancreas that controls transfusions can now be prepared with human hemoglobin the body’s uptake of glucose from blood. For many years, peo- produced in genetically modified pigs. Sheep have also been ple with insulin-dependent diabetes controlled their disease by genetically modified to produce a number of drugs in their injecting insulin obtained from the pancreases of slaughtered milk. This procedure has no apparent effect upon the sheep, animals. Obtaining this insulin is an expensive process, and and they provide a ready source of raw material for the product the insulin from animals is not as effective as human insulin. that does not require sacrificing animals. Because of the value of human insulin and the protein’s Gene therapy may eventually provide cures for some small size, producing human insulin by rDNA techniques was genetic diseases. It is possible to imagine removing some cells an early goal for the pharmaceutical industry. To produce the from a person and transforming them with a normal gene hormone, synthetic genes were first constructed for each of the to replace a defective or mutated gene. When these cells are two short polypeptide chains that make up the insulin mol- returned to the person, they should function normally. For ecule. The small size of these chains—only 21 and 30 amino example, gene therapy has been used to treat hemophilia acids long—made it possible to use synthetic genes. Follow- B and severe combined immunodeficiency. Adenoviruses ing the procedure described earlier (page 249), each of the and retroviruses are used most often to deliver genes; how- two synthetic genes was inserted into a plasmid vector and ever, some researchers are working with plasmid vectors. An linked to the end of a gene coding for the bacterial enzyme attenuated retrovirus was used as the vector when the first β-galactosidase, so that the insulin polypeptide was copro- gene therapy to treat hemophilia in humans was performed duced with the enzyme. Two different E. coli bacterial cultures in 1990. Glybera® is a gene therapy drug licensed in Europe were used, one to produce each of the insulin polypeptide to treat lipoprotein lipase deficiency. It uses an adenovirus chains. The polypeptides were then recovered from the bacte- to deliver the lipase gene to cells. Antisense DNA (page 262) ria, separated from the β-galactosidase, and chemically joined introduced into cells is also being explored. Fomivirsen is an to make human insulin. This accomplishment was one of the antisense DNA drug used in the treatment of cytomegalovirus early commercial successes of DNA technology, and it illus- retinitis. trates a number of the principles and procedures discussed in Thus far, gene therapy results have not been impressive; this chapter. there have even been a few deaths attributed to the viral vec- Another human hormone that is now being produced com- tors. A great deal of preliminary work remains to be done, but mercially by genetic modification of E. coli is somatostatin. At cures may not be possible for all genetic diseases. one time 500,000 sheep brains were needed to produce 5 mg of Gene editing is a promising new technology to correct animal somatostatin for experimental purposes. By contrast, genetic mutations at precise locations. Gene editing uses only 8 liters of a genetically modified bacterial culture are CRISPR (pronounced “crisper”), which stands for clustered now required to obtain the equivalent amount of the human regularly interspaced short palindromic repeats. CRISPR hormone. enzymes are found in archaea and bacteria, where they Subunit vaccines, consisting only of a protein portion of destroy foreign DNA. A small RNA molecule, complementary a pathogen, are being made by genetically modifying yeasts. to the desired target, binds DNA, and then the Cas9 enzyme Subunit vaccines have been produced for a number of diseases, cuts the DNA like molecular scissors. The cell’s DNA poly- notably hepatitis B. One of the advantages of a subunit vac- merase and DNA ligase reattach the ends. A researcher can cine is that there is no chance that the vaccine will cause an add template DNA for the correct gene, which can be attached infection. The protein is harvested from genetically modified by the DNA ligase. If may be possible to correct mutations in cells and purified for use as a vaccine. Animal viruses such as the human genome to treat genetic causes of disease. Gene vaccinia virus can be genetically modified to carry a gene for editing was used to repair a defective muscle protein gene in another microbe’s surface protein. When injected, the virus mice with Duchenne muscular dystrophy. A parvovirus was acts as a vaccine against the other microbe. used to deliver the gene-editing system into mice. In 2016, the 256 PART ONE Fundamentals of Microbiology TABLE 9.2 Some Pharmaceutical Products of rDNA Product Comments Cervical Cancer Vaccine Consists of viral proteins; produced by Saccharomyces cerevisiae or by insect cells Epidermal Growth Factor (EGF) Heals wounds, burns, ulcers; produced by E. coli Erythropoietin (EPO) Treatment of anemia; produced by mammalian cell culture Interferon IFN–a Therapy for leukemia, melanoma, and hepatitis; produced by E. coli and S. cerevisiae (yeast) IFN–b Treatment for multiple sclerosis; produced by mammalian cell culture IFN–g Treatment of chronic granulomatous disease; produced by E. coli Hepatitis B Vaccine Produced by S. cerevisiae that carries hepatitis-virus gene on a plasmid Human Growth Hormone (hGH) Corrects growth deficiencies in children; produced by E. coli Human Insulin Therapy for diabetes; better tolerated than insulin extracted from animals; produced by E. coli Influenza Vaccine Vaccine made from E. coli or S. cerevisiae carrying virus genes Interleukins Regulate the immune system; possible treatment for cancer; produced by E. coli Orthoclone OKT3 Monoclonal antibody used in transplant patients to help suppress the immune system, reducing the chance of Muromonab-CD3 tissue rejection; produced by mouse cells Pulmozyme (rhDNase) Enzyme used to break down mucous secretions in cystic fibrosis patients; produced by mammalian cell culture Relaxin Used to ease childbirth; produced by E. coli Superoxide Dismutase (SOD) Minimizes damage caused by oxygen free radicals when blood is resupplied to oxygen-deprived tissues; produced by S. cerevisiae and Komagataella pastoris (yeast) Taxol Plant product used for treating ovarian cancer; produced in E. coli Tissue Plasminogen Activator Dissolves the fibrin of blood clots; therapy for heart attacks; produced by mammalian cell culture Tumor Necrosis Factor (TNF) Causes disintegration of tumor cells; produced by E. coli Veterinary Use Canine Distemper Vaccine Canarypox virus carrying canine distemper virus genes Feline Leukemia Vaccine Canarypox virus carrying feline leukemia virus genes first clinical trials were approved to modify a patient’s T cells CHECK YOUR UNDERSTANDING (see page 476) to fight cancer. Gene silencing is a natural process that occurs in a wide ✓ 9-13 Explain how DNA technology can be used to treat variety of eukaryotes and is apparently a defense against viruses disease and to prevent disease. and transposons. Gene silencing is similar to miRNA (page 219) ✓ 9-14 What is gene silencing? in that a gene encoding a small piece of RNA is transcribed. Following transcription, RNAs called small interfering RNAs Genome Projects (siRNAs) are formed after processing by an enzyme called Dicer. The first genome to be sequenced was from a bacteriophage in The siRNA molecules bind to mRNA, which is then destroyed 1977. In 1995, the genome of a free-living cell—Haemophilus influ- by proteins called the RNA-induced silencing complex (RISC), enzae—was sequenced. Since then, 1000 prokaryotic genomes thus silencing the expression of a gene (Figure 9.14). and over 400 eukaryotic genomes have been sequenced. New technology called RNA interference (RNAi) holds In shotgun sequencing, small pieces of a genome of a free- promise for gene therapy for treating genetic diseases. A small living cell are sequenced, and the sequences are then assem- DNA insert encoding siRNA against the gene of interest could bled using a computer. Any gaps between the pieces then be cloned into a plasmid. When transferred into a cell, the cell have to be found and sequenced (Figure 9.15). This technique would produce the desired siRNA. Clinical trials are currently can be used on environmental samples to study the genomes being conducted to test RNAi to prevent Ebola and respiratory of microorganisms that haven’t been cultured. The study of syncytial virus infections.  CHAPTER 9   Biotechnology and DNA Technology 2 eventually be of great medical benefit, especially for the diag- CLINICAL CASE Resolved nosis and treatment of genetic diseases. T he amino acid sequence reflects the nucleotide sequence. Analysis of the amino acid signature pattern confirms that the viruses from the dentist and patients are closely Scientific Applications Recombinant DNA technology can be used to make products, but related. HIV has a high mutation rate, so HIVs from different this isn’t its only important application. Because of its ability to individuals are genetically distinct. Dr. B.’s HIV is different produce many copies of DNA, it can serve as a sort of DNA “print- from the local control and from the outlier. Dr. B.’s amino acid ing press.” Once a large amount of a particular piece of DNA is sequences and those of patients A, B, C, E, and G are distinct available, various analytic techniques, discussed in this section, from those in the control and in the outlier and from two can be used to “read” the information contained in the DNA. dental patients with known behavioral risks for HIV infection. In 2010, researchers synthesized the smallest known cellu- PCR and RFLP analyses have made it possible to track lar genome during the Minimal Genome Project. A copy of the transmission of disease between individuals, communities, Mycoplasma mycoides genome was synthesized and transplanted and countries. This tracking works best with pathogens that into an M. capricolum cell that had had its own DNA removed. have enough genetic variation to identify different strains. The modified cell produced M. mycoides proteins. This experi- *** ment showed that large-scale changes to a genome can be made Dr. B. died before the mode of transmission could be established. But in the era when he practiced dentistry, it and that an existing cell will accept this DNA. was not always the norm to wear gloves when performing DNA sequencing has produced an enormous amount of procedures. Patient interviews indicated that Dr. B. didn’t like information that has spawned the new field of bioinformatics, to wear gloves. It is likely that HIV was transmitted when a cut the science of understanding the function of genes through on the doctor’s bare hands allowed the virus to enter patients’ computer-assisted analysis. DNA sequences are stored in gums. Today the CDC and state health departments ask dental care providers to use universal precautions, including wearing gloves and masks and sterilizing equipment that is Nucleus to be reused. Had Dr. B. used standard precautions, it is DNA extremely unlikely he would have infected patients. 1 An abnormal gene, cancer 243 249 252 254 257 gene, or virus gene is transcribed in a host cell. RNA genetic material taken directly from environmental samples is transcript called metagenomics. The Human Genome Project was an international 13-year effort, formally begun in October 1990 and completed in 2003. The goal of the project was to sequence the entire human mRNA genome, approximately 3 billion nucleotide pairs, compris- ing 20,000 to 25,000 genes. Thousands of people in 18 coun- siRNA tries participated in this project. Researchers collected blood (female) or sperm (male) samples from a large number of 2 siRNA binds donors. Only a few samples were processed as DNA resources, mRNA. and the source names are protected so that neither donors nor scientists knew whose samples were used. Development of shotgun sequencing greatly speeded the process, and 99% of 3 RISC breaks the genome has been sequenced. down the RNA complex. One surprising finding was that less than 2% of the genome encodes a functional product—the other 98% includes miRNA genes, viral remnants, repetitive sequences (called short tandem repeats), introns, the chromosome ends (called telomeres), and 4 No protein expression transposons (page 231). Cytoplasm occurs. The next goal of researchers is the Human Proteome Proj- ect, which will map all the proteins expressed in human cells. Figure 9.14 Gene silencing could provide treatments for a wide range of diseases. Even before it is completed, however, it’s yielding data that are of immense value to our understanding of biology. It will also Q Does RNAi act during or after transcription? 258 PART ONE Fundamentals of Microbiology 1 Isolate DNA. 4 Sequence DNA fragments. 5 Assemble sequences. 2 Fragment DNA with restriction enzymes. 6 Edit 3 Clone DNA sequences; in a bacterial ACTGT TC fill in gaps. artificial chromosome (BAC). BAC (a) Constructing a gene library (b) Random sequencing (c) Closure phase Figure 9.15 Shotgun sequencing. In this technique, a genome is cut into pieces, and each piece is sequenced. Then the pieces are fit together. There may be gaps if a specific DNA fragment was not sequenced. Q Does this technique identify genes and their locations? web-based databases referred to as GenBank. Genomic infor- in a well at one end of a layer of agarose gel. Then an electrical mation can be searched with computer programs to find spe- current is passed through the gel. While the charge is applied, cific sequences or to look for similar patterns in the genomes of the different-sized RFLPs migrate through the gel at different different organisms. Microbial genes are now being searched to rates. The RFLPs are transferred onto a filter by blotting and identify molecules that are the virulence factors of pathogens. are exposed to a labeled probe made from the cloned gene of By comparing genomes, researchers discovered that Chlamydia interest, in this case the CF gene. The probe will hybridize to trachomatis (tra-KŌ-ma-tis) produces a toxin similar to that of this mutant gene but not to the normal gene. Fragments to Clostridium difficile (DIF-fi-sē-il). which the probe binds are identified by a colored dye. With The next goal is to identify the proteins encoded by these this method, any person’s DNA can be tested for the presence genes. Proteomics is the science of determining all of the pro- of the mutated gene. teins expressed in a cell. Genetic testing can now be used to screen for several hun- Reverse genetics is an approach to discovering the function dred genetic diseases. Such screening procedures can be per- of a gene from a genetic sequence. Reverse genetics attempts to formed on prospective parents and also on fetal tissue. Two of connect a given genetic sequence with specific effects on the the more commonly screened genes are those associated with organism. For example, if you mutate or block a gene (see the inherited forms of breast cancer and the gene responsible for earlier discussions of gene editing on page 255 and gene silenc- Huntington’s disease. Genetic testing can help a physician pre- ing on page 256), you can then look for a characteristic the scribe the correct medication for a patient. The drug herceptin, organism lost. for example, is effective only in breast cancer patients with a An example of the use of human DNA sequencing is the specific nucleotide sequence in the HER2 gene. identification and cloning of the mutant gene that causes cystic fibrosis (CF). CF is characterized by the oversecretion of mucus, Forensic Microbiology leading to blocked respiratory passageways. The sequence of the For several years, microbiologists have used RFLPs in a method mutated gene can be used as a diagnostic tool in a hybridization of identification known as DNA fingerprinting to identify technique called Southern blotting (Figure 9.16), named for Ed bacterial or viral pathogens (Figure 9.17). Southern, who developed the technique in 1975. DNA chips (see Figure 10.18, page 288) or PCR microarrays In this technique, subject DNA is digested with a restriction that can screen a sample for multiple pathogens at once are enzyme, yielding thousands of fragments of various sizes. The now being used. In a DNA chip, up to 22 primers from dif- fragments are called RFLPs (pronounced “rif-lip”), for restric- ferent microorganisms can be used to initiate the PCR. A sus- tion fragment length polymorphisms. The different fragments are pect microorganism is identified if DNA is copied from one of then separated by gel electrophoresis. The fragments are put the primers. At the Centers for Disease Control and Prevention  CHAPTER 9   Biotechnology and DNA Technology 2