Microbiology Biotechnology and DNA Technology PDF

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...

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.