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Biotechnology and DNA Technology

Learning Objectives	Check Your Understanding
9-1    Compare and contrast biotechnology, genetic modification, and recombinant DNA technology.	Differentiate biotechnology and recombinant DNA technology.
9-2    Identify the roles of a clone and a vector in making recombinant DNA.	In one sentence, describe how a vector and clone are used.
9-3    Compare selection and mutation.	How are selection and mutation used in biotechnology?
9-4    Define restriction enzymes, and outline how they are used to make recombinant DNA.	What is the value of restriction enzymes in recombinant DNA technology?
9-5    List the four properties of vectors.	What criteria must a vector meet?
9-6    Describe the use of plasmid and viral vectors.	Why is a vector used in recombinant DNA technology?
9-7    Outline the steps in PCR, and provide an example of its use.	For what is each of the following used in PCR: primer, DNA polymerase, 94°C?
9-8    Describe five ways of getting DNA into a cell.	Contrast the five ways of putting DNA into a cell.
9-9    Describe how a genomic library is made.	What is the purpose of a genomic library?
9-10  Differentiate cDNA from synthetic DNA.	Why isn’t cDNA synthetic?
9-11  Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products.	How are recombinant clones identified?
9-12  List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, plant cells.	What types of cells are used for cloning rDNA?
9-13  List at least five applications of DNA technology.	Explain how DNA technology can be used to treat disease and to prevent disease.
9-14  Define RNAi.	What is gene silencing?
9-15  Discuss the value of genome projects.	How are shotgun sequencing, bioinformatics, and proteomics related to genome projects?
9-16  Define the following terms: random shotgun sequencing, bioinformatics, and proteomics.
9-17  Diagram the Southern blotting procedure, and provide an example of its use.	What is Southern blotting?
9-18  Diagram DNA fingerprinting, and provide an example of its use.	Why do RFLPs result in a DNA fingerprint?
9-19  Outline genetic engineering with Agrobacterium.	Of what value is the plant pathogen Agrobacterium?
9-20  List the advantages of, and problems associated with, the use of genetic modification techniques.	Identify two advantages and two problems associated with genetically modified organisms.

 

Chapter Summary

Introduction to Biotechnology (pp. 239–241)

ASM 4.5: Cell genomes can be manipulated to alter cell function.

1. Biotechnology is the use of microorganisms, cells, or cell components to make a product.

Recombinant DNA Technology (p. 239)

2. Closely related organisms can exchange genes in natural recombination.
3. Genes can be transferred among unrelated species via laboratory manipulation, called recombinant DNA technology.
4. Recombinant DNA is DNA that has been artificially manipulated to combine genes from two different sources.

An Overview of Recombinant DNA Procedures (pp. 239–241)

5. A desired gene is inserted into a DNA vector, such as a plasmid or a viral genome.
6. The vector inserts the DNA into a new cell, which is grown to form a clone.
7. Large quantities of the gene product can be harvested from the clone.

Tools of Biotechnology (pp. 241–244)

Selection (p. 241)

1. Microbes with desirable traits are selected for culturing by artificial selection.

Mutation (p. 241)

2. Mutagens are used to cause mutations that might result in a microbe with desirable traits.
3. Site-directed mutagenesis is used to change a specific codon in a gene.

Restriction Enzymes (pp. 241–242)

4. Prepackaged kits are available for rDNA techniques.
5. A restriction enzyme recognizes and cuts only one particular nucleotide sequence in DNA.
6. Some restriction enzymes produce sticky ends, short stretches of single-stranded DNA at the ends of the DNA fragments.
7. Fragments of DNA produced by the same restriction enzyme will spontaneously join by base pairing. DNA ligase can covalently link the DNA backbones.

Vectors (pp. 242–243)

8. Vectors are DNA used to transfer other DNA between cells.
9. A plasmid containing a new gene can be inserted into a cell by transformation.
10. A virus containing a new gene can insert the gene into a cell.

Polymerase Chain Reaction (pp. 243–244)

11. The polymerase chain reaction (PCR) is used to make multiple copies of a desired piece of DNA enzymatically.
12. PCR can be used to increase the amounts of DNA in samples to detectable levels. This may allow sequencing of genes, the diagnosis of genetic diseases, or the detection of viruses.

     

PCR Links to an external site.

 

Techniques of Genetic Modification (pp. 244–250)

Inserting Foreign DNA into Cells (pp. 245–246)

1. Cells can take up naked DNA by transformation. Chemical treatments are used to make cells that are not naturally competent take up DNA.
2. Pores made in protoplasts and animal cells by electric current in the process of electroporation can provide entrance for new pieces of DNA.
3. Protoplast fusion is the joining of cells whose cell walls have been removed.
4. Foreign DNA can be introduced into plant cells by shooting DNA-coated particles into the cells.
5. Foreign DNA can be injected into animal cells by using a fine glass micropipette.

 

Obtaining DNA (pp. 246–248)

6. Genomic libraries can be made by cutting up an entire genome with restriction enzymes and inserting the fragments into bacterial plasmids or phages.
7. Complementary DNA (cDNA) made from mRNA by reverse transcription can be cloned in genomic libraries.
8. Synthetic DNA can be made in vitro by a DNA synthesis machine.

Selecting a Clone (pp. 248–249)

9. Antibiotic-resistance markers on plasmid vectors are used to identify cells containing the engineered vector by direct selection.
10. In blue-white screening, the vector contains the genes for ampR and β-galactosidase.
11. The desired gene is inserted into the β-galactosidase gene site, destroying the gene.
12. Clones containing the recombinant vector will be resistant to ampicillin and unable to hydrolyze X-gal (white colonies). Clones containing the vector without the new gene will be blue. Clones lacking the vector will not grow.
13. Clones containing foreign DNA can be tested for the desired gene product.
14. A short piece of labeled DNA called a DNA probe can be used to identify clones carrying the desired gene.

 

Making a Gene Product (pp. 249–250)

15. E. coli is used to produce proteins using rDNA because E. coli is easily grown and its genomics are well understood.
16. Efforts must be made to ensure that E. coli’s endotoxin does not contaminate a product intended for human use.
17. To recover the product, E. coli must be lysed, or the gene must be linked to a gene that produces a naturally secreted protein.
18. Yeasts can be genetically modified and are likely to secrete a gene product continuously.
19. Genetically modified mammalian cells can be grown to produce proteins such as hormones for medical use.
20. Genetically modified plant cells can be grown and used to produce plants with new properties.

Applications of DNA Technology (pp. 250–258)

ASM 6.3: Humans utilize and harness microorganisms and their products.

ASM 4.5: Cell genomes can be manipulated to alter cell function.

ASM 6.2: Microorganisms provide essential models that give us fundamental knowledge about life processes.

1. Cloned DNA is used to produce products, study the cloned DNA, and alter the phenotype of an organism.

Therapeutic Applications (pp. 251–252)

2. Synthetic genes linked to the β-galactosidase gene (lacZ) in a plasmid vector were inserted into E. coli, allowing E. coli to produce and secrete the two polypeptides used to make human insulin.
3. Cells and viruses can be modified to produce a pathogen’s surface protein, which can be used as a vaccine.
4. DNA vaccines consist of rDNA cloned in bacteria.
5. Gene therapy can be used to cure genetic diseases by replacing the defective or missing gene.
6. RNAi may be useful to prevent expression of abnormal proteins.

Genome Projects (p. 252–254)

7. Nucleotide sequences of the genomes of over 1,000 organisms, including humans, have been completed.
8. This leads to determining the proteins produced in a cell.

Scientific Applications (pp. 254–256)

9. DNA can be used to increase understanding of DNA, for genetic fingerprinting, and for gene therapy.
10. DNA sequencing machines are used to determine the nucleotide base sequence of restriction fragments in shotgun sequencing.
11. Bioinformatics is the use of computer applications to study genetic data; proteomics is the study of a cell’s proteins.
12. Southern blotting can be used to locate a gene in a cell.
13. DNA probes can be used to quickly identify a pathogen in body tissue or food.
14. Forensic microbiologists use DNA fingerprinting to identify the source of bacterial or viral pathogens.
15. Bacteria may be used to make nano-sized materials for nanotechnology machines.

Agricultural Applications (pp. 256–258)

16. Cells from plants with desirable characteristics can be cloned to produce many identical cells. These cells can then be used to produce whole plants from which seeds can be harvested.
17. Plant cells can be modified by using the Ti plasmid vector. The tumor-producing T genes are replaced with desired genes, and the recombinant DNA is inserted into
    Agrobacterium. The bacterium naturally transforms its plant hosts.
18. Antisense DNA can prevent expression of unwanted proteins.

Safety Issues and the Ethics of Using DNA Technology (pp. 258–260)

1. Strict safety standards are used to avoid the accidental release of genetically modified microorganisms.
2. Some microbes used in rDNA cloning have been altered so that they cannot survive outside the laboratory.
3. Microorganisms intended for use in the environment may be modified to contain suicide genes so that the organisms do not persist in the environment.
4. Genetic testing raises a number of ethical questions: Should employers and insurance companies have access to a person’s genetic records? Will some people be targeted for either breeding or sterilization? Will genetic counseling be available to everyone?
5. Genetically modified crops must be safe for consumption and for release in the environment.

Classification of Microorganisms

Learning Objectives	Check Your Understanding
10-1    Define taxonomy, taxon, and phylogeny.	Of what value are taxonomy and systematics?
10-2    Discuss the limitations of a two-kingdom classification system.	Why shouldn’t bacteria be placed in the plant kingdom?
10-3    Identify the contributions of Linnaeus, von Nägeli, Chatton, Whittaker, and Woese.
10-4    Discuss the advantages of the three-domain system.	What evidence supports classifying organisms into three domains?
10-5    List the characteristics of the Bacteria, Archaea, and Eukarya domains.	Compare archaea and bacteria; bacteria and eukarya; and archaea and eukarya.
10-6    Explain why scientific names are used.	Using Escherichia coli and Entamoeba coli as examples, explain why the genus name must always be written out for the first use. Why is binomial nomenclature preferable to the use of common names?
10-7    List the major taxa.	Find the gram-positive bacteria Staphylococcusin Appendix F. To which bacteria is this genus more closely related: Bacillus or Streptococcus?
10-8    Differentiate culture, clone, and strain.	Use the terms species, culture, clone, and strain in one sentence to describe growing methicillin-resistant Staphylococcus aureus (MRSA).
10-9    List the major characteristics used to differentiate the three kingdoms of multicellular Eukarya.	Assume you discovered a new organism: it is multicellular, is nucleated, is heterotrophic, and has cell walls. To what kingdom does it belong?
10-10  Define protist.	Write your own definition of protist.
10-11  Differentiate eukaryotic, prokaryotic, and viral species.	Why wouldn’t the definition of a viral species work for a bacterial species?
10-12  Compare and contrast classification and identification.	Classification groups living organisms according to similar characteristics. Identification is a means to identify an unknown organism. Is a cladogram used for identification or classification?
10-13  Explain the purpose of Bergey’s Manual.	What is in Bergey’s Manual?
10-14  Describe how staining and biochemical tests are used to identify bacteria.	Design a rapid test for a Staphylococcus aureus. (Hint: See Figure 6.10, page 166.)
10-15  Differentiate Western blotting from Southern blotting.	What is tested in Western blotting and Southern blotting?
10-16  Explain how serological tests and phage typing can be used to identify an unknown bacterium.	What is identified by phage typing?
10-17  Describe how a newly discovered microbe can be classified by DNA base composition, DNA fingerprinting, and PCR.	Why does PCR identify a microbe?
10-18  Describe how microorganisms can be identified by nucleic acid hybridization, Southern blotting, DNA chips, ribotyping, and FISH.	Which techniques involve nucleic acid hybridization?
10-19  Differentiate a dichotomous key from a cladogram.	Is a cladogram used for identification or classification?

 

Chapter Summary

Introduction (p. 264)

ASM 1.5: The evolutionary relatedness of organisms is best reflected in phylogenetic trees.

1. Taxonomy is the science of the classification of organisms. Its goal is to show relationships among organisms.
2. Taxonomy also provides a means of identifying organisms.

The Study of Phylogenetic Relationships (pp. 265–268)

1. Phylogeny is the evolutionary history of a group of organisms.
2. The taxonomic hierarchy shows evolutionary, or phylogenetic, relationships among organisms.
3. Bacteria were separated into the Kingdom Prokaryotae in 1968.
4. Living organisms were divided into five kingdoms in 1969.

The Three Domains (pp. 265–268)

5. Living organisms are currently classified into three domains. A domain can be divided into kingdoms.
6. In this system, plants, animals, and fungi belong to the Domain Eukarya.
7. Bacteria (with peptidoglycan) form a second domain.
8. Archaea (with unusual cell walls) are placed in the Domain Archaea.

A Phylogenetic Tree (pp. 268–269)

9. Organisms are grouped into taxa according to phylogenetic relationships (from a common ancestor).
10. Some of the information for eukaryotic relationships is obtained from the fossil record.
11. Prokaryotic relationships are determined by rRNA sequencing.

Classification of Organisms (pp. 269–272)

ASM 1.4: The traditional concept of species is not readily applicable to microbes due to asexual reproduction and the frequent occurrence of horizontal gene transfer.

1. According to scientific nomenclature, each organism is assigned two names, or a binomial: a genus and a specific epithet, or species.

The Taxonomic Hierarchy (p. 270)

2. A eukaryotic species is a group of organisms that interbreed with each other but do not breed with individuals of another species.
3. Similar species are grouped into a genus; similar genera are grouped into a family; families, into an order; orders, into a class; classes, into a phylum; phyla, into a kingdom; and kingdoms, into a domain.

Classification of Prokaryotes (p. 270)

4. Bergey’s Manual of Systematic Bacteriology is the standard reference on bacterial classification.
5. A group of bacteria derived from a single cell is called a strain.
6. Closely related strains constitute a bacterial species.

Classification of Eukaryotes (pp. 270–271)

7. Eukaryotic organisms may be classified into the Kingdom Fungi, Plantae, or Animalia.
8. Protists are mostly unicellular organisms; these organisms are currently being assigned to kingdoms.
9. Fungi are absorptive chemoheterotrophs that develop from spores.
10. Multicellular photoautotrophs are placed in the Kingdom Plantae.
11. Multicellular ingestive heterotrophs are classified as Animalia.

Classification of Viruses (pp. 271–272)

12. Viruses are not placed in a kingdom. They are not composed of cells and cannot grow without a host cell.
13. A viral species is a population of viruses with similar characteristics that occupies a particular ecological niche.

Methods of Classifying and Identifying Microorganisms (pp. 272–285)

ASM 8.3: Use appropriate methods to identify microorganisms (media-based, molecular, and serological).

1. Bergey’s Manual of Determinative Bacteriology is the standard reference for laboratory identification of bacteria.
2. Morphological characteristics are useful in identifying microorganisms, especially when aided by differential staining techniques.
3. The presence of various enzymes, as determined by biochemical tests, is used in identifying bacteria and yeasts.
4. Serological tests, involving the reactions of microorganisms with specific antibodies, are useful in determining the identity of strains and species, as well as relationships among organisms. ELISA and Western blotting are examples of serological tests.
5. Phage typing is the identification of bacterial species and strains by determining their susceptibility to various phages.
6. Fatty acid profiles can be used to identify some organisms.
7. Flow cytometry measures physical and chemical characteristics of cells.
8. The percentage of GC base pairs in the nucleic acid of cells can be used in the classification of organisms.
9. The number and sizes of DNA fragments, or DNA fingerprints, produced by restriction enzymes are used to determine genetic similarities.
10. NAATs can be used to amplify a small amount of microbial DNA in a sample. The presence or identification of an organism is indicated by amplified DNA.
11. Single strands of DNA, or of DNA and RNA, from related organisms will hydrogen-bond to form a double-stranded molecule; this bonding is called nucleic acid hybridization.
12. Southern blotting, DNA chips, and FISH are examples of nucleic acid hybridization techniques.
13. The sequence of bases in ribosomal RNA can be used in the classification of organisms.
14. Dichotomous keys are used for the identification of organisms. Cladograms show
    phylogenetic relationships among organisms.

 

 

         

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