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DNA Cloning

DNA cloning can isolate a gene and produce many copies of it. The gene can be studied in the laboratory or inserted into a bacterium, plant, or animal. Then, this gene may be transcribed and translated to produce a protein, which can become a commercial product or used as a medicine.

Two methods are currently available for making copies of DNA: recombinant DNA technology and the polymerase chain reaction (PCR). Recombinant DNA contains DNA from two different sources. A restriction enzyme is used to cleave plasmid DNA and to cleave foreign DNA. The resulting “sticky ends” facilitate the insertion of foreign DNA into vector DNA. The foreign gene is sealed into the vector DNA by DNA ligase. Both bacterial plasmids and viruses can be used as vectors to carry foreign genes into bacterial host cells.

PCR uses the enzyme DNA polymerase to quickly make multiple copies of a specific piece (target) of DNA. PCR is a chain reaction because the targeted DNA is replicated over and over again. Analysis of DNA segments following PCR has all sorts of uses from assisting genomic research to doing DNA fingerprinting for the purpose of identifying individuals and their paternity.

Biotechnology Products

Transgenic organisms have had a foreign gene inserted into them. Genetically modified bacteria, agricultural plants, and farm animals now produce commercial products of interest to humans, such as hormones and vaccines. Bacteria usually secrete the product. The seeds of plants and the milk of animals contain the product.

Transgenic bacteria have also been engineered to promote the health of plants, perform bioremediation, extract minerals, and produce chemicals. Transgenic crops, engineered to resist herbicides and pests, are commercially available. Transgenic animals have been given various genes, in particular the one for bovine growth hormone (bGH). Cloning of animals is now possible.

Gene Therapy

Gene therapy, by either ex vivo or in vivo methods, is used to correct the genotype of humans and to cure various human ills. Ex vivo gene therapy has apparently helped children with SCID lead normal lives. In vivo treatment for cystic fibrosis has been less successful. A number of in vivo therapies are being employed in the war against cancer and other human illnesses, such as cardiovascular disease.


Researchers now know the sequence of all the base pairs along the length of the human chromosomes. So far, researchers have found only 23,000 genes that code for proteins; the rest of our DNA consists of regions that do not code for a protein. Currently, researchers are placing an emphasis on functional and comparative genomics.

Genes only comprise 1.5% of the human genome. The rest of this DNA is surprisingly more active than once thought. About half of this DNA consists of repetitive DNA elements, which may be in tandem or interspersed throughout several chromosomes. Some of this DNA is made up of mobile DNA sequences called transposons, which are a driving evolutionary force within the genome. The remaining half of the genome remains unclassified, but even these unknown DNA sequences may play an important role in regulation of gene expression, and challenging the classical definition of the gene. Functional genomics aims to understand the function of protein coding regions and noncoding regions of our genome. To that end, researchers are utilizing new tools such as DNA microarrays. Microarrays can also be used to create an individual's genetic profile, which can be helpful in predicting illnesses and how a person will react to particular medications.

Comparative genomics has revealed that there is little difference between the DNA sequence of our bases and those of many other organisms. Genome comparisons have revolutionized our understanding of evolutionary relations by revealing previously unknown relationships between organisms.

Proteomics is the study of which genes are active in producing proteins in which cells under which circumstances. Bioinformatics is the use of the computer to assist proteomics and functional and comparative genomics.

understanding the terms

Match the terms to these definitions:

  1. Bacterial agent that stops viral reproduction by cleaving viral DNA; used to cut DNA at specific points during production of recombinant DNA.

  2. Free-living organism in the environment that has had a foreign gene inserted into it.

  3. Use of animal organs, instead of human organs, in human transplant patients.

  4. Production of identical copies; in genetic engineering, the production of many identical copies of a gene.

  5. Biotechnology method that can quickly produce many duplicate copies of a piece of DNA.

reviewing this chapter
  1. What is the methodology for producing recombinant DNA so useful for gene cloning? 250

  2. What is the polymerase chain reaction (PCR), and how is it carried out to produce multiple copies of a DNA segment? 25152

  3. How does STR profiling produce a DNA fingerprint? 25152

  4. What are some practical applications of DNA segment analysis following PCR? 252

  5. For what purposes have bacteria, plants, and animals been genetically altered? 25254

  6. Explain and give examples of ex vivo and in vivo gene therapies in humans. 25455

  7. What was the purpose of the Human Genome Project? What is the goal of functional genomics? 25558

  8. What insights into evolutionary relationships between organisms are arising from comparative genomics? 258

  9. Describe the various types of intergenic DNA sequences found within the genome. 25657

  10. What are the goals of proteomics and bioinformatics? 25860

testing yourself

Choose the best answer for each question.

  1. Using this key, put the phrases in the correct order to form a plasmid-carrying recombinant DNA.


    1. use restriction enzymes

    2. use DNA ligase

    3. remove plasmid from parent bacterium

    4. introduce plasmid into new host bacterium

      1. 1, 2, 3, 4

      2. 4, 3, 2, 1

      3. 3, 1, 2, 4

      4. 2, 3, 1, 4

  2. Restriction enzymes found in bacterial cells are ordinarily used

    1. during DNA replication.

    2. to degrade the bacterial cell's DNA.

    3. to degrade viral DNA that enters the cell.

    4. to attach pieces of DNA together.

  3. A genetic profile can

    1. assist in maintaining good health.

    2. be accomplished utilizing bioinformatics.

    3. show how many genes are normal.

    4. be accomplished utilizing a microarray.

    5. Both a and d are correct.

  4. Bacteria are able to successfully transcribe and translate human genes because

    1. both bacteria and humans contain plasmid vectors.

    2. bacteria can replicate their DNA, but humans cannot.

    3. human and bacterial ribosomes are vastly different.

    4. the genetic code is nearly universal.

  5. Bioinformatics can

    1. assist genomics and proteomics.

    2. compare our genome to that of a monkey.

    3. depend on computer technology.

    4. match up genes with proteins.

    5. All of these are correct.

  6. The polymerase chain reaction

    1. uses RNA polymerase.

    2. takes place in huge bioreactors.

    3. uses a temperature-insensitive enzyme.

    4. makes lots of nonidentical copies of DNA.

    5. All of these are correct.

  7. Which is a true statement?

    1. Genomics would be slow going without bioinformatics.

    2. Genomics is related to the field of proteomics.

    3. Genomics has now moved on to functional and comparative genomics.

    4. Genomics shows that we are related to all other organisms tested so far.

    5. All of these are correct.

  8. DNA amplified by PCR and then used for fingerprinting could come from

    1. any diploid or haploid cell.

    2. only white blood cells that have been karyotyped.

    3. only skin cells after they are dead.

    4. only purified animal cells.

    5. Both b and d are correct.

  9. Which was used to find the function of the cystic fibrosis gene?

    1. microarray

    2. proteomics

    3. comparative genomics and bioinformatics

    4. sequencing the gene

  10. Which of these pairs is incorrectly matched?

    1. DNA ligase—mapping human chromosomes

    2. protoplast—plant cell engineering

    3. DNA fragments—DNA fingerprinting

    4. DNA polymerase—PCR

  11. Page 263
  12. Which matches best to proteomics?

    1. Start with known gene sequences and build proteins.

    2. Use a microarray to discover what proteins are active in particular cells.

    3. Use bioinformatics to discover the proteins in the cells of other organisms.

    4. Match up known proteins with known genes.

  13. Which is not a correct association with regard to bioengineering?

    1. plasmid as a vector—bacteria

    2. protoplast as a vector—plants

    3. RNA virus as a vector—human stem cells

    4. All of these are correct.

  14. Proteomics is used to discover

    1. what genes are active in what cells.

    2. what proteins are active in what cells.

    3. the structure and function of proteins.

    4. how proteins interact.

    5. All but a are correct.

  15. Which of these is an incorrect statement?

    1. Bacteria usually secrete the biotechnology product into the medium.

    2. Plants are being engineered to have human proteins in their seeds.

    3. Animals are engineered to have a human protein in their milk.

    4. Animals can be cloned, but plants and bacteria cannot.

  16. Repetitive DNA elements

    1. may be a tandem or spread across several chromosomes.

    2. are found in centromeres and telomeres.

    3. make up nearly half of human chromosomes.

    4. may be present just a few too many thousands of copies.

    5. All of these are correct.

  17. Because of the Human Genome Project, we now know

    1. the sequence of the base pairs of our DNA.

    2. the sequence of all genes along the human chromosomes.

    3. all the mutations that lead to genetic disorders.

    4. All of these are correct.

    5. Only a and c are correct.

  18. Which of the following delivery methods is not used in gene therapy?

    1. virus

    2. nasal sprays

    3. liposomes

    4. electric currents

  19. The restriction enzyme called EcoRI has cut double-stranded DNA in the following manner. The piece of foreign DNA to be inserted has what bases from the left and from the right?

  20. Which of these is a true statement?

    1. Plasmids can serve as vectors.

    2. Plasmids can carry recombinant DNA, but viruses cannot.

    3. Vectors carry only the foreign gene into the host cell.

    4. Only gene therapy uses vectors.

    5. Both a and d are correct.

  21. Gene therapy

    1. is sometimes used in medicine today.

    2. is always successful.

    3. is only used to cure genetic disorders, such as SCID and cystic fibrosis.

    4. makes use of viruses to carry foreign genes into human cells.

    5. Both a and d are correct.

thinking scientifically
  1. Before the human genome was sequenced, gene discovery was accomplished through the use of DNA libraries. A genomic library is a set of cloned DNA segments that altogether are representative of the genome of an organism, whereas a cDNA library contains only expressed DNA sequences for a particular cell. How might these libraries be used to map the introns and exons of a gene within the genome?

  2. The Science Focus on page 260 describes copy number variations within the genome. Copy number variations do not always contain genes. How might having extra or missing copies of intergenic DNA sequences be beneficial? How might it be harmful?

bioethical issue
Transgenic Crops

Transgenic plants can possibly allow crop yields to keep up with the ever-increasing worldwide demand for food. And some of these plants have the added benefit of requiring less fertilizer and/or pesticides, which are harmful to human health and the environment.

But some scientists believe transgenic crops pose their own threat to the environment, and many activists believe transgenic plants are themselves dangerous to our health. Studies have shown that wind-carried pollen can cause transgenic crops to hybridize with nearby weedy relatives. Although it has not happened yet, some fear that characteristics acquired in this way might cause weeds to become uncontrollable pests. Or perhaps a toxin produced by transgenic crops could possibly hurt other organisms in the field. Many researchers are conducting tests to see if this might occur. And although transgenic crops have not caused any illnesses in humans so far, some scientists concede the possibility that people could be allergic to the transgene's protein product. After unapproved genetically modified corn was detected in Taco Bell taco shells several years ago, a massive recall pulled about 2.8 million boxes of the product from grocery stores.

Already, transgenic plants must be approved by the Food and Drug Admin i stration before they are consid ered safe for human consumption, and they must meet certain Environmental Protection Administration standards. Some people believe safety standards for transgenic crops should be further strength ened, while others fear stricter standards will result in less food produced. Another possibility is to retain the current standards but require all biotech foods to be clearly labeled so the buyer can choose whether or not to eat them. Which approach do you prefer?

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