CHAPTER
12
DNA Technology and Genomics
Lecture Outline
I. Introduction
1. Hawaii’s papaya industry seemed doomed just a few decades ago.
a. A deadly pathogen called papaya ringspot virus (PRV) had spread throughout the islands.
b.It appeared poised to completely decimate the papaya plant population.
2. Scientists from the University of Hawaii were able to rescue the industry by creating new, genetically engineered PRV-resistant strains of papaya.
3. Today, the papaya industry is once again vibrant, and the vast majority of Hawaii’s papayas are genetically modified organisms (GMOs).
4. Some critics have raised safety concerns a. for the people who eat them and b.for the environment.
5. Should we, in fact, be concerned about the safety of GMO crops?
a. This question continues to foster considerable debate and disagreement.
6. In addition to GMOs in our diet, DNA technologies affect our lives in many other ways.
a. Gene cloning is used to produce medical and industrial products. b.DNA profiling has changed the field of forensic science.
c. New technologies produce valuable data for biological research. d.DNA can be used to investigate historical questions.
II. Gene Cloning
A.12.1 Genes can be cloned in recombinant plasmids
1. Biotechnology is the manipulation of organisms or their components to make useful products.
2. For thousands of years, humans have
a. used microbes to make wine and cheese and b.selectively bred stock, dogs, and other animals.
3. DNA technology is the set of modern techniques used to study and manipulate genetic material.
4. Genetic engineering involves manipulating genes for practical purposes.
6. Recombinant DNA is formed by joining nucleotide sequences from two different sources and often different species.
a. One source contains the gene that will be cloned. b.Another source is a gene carrier called a vector.
c. Plasmids are small, circular DNA molecules that replicate separately from the much larger bacterial chromosome; they are often used as vectors.
7. The following are the steps in cloning a gene: a. Plasmid DNA is isolated.
b.DNA containing the gene of interest is isolated.
c. Plasmid DNA is treated with a restriction enzyme that cuts in one place, opening the circle.
d.DNA with the target gene is treated with the same enzyme and many fragments are produced.
e. Plasmid and target DNA are mixed and associate with each other.
f. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together.
g. The recombinant plasmid containing the target gene is taken up by a bacterial cell. h.The bacterial cell reproduces to form a clone, a group of genetically identical cells
descended from a single ancestral cell.
8. Gene cloning can be used to produce a variety of desirable products.
B. 12.2 VISUALIZING THE CONCEPT: Enzymes are used to “cut and paste” DNA 1. Restriction enzymes recognize a particular short DNA sequence, called a
a. restriction site, and
b.cut both strands of the DNA at precise points in the sequence, yielding pieces of DNA called restriction fragments.
2. Once cut, fragments of DNA can be pasted together by the enzyme DNA ligase. C.12.3 Cloned genes can be stored in genomic libraries
1. A genomic library is a collection of all of the cloned DNA fragments from a target genome.
2. Genomic libraries can be constructed with different types of vectors. a. In a plasmid library, genomic DNA is carried by plasmids.
b.In a bacteriophage (phage) library, genomic DNA is incorporated into bacteriophage DNA.
D.12.4 Reverse transcriptase can help make genes for cloning
1. A researcher can focus on the genes expressed in a particular kind of cell by using its mRNA as the starting material for cloning.
2. In this process,
a. mRNA from a specific cell type is the template,
b. reverse transcriptase produces a DNA strand from mRNA, c. DNA polymerase produces the second DNA strand, and
d. the DNA that results from such a procedure, called complementary DNA (cDNA), represents only the subset of genes that had been transcribed into mRNA in the starting cells.
3. Advantages of cloning with cDNA include the ability to
a. study genes responsible for specialized characteristics of a particular cell type and b.obtain gene sequences that are smaller in size, easier to handle, and do not have
E. 12.5 Nucleic acid probes identify clones carrying specific genes 1. Nucleic acid probes bind very selectively to cloned DNA.
a. Probes can be DNA or RNA sequences complementary to a portion of the gene of interest.
b.A probe binds to a gene of interest by base pairing.
c. Probes are labeled with a radioactive isotope or fluorescent tag for detection. 2. One way to screen a gene library is as follows:
a. Bacterial clones are transferred to filter paper.
b.A chemical treatment is used to break open the cells and separate the DNA strands. c. A probe solution is added, and any bacterial colonies carrying the gene of interest
will be tagged on the filter paper.
d.The clone carrying the gene of interest is grown for further study.
III. Genetically Modified Organisms
A.12.6 Recombinant cells and organisms can mass-produce gene products
1. Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins.
2. Bacteria are often the best organisms for manufacturing a protein product because bacteria
a. have plasmids and phages available for use as gene-cloning vectors, b.can be grown rapidly and cheaply,
c. can be engineered to produce large amounts of a particular protein, and d.often secrete the proteins directly into their growth medium.
3. Yeast cells a. are eukaryotes, b. are easy to grow,
c. have long been used to make bread and beer,
d.can take up foreign DNA and integrate it into their genomes, and
e. are often better than bacteria at synthesizing and secreting eukaryotic proteins. 4. Mammalian cells must be used to produce proteins with chains of sugars. Examples
include
a. human erythropoietin (EPO), which stimulates the production of red blood cells, b.factor VIII to treat hemophilia, and
c. tissue plasminogen activator (TPA), used to treat heart attacks and strokes. 5. Pharmaceutical researchers are currently exploring the mass production of gene
products by
a. whole animals or b.plants.
6. Recombinant animals
a. are difficult and costly to produce and
b.must be cloned to produce more animals with the same traits.
B. 12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine
1. DNA technology, including gene cloning, is widely used to produce medicines and to diagnose diseases.
2. Therapeutic hormones produced by DNA technology include a. insulin to treat diabetes,
c. tissue plasminogen activator (TPA), a protein that helps dissolve blood clots and re-duces the risk of subsequent heart attacks.
3. DNA technology is used to a. test for inherited diseases,
b.detect infectious agents such as HIV, and
c. produce vaccines, harmless variants (mutants) or derivatives of a pathogen that stimulate the immune system to mount a lasting defense against that pathogen, thereby preventing disease.
C.12.8 CONNECTION: Genetically modified organisms are transforming agriculture 1. Since ancient times, people have selectively bred agricultural crops to make them
more useful.
2. DNA technology is quickly replacing traditional breeding programs to improve the productivity of agriculturally important plants and animals.
3. Genetically modified organisms (GMOs) contain one or more genes introduced by artificial means.
4. Transgenic organisms contain at least one gene from another species. 5. The most common vector used to introduce new genes into plant cells is
a. a plasmid from the soil bacterium Agrobacterium tumefaciens
b.called the Ti plasmid.
6. GMO crops may be able to help a great many hungry people by improving a. food production,
b.shelf life,
c. pest resistance, and
d.the nutritional value of crops.
7. Golden Rice, a transgenic variety created in 2000 with a few daffodil genes, produces yellow grains containing beta-carotene, which our body uses to make vitamin A. 8. Genetic engineers are now creating plants that make human proteins for medical use. 9. Pharmaceutical trails currently under way involve using modified
a. rice to treat infant diarrhea, b.corn to treat cystic fibrosis, c. safflower to treat diabetes, and d.duckweed to treat hepatitis.
10.Although promising, no plant-made drugs intended for use by humans have been approved or sold.
11.Agriculture researchers are producing transgenic animals by injecting cloned genes directly into the nuclei of fertilized eggs.
a. Genetically modified pigs convert less healthy fatty acids to omega-3 fatty acids, pro-ducing meat with four to five times as much healthy omega-3 fat as regular pork. b.Atlantic salmon have been genetically modified to mature in half the time of
conventional salmon and grow to twice the size.
D.12.9 SCIENTIFIC THINKING: Genetically modified organisms raise health concerns 1. Scientists use safety measures to guard against production and release of new
pathogens.
2. To guard against rogue microbes, scientists developed a set of guidelines, including a. strict laboratory safety and containment procedures,
c. a prohibition on certain obviously dangerous experiments.
3. Today, most public concern centers on whether GMOs are safe for humans and the en-vironment.
4. GMOs are used in crop production because they are a. more nutritious or
b.cheaper to produce.
5. But do these advantages come at a cost to the health of people consuming GMOs? 6. A 2012 animal study involved 104 pigs that were divided into two groups.
a. One group was fed a diet containing 39% GMO corn. b.Another group was fed a closely related non-GMO corn.
7. The health of the pigs—in terms of growth, organ structure, and immune response against foreign DNA—was measured in the short term (31 days), in the medium term (110 days), and over the normal generational life span.
8. The researchers reported no significant differences between the two groups and no traces of foreign DNA in the slaughtered pig.
9. Although pigs are a good model organism for human digestion, critics argue that human data are required to draw conclusions about the safety of dietary GMOs in peo-ple.
10.A human study of Golden Rice
a. was conducted jointly by Chinese and American scientists and published in 2012 and b.concluded that GMO rice can indeed be effective in preventing vitamin A
deficiency among children who rely on rice as a staple food.
11.To date, no study has documented health risks in humans from GMO foods and there is general agreement among scientists that the GMO foods on the market are safe. 12.On the other hand, because they are new, it is not yet possible to measure the
long-term effects (if any) of GMOs on human health. 13.Concerns remain that transgenic plants might
a. pass their new genes to related species in nearby wild areas and b.disturb the composition of the natural ecosystem.
14.Although the majority of several staple crops that are grown in the United States—in-cluding corn and soybeans—are genetically modified, products made from GMOs are not required to be labeled in any way.
a. In 2012, citizens of California voted down (53% to 47%) a ballot measure requiring GMO labeling of food and drink in that state.
15.In the case of GMO crops, zero risk is probably unattainable.
16.Scientists and the public need to weigh the possible benefits versus risks on a case-by-case basis.
17.The best scenario would be to proceed with caution, basing our decisions on sound scientific information rather than on either irrational fear or blind optimism.
E. 12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases 1. Gene therapy is the alteration of a diseased individual’s genes for therapeutic
purposes.
2. One possible procedure is the following:
a. A gene from a healthy person is cloned, converted to an RNA version, and then inserted into the RNA genome of a harmless virus.
c. The virus inserts a DNA version of its genome, including the normal human gene, into the cell’s DNA.
d.The engineered cells are then injected back into the patient.
3. The promise of gene therapy thus far exceeds actual results, but there have been some successes in the treatment of
a. severe combined immunodeficiency (SCID) and b.Leber’s congenital amaurosis (LCA).
4. The use of gene therapy raises ethical questions.
a. Some critics suggest that tampering with human genes in any way will inevitably lead to the practice of eugenics, the deliberate effort to control the genetic makeup of human populations.
b.Other observers see no fundamental difference between the transplantation of genes into somatic cells and the transplantation of organs.
IV. DNA Profiling
A.12.11 The analysis of genetic markers can produce a DNA profile
1. DNA profiling is the analysis of DNA samples to determine whether they come from the same individual.
2. In a typical investigation involving a DNA profile:
a. DNA samples are isolated from the crime scene, suspects, victims, or other evidence,
b.selected markers from each DNA sample are amplified (copied many times), producing a large sample of DNA fragments, and
c. the amplified DNA markers are compared, providing data about which samples are from the same individual.
B. 12.12 The PCR method is used to amplify DNA sequences
1. Polymerase chain reaction (PCR) is a technique by which a specific segment of a DNA molecule can be targeted and quickly amplified in the laboratory.
2. PCR relies on a pair of primers, which are chemically synthesized, single-stranded DNA molecules with sequences that are complementary to sequences at each end of the target sequence.
a. One primer is complementary to one strand at one end of the target sequence. b.The second primer is complementary to the other strand at the other end of the
sequence.
c. The primers thus bind to sequences that flank the target sequence, marking the start and end points for the segment of DNA being amplified.
3. The basic steps of PCR are as follows:
a. The reaction mixture is heated to separate the strands of the DNA double helices. b.The strands are cooled. As they cool, primer molecules hydrogen-bond to their
target sequences on the DNA.
c. A heat-stable DNA polymerase builds new DNA strands by extending the primers in the 5′→3′ direction.
4. These three steps are repeated over and over, doubling the amount of DNA after each three-step cycle.
5. The advantages of PCR include
a. the ability to amplify DNA from a small sample, b.rapid results, and
6. Devised in 1985, PCR has had a major impact on biological research and biotechnology. PCR has been used to amplify DNA from
a. fragments of ancient DNA from a mummified human, b.a 40,000-year-old frozen wooly mammoth,
c. a 30-million-year-old plant fossil, and
d.DNA from fingerprints or from tiny amounts of blood, tissue, or semen found at crime scenes.
C.12.13 Gel electrophoresis sorts DNA molecules by size
1. Many DNA technology applications rely on gel electrophoresis, a method that separates macromolecules, usually proteins or nucleic acids, on the basis of size, electrical charge, or other physical properties.
2. Gel electrophoresis can be used to separate DNA molecules based on size as follows: a. A DNA sample is placed at one end of a porous gel.
b.Current is applied, and DNA molecules move from the negative electrode toward the positive electrode.
c. Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel.
d.DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence.
e. Each band is a collection of DNA molecules of the same length. D.12.14 Short tandem repeat analysis is commonly used for DNA profiling
1. Repetitive DNA consists of nucleotide sequences that are present in multiple copies in the genome.
2. Short tandem repeats (STRs) are short nucleotide sequences that are repeated in tandem, composed of different numbers of repeating units in individuals, that are used in DNA profiling.
3. STR analysis
a. compares the lengths of STR sequences at specific sites in the genome and b.typically analyzes 13 sites scattered in the genome.
E. 12.15 CONNECTION: DNA profiling has provided evidence in many forensic investigations 1. DNA profiling is used to
a. determine guilt or innocence in a crime, b.settle questions of paternity,and
c. probe the origin of nonhuman materials.
2. Lawyers at the Innocence Project, a nonprofit organization dedicated to overturning wrongful convictions, used DNA technology and legal work to exonerate more than 300 convicted criminals since 1989, including 17 who were on death row.
3. One of the strangest cases of DNA profiling is that of Cheddar Man, a 9,000-year-old skeleton found in a cave near Cheddar, England.
a. DNA was extracted from his tooth and analyzed.
b.The DNA profile showed that Cheddar Man was a direct ancestor—through approximately 300 generations—of a present-day schoolteacher who lived only a half mile from the cave!
F. 12.16 RFLPs can be used to detect differences in DNA sequences
1. Geneticists have cataloged many single-base-pair variations in the genome.
3. SNPs occur on average about once in 100 to 300 base pairs in the human genome, a. in the coding sequences of genes and
b.in noncoding sequences between genes.
4. SNPs may alter a restriction site—the sequence recognized by a restriction enzyme. a. Such alterations change the lengths of the restriction fragments formed by that
enzyme when it cuts the DNA.
b.A sequence variation of this type is called a restriction fragment length polymorphism (RFLP, pronounced “rif-lip”).
c. Thus, RFLPs can serve as genetic markers for particular loci in the genome.
V. Genomics
A.12.17 Genomics is the scientific study of whole genomes
1. Genomics is the study of an organism’s complete set of genes and their interactions. a. Initial studies focused on prokaryotic genomes.
b.Many eukaryotic genomes have since been investigated.
c. As of 2013, the genomes of nearly 7,000 species have been completed, and thousands more are in progress.
2. Genomics allows another way to examine evolutionary relationships.
a. Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans.
b.Functions of human disease-causing genes have been determined by comparing hu-man genes to similar genes in yeast.
B. 12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes
1. The goals of the Human Genome Project (HGP) included
a. determining the nucleotide sequence of all DNA in the human genome and b.identifying the location and sequence of every human gene.
2. Results of the HGP indicate that
a. humans have about 21,000 genes in 3 billion nucleotide pairs, b.only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs, and c. the remaining 98.5% of the DNA is noncoding DNA.
3. Much of the DNA between genes consists of repetitive DNA, nucleotide sequences present in many copies in the genome.
4. Stretches of DNA with thousands of short repetitions are also prominent at a. the centromeres and
b.ends of chromosomes—called telomeres.
5. In a second main type of repetitive DNA, each repeated unit is hundreds of nucleo-tides long, and the copies are scattered around the genome.
6. Most of these sequences seem to be associated with transposable elements (“jumping genes”), DNA segments that can move or be copied from one location to another in a chromosome and even between chromosomes.
C.12.19 The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly
1. The HGP proceeded through three stages that provided progressively more detailed views of the human genome.
b.A physical map was constructed from nucleotide distances between the linkage map markers.
c. DNA sequences for the mapped fragments were determined.
2. The whole-genome shotgun method was proposed in 1992 by molecular biologist J. Craig Venter.
3. He used restriction enzymes to produce fragments that were cloned and sequenced in just one stage and ran high-performance computer analyses to assemble the sequence by aligning overlapping regions.
4. Today, this whole-genome shotgun approach is the method of choice for genomic researchers because it is
a. relatively fast and b.inexpensive.
5. However, limitations of the whole-genome shotgun method suggest that a hybrid approach using the genome shotgunning and physical maps may prove to be the most useful.
D.12.20 Proteomics is the scientific study of the full set of proteins encoded by a genome 1. Proteomics
a. is the study of the full protein sets encoded by genomes and b.investigates protein functions and interactions.
2. The human proteome includes about 100,000 proteins.
3. Genomics and proteomics are helping biologists study life from an increasingly holistic approach.
E. 12.21 EVOLUTION CONNECTION: Genomes hold clues to human evolution 1. Human and chimp genomes differ by
a. 1.2% in single-base substitutions and
b.2.7% in insertions and deletions of larger DNA sequences. 2. Genes showing rapid evolution in humans include
a. genes for defense against malaria and tuberculosis, b.a gene regulating brain size, and
c. the FOXP2 gene, which is involved with speech and vocalization. 3. Neanderthals
a. first appeared at least 300,000 years ago, b.were humans’ closest relatives,
c. were a separate species, d.also had the FOXP2 gene,
