Biotechnology - Advanced

Douglas Wilkin, Ph.D.

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AUTHORDouglas Wilkin, Ph.D.

www.ck12.org Chapter 1. Biotechnology - Advanced

CHAPTER 1Biotechnology - Advanced

CHAPTER OUTLINE

1.1 Biotechnology - Advanced

1.2 Gene Cloning - Advanced

1.3 The Polymerase Chain Reaction - Advanced

1.4 The Human Genome Project - Advanced

1.5 Biotechnology and Medicine - Advanced

1.6 Biotechnology and Agriculture - Advanced

1.7 Cloning - Advanced

1.8 Biotechnology and Forensic Science - Advanced

1.9 Ethical, Legal, and Social Issues of Biotechnology - Advanced

1.10 References

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Introduction

Biotechnology. Gene Therapy. Designer plants. Designer babies. Reality or fiction? During your lifetime, genetherapy may be mainstream medicine. Here we see a representation of the insertion of DNA into the nucleus of acell. Is this possible? Yes. In these concepts, you will learn how human, animal and plant chromosomes and genesare manipulated to make our lives better. But what does it mean "to make our lives better." Does this not just includecuring genetic diseases, but also improving crops? Biotechnology has created a number of very important ethicalissues that will be discussed for many years to come. Some uses of biotechnology are fact, others will always befiction.

"Without question, man’s knowledge of man is undergoing the greatest revolution since Leonardo. In many ways,personalized medicine is already here." Dr. Francis Collins, Director, The National Institutes of Health, Bethesda,Maryland.

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1.1 Biotechnology - Advanced

• Describe what is meant by Biotechnology and DNA technology?• Describe DNA sequencing.

So how does a scientist work with DNA?

It usually starts with the sequence of As, Cs, Gs and Ts. Once the sequence is known, so much more can be done.Specific regions can be isolated, cloned, amplified, and analyzed. In fact, the ability to amplify a specific region ofDNA has revolutionized biological research. And all of this is done to directly or indirectly help us.

Biotechnology

Is it really possible to clone people? Another question is, should we clone people? Are scientific fantasies, suchas depicted on TV shows such as Star Trek or in the movie GATTACA, actually a possibility? Who can really say?How, really, will science affect our future? The answers partially lie in the field of biotechnology.

Biotechnology is technology based on biological applications. These applications are increasingly used in medicine,agriculture and food science. Biotechnology combines many features of biology, including genetics, molecularbiology, biochemistry, embryology, and cell biology. Many aspects of biotechnology center around DNA and itsapplications, otherwise known as DNA technology. We could devote a whole textbook to current applications ofbiotechnology, however, we will focus on the applications towards medicine and agriculture, and the extension intothe forensic sciences. First, though, we need to understand DNA technology.

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DNA Technology

What is DNA technology? Is it using and manipulating DNA to help people? Is it using DNA to make bettermedicines and individualized treatments? Is it analyzing DNA to determine predispositions to genetic diseases? Theanswers to these questions, and many more, is yes. And the answers to many of these issues begin with the HumanGenome Project.

Scientists have sequenced a consensus version of the human genome. Now what? Do we know what all the genesare or what they do? Not yet. Do we know what phenotypes are associated with mutations in the genes? Formany genes, or even most genes, we do not. Do we even know exactly how many genes we have? Not exactly.And we are far away from knowing what, at the genomic level, makes us all unique. So how does this informationhelp us? The Human Genome Project has been labeled a landmark scientific event. But what can we do with thisinformation? More on the Human Genome Project will be discussed in the DNA Technology: The Human GenomeProject (Advanced) concept.

There are many applications of genetic information, including applications in medicine and agriculture. The appli-cations of genetics to forensic science have become one of the most important aspects of the criminal justice system.And of course, these applications raise many ethical questions. See Genes can be moved between species at http://www.dnaftb.org/34/animation.html to see Stanley Cohen and Herbert Boyer speak about developing recombinantDNA technology. See Different genes are active in different kinds of cells at http://www.dnaftb.org/36/animation.html for more recent molecular technologies.

Biotechnology: The Invisible Revolution can be seen at http://www.youtube.com/watch?v=OcG9q9cPqm4 .

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/162

DNA Sequencing

For years, arguably beginning with the rediscovery of Mendel’s work in the early 1900s, scientists have knownabout heritable factors or genes. Then in the 1940s and 1950s, when it was proved that DNA is the genetic materialand has a triplet code made of just four bases, understanding the sequence of this code became the next significantendeavor. How do you sequence the ACGTs? The genetic code told us of the amino acids coded for by each codon,but it also told us that multiple codons could code for the same amino acid. Would sequencing the human genomeexplain to us what it means to be human? Maybe at a molecular level. But even prior to undertaking such a largeeffort, sequencing small parts, even just small segments of a gene, would form the basis of DNA analysis and DNAtechnology. But first methods to sequence DNA had to be developed.

Sequencing of DNA would allow the analysis of genes to become more feasible. How big is a gene? Where are thestart and stop codons? Where are the intron and exon junctions? What is the gene’s consensus sequence? What aresignificant base changes within a gene? These questions could start to be answered.

DNA sequencing was built upon earlier knowledge of DNA polymerases and DNA replication. The chain-terminationmethod, which makes use of a "defective" DNA nucleotide, is the basis of DNA sequencing. This method is alsoknown as the Sanger method, named after its developer Frederick Sanger. In this process, one strand of DNA is usedas a template, just as in DNA replication. The nucleotide chain elongates as a deoxynucleotide (dNTP) is attachedto the 3’ carbon end of the chain based on the base-pair rules. At the chain termination method, dideoxynucleotides(ddNTPs) are utilized in the sequencing reaction. ddNTPs lack a 3’-OH group necessary for elongation. Whena ddNTP is incorporated into the elongating chain, the elongation process stops at that base. When four separate

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sequencing reactions are done, one each in the presence of a particular ddNTP, and the products are separated by sizethrough gel electrophoresis, the DNA sequence can be read. In A gene is a discrete sequence of DNA nucleotides athttp://www.dnaftb.org/23/animation.html , Fred Sanger talks about developing DNA sequencing procedures.

Vocabulary

• biotechnology: Technology based on biological applications.

• chain-termination method: The method of DNA sequencing using dideoxynucleotide triphosphates (ddNTPs)as DNA chain terminators.

• DNA sequencing: The method of determining the order of the DNA nucleotide bases.

• DNA technology: Biotechnology focusing on DNA-based technology.

• gel electrophoresis: An analytical technique used to separate DNA fragments by size and charge; can also beused to separate RNA and proteins.

• Human Genome Project: A project to understand the genetic make-up of the human species by determiningthe DNA sequence of the human genome and the genome of a few model organisms.

• recombinant DNA: DNA engineered through the combination of two or more DNA strands; combines DNAsequences which would not normally occur together.

Summary

• Biotechnology is technology based on biological applications, combining many features of Biology includinggenetics, molecular biology, biochemistry, embryology, and cell biology.

• The goal of the Human Genome Project is to understand the genetic make-up of the human species bydetermining the DNA sequence of the human genome and the genome of a few model organisms.

• Sequencing of DNA is necessary for the analysis of genes and the genome.

Practice

Use this resource to answer the questions that follow.

• Clone Zone: http://learn.genetics.utah.edu/content/tech/cloning/clonezone/ .

1. In 1885-Seeing Double? Sea Urchins Cloned, what did Hans Adolf Edward Dreisch show? What did this tellus?

2. In 1952- Frogs Galore! Nuclear Transfer Becomes a Reality, what did Robert Briggs and Thomas King show?What did this tell us?

3. In 1996- Hello, Dolly! What did Ian Wilmut and Keith Campbell show? What did this tell us?

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Practice Answers

1. Hans Adolf Edward Dreisch showed that by merely shaking two-celled sea urchin embryos, it was possible toseparate the cells. Once separated, each cell grew into a complete sea urchin. This tells us that each cell in theembryo has its own compelete set of genetic instructions and can grow into a full organism.

2. Robert Briggs and Thomas King showed that a cell’s nucleus is in charge of directing growth and division.They created normal tadpole clones of the frog embryos that donated the nuclei, but cloning was less successfulwith donor This tells us that nuclear transfer was a viable cloning technique, the nucleus directs cell growthand, ultimately, an organism’s development, and embryonic cells early in development are better for cloningthan cells at later stages.

3. Ian Wilmut and Keith Campbell showed that by using electric shock techniques, they were able to fuse uddercells from a female adult sheep with enucleated sheep egg cells. This produced an embryo that was carried toterm in a surrogate mother. This famous lamb was named Dolly. This tells us that opportunities and potentialimplications of cloning using a donor nucleus from an adult somatic cell.

Review

1. What is the difference between biotechnology and DNA technology?2. What are the goals of the Human Genome Project?3. Explain the concept of DNA sequencing and the chain-termination method.

Review Answers

1. Biotechnology is technology based on biological applications; genetics, molecular biology, embryology andcell biology. DNA technology are technology specifically based on DNA applications.

2. The goals of the Human Genome Project is to understand the genetic make-up of the human species bydetermining the DNA sequence of the human genome and the genome of a few model organisms.

3. DNA sequencing is the method of determining the order of the DNA nucleotide bases. The basis of DNAsequencing is the chain-termination method, which makes use of a defective DNA nucleotide.

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1.2 Gene Cloning - Advanced

• Describe gene cloning and the processes involved.

What does it mean to clone?

A genetically exact copy. A clone can be a gene, a cell, an animal or plant. And these clones are produced all thetime. Theoretically, a clone could also be a human. But would that be a smart thing to do? Is it ethical? Is it evenlegal?

Gene Cloning

You probably have heard of cloning. A clone is a genetically exact copy. It can be a clone of a gene, a cell oran organism. Even a human. However, whereas cloning of humans has many ethical issues associated with it andis illegal throughout most of the world, the cloning of genes has been ongoing for well over 30 or 40 years, withcloning of animals occurring more recently. Gene cloning, also known as molecular cloning, refers to the processof isolating a DNA sequence of interest for the purpose of making multiple copies of it. The identical copies areclones. In 1973, Stanley Cohen and Herbert Boyer developed techniques to make recombinant DNA, a form ofartificial DNA. They talk about their work in Genes can be moved between species at http://www.dnaftb.org/34/animation.html .

Recombinant DNA is engineered through the combination of two or more DNA strands, combining DNA sequenceswhich would not normally occur together. In other words, selected DNA (or the DNA of "interest") is inserted intoan existing organismal genome, such as a bacterial plasmid DNA, or some other sort of vector. The recombinant

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DNA can then be inserted into another cell, such as a bacterial cell, for amplification and possibly production of theresulting protein. This process is called transformation, the genetic alteration of a cell resulting from the uptake,incorporation, and expression of foreign genetic material. Recombinant DNA technology was made possible by thediscovery of restriction endonucleases.

In The RNA message is sometimes edited at http://www.dnaftb.org/24/animation.html , Rich Rioberts and Phil Sharpdiscuss the development of tools and techniques to analyze DNA.

Restriction Enzyme Digestion and Ligation

Restriction enzymes or restriction endonucleases are prokaryotic enzymes that recognize and cut DNA at specificsequences, called restriction sites. It is believed that they evolved as a defense mechanism against foreign DNA,such as viral DNA. Over 3,000 restriction enzymes have been identified. Some of the more common restrictionenzymes are shown in the table below, where up and down arrows show the sites of cleavage. Restriction enzymesare named based on the prokaryotic organism they are isolated from. For example, those isolated from Escherichiacoli would begin with Eco. As the Table 1.1 shows, digestion with the restriction enzymes will result in overlappingor blunt ends. EcoRI produces overlapping "sticky" ends: the enzyme cleaves between the G and A on both strands.On the other hand, SmaII restriction enzyme cleavage produces "blunt" ends. The enzyme cleaves between the Gand C on both strands.

TABLE 1.1: Common Restriction Endonucleases

Enzyme Source Recognition Sequence Restriction DigestEcoRI Escherichia coli

5’GAATTC3’CTTAAG

5’—G↓AATTC—3’3’—CTTAA↑G—5’

BamHI Bacillus amyloliquefa-ciens

5’GGATCC3’CCTAGG

5’—G↓GATCC—3’3’—CCTAG↑G—5’

TaqI Thermus aquaticus

5’TCGA3’AGCT

5’—T↓CGA—3’3’—AGC↑T—5’

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TABLE 1.1: (continued)

Enzyme Source Recognition Sequence Restriction Digest

HinfI Haemophilus influenzae

5’GANTCA3’CTNAGT

5’—G↓ANTC—3’3’—CTNA↑G—5’

Sau3A Staphylococcus aureus

5’GATC3’CTAG

5’—↓GATC—3’3’—CTAG↑—5’

PvuII Proteus vulgaris

5’CAGCTG3’GTCGAC

5’—CAG↓CTG—3’*3’—GTC↑GAC—5’

SmaI Serratia marcescens

5’CCCGGG3’GGGCCC

5’—CCC↓GGG—3’*3’—GGG↑CCC—5’

HaeIII Haemophilus aegyptius

5’GGCC3’CCGG

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TABLE 1.1: (continued)

Enzyme Source Recognition Sequence Restriction Digest5’—GG↓CC—3’*3’—CC↑GG—5’

Key: N = C or G or T or A

• = blunt ends

Cloning of a segment of DNA of interest can easily be carried out with restriction enzyme digestion, followedby ligation and transformation or transfection. In the classical restriction enzyme digestion and ligation cloningprotocols, cloning of any DNA fragment essentially involves four steps:

1. isolation of the DNA of interest (or target DNA),2. ligation,3. transfection (or transformation), and4. a screening/selection procedure.

For an overview of cloning, see https://www.youtube.com/watch?v=VRpfOfAadu8 .

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/140956

Isolation of DNA

Initially, the DNA fragment to be cloned must be isolated. This DNA of interest may be a gene, part of a gene,a promoter, or another segment of DNA, and is frequently isolated by the polymerase chain reaction (PCR) orrestriction enzyme digestion. As discussed above, a restriction enzyme is an enzyme that cuts double-stranded DNAat a specific sequence. The enzyme makes two incisions, one through each strand of the double helix, withoutdamaging the nitrogenous bases. This produces either overlapping ends (also known as sticky ends) or bluntends. The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discoveryof restriction endonucleases. The first practical use of their work was the manipulation of E. coli bacteria to producehuman insulin for diabetics.

Ligation

Once the DNA of interest is isolated, a ligation procedure is necessary to insert the amplified fragment into a vectorto produce the recombinant DNA molecule. Restriction fragments (or a fragment and a plasmid/vector) can bespliced together, provided their sticky ends are complementary. Blunt end ligation is also possible.

The plasmid or vector (which is usually circular) is digested with restriction enzymes, opening up the vector to allowinsertion of the target DNA. If the isolated DNA of interest and the plasmid or vector are digested with the same

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restriction enzyme, their sticky ends will be complementary. The two DNAs are then incubated with DNA ligase,an enzyme that can attach together strands of DNA with double strand breaks. This produces a recombinant DNAmolecule. Figure 1.1 depicts a plasmid with two additional segments of DNA ligated into the plasmid, producingthe recombinant DNA molecule. Figure 1.1 depicts DNA before and after ligation.

FIGURE 1.1(left) This image shows a line drawingof a plasmid. The plasmid is drawn astwo concentric circles that are very closetogether representing the two strandsof DNA, with two large segments andone small segment depicted. The twolarge segments (blue and green) indicateantibiotic resistances usually used in ascreening procedure, and the small seg-ment (red) indicates an origin of replica-tion, used in DNA replication. The result-ing DNA is a recombinant DNA molecule.(right) Sticky ends produced by restrictionenzyme digestion can be joined with theenzyme DNA ligase.

Transfection and Selection

Following ligation, the recombinant DNA is placed into a host cell, usually bacterial, in a process called transfectionor transformation. Finally, the transfected cells are cultured. Many of these cultures may not contain a plasmid withthe target DNA as the transfection process is not usually 100% successful, so the appropriate cultures with the DNAof interest must be selected. Many plasmids/vectors include selectable markers - usually some sort of antibioticresistance ( Figure 1.1). When cultures are grown in the presence of an antibiotic, only bacteria transfected with thevector containing resistance to that antibiotic should grow. However, these selection procedures do not guaranteethat the DNA of insert is present in the cells. Further analysis of the resulting colonies is required to confirm thatcloning was successful. This may be accomplished by means of a process PCR or restriction fragment analysis, bothof which need to be followed by gel electrophoresis and/or DNA sequencing (DNA sequence analysis).

DNA sequence analysis, PCR, or restriction fragment analysis will all determine if the plasmid/vector contains theinsert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If theisolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gelelectrophoresis will separate DNA molecules based on size and charge. Examples are shown in Figure 1.2.

Gel Electrophoresis

Gel electrophoresis is an analytical technique used to separate DNA fragments by size and charge. Notice inFigure 1.2 that the "gels" are rectangular in shape. The gels are made of a gelatin-like material of either agaroseor polyacrylamide. An electric field, with a positive charge applied at one end of the gel, and a negative charge atthe other end, forces the fragments to migrate through the gel. DNA molecules migrate from negative to positivecharges due to the net negative charge of the phosphate groups in the DNA backbone. Longer molecules migratemore slowly through the gel matrix. After the separation is completed, DNA fragments of different lengths can be

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visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The resulting stained gel showsbands correspond to DNA molecules of different lengths, which also correspond to different molecular weights.Band size is usually determined by comparison to DNA ladders containing DNA fragments of known length. Gelelectrophoresis can also be used to separate RNA molecules and proteins.

FIGURE 1.2(left) DNA samples in a blue track-ing dye are being loaded into wells ofan agarose gel prior to electrophoresis.(right) Agarose gel following agarose gelelectrophoresis on an UV light box.

Recombinant DNA technology is discussed in the following videos and animations: http://www.youtube.com/watch?v=x2jUMG2E-ic (4:36)

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/160

http://www.youtube.com/watch?v=Jy15BWVxTC0 (0:50)

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/22668

http://www.youtube.com/watch?v=sjwNtQYLKeU (7:20)

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/161

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Vocabulary

• clone: A genetically identical copy; may be a gene, a cell or an organism; an organism that is geneticallyidentical to its parent.

• DNA ligase: Enzyme that can attach together (ligates) strands of DNA with double strand breaks.

• gel electrophoresis: An analytical technique used to separate DNA fragments by size and charge; can also beused to separate RNA and proteins.

• gene cloning: The process of isolating a DNA sequence of interest for the purpose of making multiple copiesof it.

• ligation: The process of forming a bond to join two nucleotide pairs; joining DNA with double strand breaks.

• plasmid: A small circular piece of DNA that is physically separate from, and can replicate independently of,chromosomal DNA within a cell.

• polymerase chain reaction (PCR): A repeating series of cycles used to amplify millions of times specificregions of a DNA strand.

• recombinant DNA: DNA engineered through the combination of two or more DNA strands; combines DNAsequences which would not normally occur together.

• restriction endonuclease: An enzyme that cuts double-stranded DNA; also known as a restriction enzyme.

• restriction enzyme: An enzyme that cuts double-stranded DNA; also known as a restriction endonuclease.

• restriction site: DNA sequence recognized and digested by a restriction enzyme.

• transfection: The insertion of foreign DNA into a host cell.

• transformation: The change in genotype and phenotype of a cell/organism due to the assimilation of externalDNA (heredity material) by a cell.

Summary

• Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interestfor the purpose of making multiple copies of it.

• Classic gene cloning involves the following steps:

1. Restriction enzyme digestion and ligation.2. Isolation of DNA.3. Ligation.4. Transfection and Selection.5. Gel electrophoresis.

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Practice

Use this resource to answer the questions that follow.

• Gene Cloning at http://agbiosafety.unl.edu/education/clone.htm

1. What is gene cloning?2. What is a gene library?3. How do restriction enzymes function?4. What is a plasmid and how is it involved in producing recombinant DNA?5. Describe how a gene library is screened.

Practice Answers

1. Gene cloning is the process in which a gene of interest is located and copied (cloned) out of DNA extractedfrom an organism.

2. A gene library is a collection of living bacteria colonies that have been transformed with different pieces ofDNA from the organism that is the source of the gene of interest.

3. Restriction enzymes read the nucleotide sequence of the DNA and recognize specific sequences. The enzymesthen cut the DNA sequence by breaking the bonds between nucleotides in a DNA strand.

4. Plasmids are small circles of DNA in bacterial cells that are naturally present in addition to the bacteria’s otherDNA. After the bacterial DNA and plasmid DNA are cut by restriction enzymes, they are combined into onetest tube. Some of the plasmid DNA will be inserted into the bacterial DNA, forming recombinant DNA.

5. Library screening identifies colonies, which have that particular gene by detecting the DNA sequence of thecloned gene, detecting a protein that the gene encodes, or the use of linked DNA markers.

Review

1. What are restriction endonucleases?2. How are gene cloning and recombinant DNA related?3. Describe the process of gene cloning.4. How does gel electrophoresis analyze DNA?

Review Answers

1. Restriction Endonuclease an enzyme that cuts double-stranded DNA; combines DNA sequences which wouldnot normally occur together.

2. Gene cloning is the process of inserting a recombinant DNA sequence, artificial DNA, into an existingorganismal genome, which is inserted into another cell for amplification and possibly production of theresulting protein.

3. The process of gene cloning is (1) isolation of the DNA of interest, (2) ligation, (3) transfection, and (4) ascreening/selection procedure.

4. Gel electrophoresis is an analytical technique used to separate DNA fragments by size and charge. An electricfield, with a positive charge applied at one end of the gel, and a negative charge at the other end, forces thefragments to migrate through the gel. DNA molecules migrate from negative to positive charges due to thenet negative charge of the phosphate groups in the DNA backbone. Longer molecules migrate more slowlythrough the gel matrix. The resulting stained gel shows bands correspond to DNA molecules of differentlengths, which also correspond to different molecular weights.

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1.3 The Polymerase Chain Reaction - Ad-vanced

• What is PCR?• Describe the processes involved in PCR.

How do you make a scientific process easier?

Use a machine. The polymerase chain reaction has revolutionized biological and biomedical research and appli-cations. Luckily many machines have been developed that allow this process to be performed rapidly and withprecision.

The Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is used to amplify specific regions of a DNA strand millions of times. Aregion may be a number of loci, a single gene, a part of a gene, or a non-coding sequence. This technique producesa useful quantity of DNA for analysis, be it medical, forensic or some other form of analysis. Amplification of DNAfrom as little as a single cell is possible. Whole genome amplification is also possible.

PCR utilizes a heat stable DNA polymerase, Taq polymerase (or Taq DNA polymerase), named after the ther-mophilic bacterium Thermus aquaticus, from which it was originally isolated. T. aquaticus is a bacterium that livesin hot springs and hydrothermal vents, and Taq polymerase is able to withstand the high temperatures required todenature DNA during PCR (discussed below). Taq polymerase’s optimum temperature for activity is between 75◦Cand 80◦C. Recently other DNA polymerases have also been used for PCR.

A basic PCR involves a series of repeating cycles involving three main steps (see Figure 1.3):

1. Denaturation of the double stranded DNA.2. Annealing of specific oligonucleotide primers.3. Extension of the primers to amplify the region of DNA of interest.

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These steps will be discussed in additional detail below.

The oligonucleotide primers are single stranded pieces of DNA that correspond to the 5’ and 3’ ends of the DNAregion to be amplified. These primers will anneal to the corresponding segment of denatured DNA. Taq Polymerase,in the presence of free deoxynucleotide triphosphates (dNTPs), will extend the primers to create double strandedDNA. After many cycles of denaturation, annealing and extension, the region between the two primers will begreatly amplified.

The PCR is commonly carried out in a thermal cycler, a machine that automatically allows heating and cooling of thereactions to control the temperature required at each reaction step (see below). The PCR usually consists of a seriesof about 30 to 35 cycles. Most commonly, PCR is carried out in three repeating steps, with some modifications forthe first and last step.

PCR is usually performed in small tubes or wells in a tray, each often beginning with the complete genome of thespecies being studied. As only a specific sequence from that genome is of interest, the sequence specific primersare targeted to that sequence. PCR is done with all the building blocks necessary to create DNA: template DNA,primers, dNTPs, and a DNA polymerase.

The three basic steps of PCR ( Figure 1.3) are:

• Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94 -98◦C for 30 to 60 seconds. It disrupts the hydrogen bonds between complementary bases of the DNA strands,yielding single strands of DNA.

• Annealing step: The reaction temperature is lowered to 50-65◦C for 30 to 60 seconds, allowing annealingof the primers to the single-stranded DNA template. Stable hydrogen bonds form between the DNA strand(the template) and the primers when the primer sequence very closely matches the complementary templatesequence. Primers are usually 17 - 22 nucleotides long and are carefully designed to bind to only one site inthe genome. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

• Extension step: A temperature of around 72◦C is used for this step, which is close to the optimum temperatureof Taq polymerase. At this step the Taq polymerase extends the primer by adding dNTPs, using one DNAstrand as a template to create a the other (new) DNA strand. The extension time depends on the length ofthe DNA fragment to be amplified. As a standard, at its optimum temperature, the DNA polymerase willpolymerize a thousand bases in one minute.

Utilizing PCR, DNA can be amplified millions of times to generate quantities of DNA that can be used for a numberof purposes. These include the use of DNA for prenatal or genetic testing, such as testing for a specific mutation.PCR has revolutionized the fields of biotechnology, human genetics, and a number of other sciences. Many of theapplications will be discussed in the additional concepts. PCR was developed in 1983 by Kary Mullis. Due to theimportance of this process and the significance it has had on scientific research, Dr. Mullis was awarded the NobelPrize in Chemistry in 1993, just 10 years after his discovery. See http://www.dnalc.org/resources/spotlight/ foranimations of Dr. Mullis and PCR.

To say that PCR, molecular cloning and the Human Genome Project has revolutionized biology and medicine wouldbe an understatement. These efforts have led to numerous accolades, including Nobel prizes, and more may follow.Some of the ways that these discoveries have shaped our lives are the focus of the Concept Biotechnology (Advanced)concepts.

Vocabulary

• annealing step: Second step of a PCR cycle; allows oligonucleotide primers to bind to their specific site onsingle-strnaded DNA.

• denaturation step: First step of a PCR cycle; disrupts the hydrogen bonds between complementary bases ofthe DNA strands, yielding single strands of DNA.

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FIGURE 1.3The Polymerase Chain Reaction. Thepolymerase chain reaction involves threesteps. High temperatures are needed forthe process to work. The enzyme Taqpolymerase is used in step 3 because itcan withstand high temperatures.

• extension step: Third step of a PCR cycle; allows Taq polymerase to extend primer, forming double-strandedDNA.

• oligonucleotide primer: Short single-stranded piece of DNA; hydrogen bonds to a DNA strand to serve as ainitiation segment for DNA polymerase to extend.

• Polymerase Chain Reaction (PCR): A repeating series of cycles used to amplify millions of times specificregions of a DNA strand.

• Taq polymerase: A DNA polymerase named after the thermophilic bacterium Thermus aquatics from whichit was originally isolated; the heat-stable polymerase used in the PCR reaction.

Summary

• The Polymerase Chain Reaction (PCR) is used to amplify millions of times specific regions of a DNA strand.This can be a number of loci, a single gene, a part of a gene, or a non-coding sequence.

• PCR usually involves the following steps:

1. Denaturation step.

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2. Annealing step.3. Extension step.

Explore More

Use these resources to answer the questions that follow.

• PCR at http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml

1. What is PCR?2. Why does PCR only amplify a specific region of DNA?3. What is an amplicon?4. Define the following:

a. DNA Template.b. Taq DNA polymerase.c. Primer.

• Polymerase Chain Reaction at http://www.genome.gov/10000207

1. In your own words, answer the following:

a. What is PCR?b. What is PCR used for?c. How does PCR work?

Explore More Answers

PCR

1. PCR is a method developed by Kary Mullis based on using the ability of DNA polymerase to synthesize newstrand of DNA complementary to the offered template strand.

2. PCR can only amplify a specific region of DNA because DNA polymerase can only add a nucleotide onlyonto a preexisting 3’-OH group, therefore it needs a primer to which it can add the first nucleotide. Thisrequirement makes it possible to delineate a the target sequence.

3. An amplicon is the specific DNA product generated by PCR using one pair of PCR primers.4. Sample answers:

a. DNA template is the sample DNA that contains the target sequence.b. Taq DNA polymerase is a type of enzyme from Thermis aquaticus that synthesizes new strands of DNA

complementary to the target sequence.c. Primer is a short pieces of single-stranded DNA that are complementary to the target sequence.

Polymerase Chain Reaction

1. Sample answers:

a. PCR is a technique used to amplify small segments of DNA.b. PCR is valuable in a number of newly emerging laboratory and clinical techniques, including finger-

printing, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders.c. PCR works by heating DNA so it denatures and then using the Taq polyermase to synthesize two new

strands of DNA. Then these strands can be used to create two new copies, and is repeated 30 or 40 times,leading to more than one billion exact copies of the original DNA segment.

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Explore More II

• PCR Virtual Lab at http://learn.genetics.utah.edu/content/labs/pcr/

Review

1. What is PCR?2. What allows PCR to be done at high temperatures?3. Describe the PCR process.4. Illustrate the PCR process.

Review Answers

1. PCR is a repeating series of cycles used to amplify a specific region of a DNA strand millions of times.2. The ability of the Taq polymerase to withstand high temperatures, which is required to denature DNA, allows

PCR to be done at high temperatures.3. PCR is consists of three main steps; (1) denaturation of the double stranded DNA, (2) annealing of specific

oligonucleotide primers, and (3) extension of the primers to amplify the region of DNA of interest.

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4.

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1.4 The Human Genome Project - Advanced

• What is the Human Genome Project?• Describe the goals and the importance of the Human Genome Project.

Why is understanding the whole human genome important?

That’s over 3,000,000,000 pieces of information.

The Human Genome Project was one of the great feats of exploration in history - an inward voyage of discoveryrather than an outward exploration of the planet or the cosmos; an international research effort to sequence andmap all of the genes - together known as the genome - of members of our species, Homo sapiens. Completed in April2003, the HGP gave us the ability to, for the first time, to read nature’s complete genetic blueprint for building ahuman being. - National Human Genome Research Institute (http://www.genome.gov/10001772 ).

The Human Genome Project

The Human Genome

All the DNA of the human species makes up the human genome. This DNA consists of about 3 billion base pairsand is divided into thousands of genes on 23 pairs of chromosomes. The human genome also includes regulatoryand noncoding sequences of DNA, as shown in Figure 1.4.

In A genome is an entire set of genes at http://www.dnaftb.org/39/animation.html , James Watson discusses thehuman genome and the Human Genome Project.

The Human Genome Project

Thanks to the Human Genome Project (HGP), scientists now know a consensus DNA sequence of the entire humangenome. The Human Genome Project was an international project that includes scientists from around the world. It

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FIGURE 1.4Human Genome, Chromosomes, andGenes. Each chromosome of the humangenome contains many genes as wellas noncoding intergenic (between genes)regions. Each pair of chromosomes isshown here in a different color.

began in 1990, and by 2003, scientists had sequenced all 3 billion base pairs of human DNA. Now they are trying toidentify and characterize all the genes in the genome.

If we are all 99.9% genetically identical, what makes us different? How does that 0.1% make us tall or short, light ordark, develop cancer or not? To understand that 0.1%, we also need to understand the other 99.9%. Understandingthe human genome is the goal of the Human Genome Project. This project, publicly funded by the United StatesDepartment of Energy (DOE) ( Figure 1.5); and the National Human Genome Research Institute (NHGRI), partof the National Institutes of Health (NIH), may be one of the landmark scientific events of our lifetime.

As stated above, the goal of the HGP is to understand the genetic make-up of the human species by determining theDNA sequence of the human genome ( Figure 1.5) and the genome of a few model organisms. However, it is notjust determining the 3 billion bases; it is understanding what they mean. How many human genes are there? Dothese genes encode transcription factors, transport proteins, growth factors, structural proteins, or oncogenes? Orany of the other various types of proteins? Does a base change in the consensus sequence of a gene cause a geneticdisease? What is the phenotype associated with mutations in a specific gene? Because of the HGP, these questionscan be addressed.

The genes in the genome are in the process of being identified and characterized, as are the proteins associated withthose genes. A preliminary estimate of the number of genes in the human genome is around 22,000 to 23,000.

The sequence of the human DNA is stored in databases available to anyone on the Internet. The U.S. NationalCenter for Biotechnology Information (NCBI), part of the NIH, as well as comparable organizations in Europeand Japan, maintain the genomic sequences in a database known as GenBank. Protein sequences are also maintainedin this database. The sequences in these databases are the combined sequences of anonymous donors, and as such donot yet address the individual differences that make us unique. However, the known sequence does lay the foundationto identify the unique differences among all of us. Most of the currently identified variations among individuals willbe single nucleotide polymorphisms, or SNPs. A SNP (pronounced "snip") is a DNA sequence variation occurringat a single nucleotide in the genome. For example, two sequenced DNA fragments from different individuals,GGATCTACCGAA to GGATTTACCGAA, contain a difference in a single nucleotide. If this base change occurs ina gene, the base change then results in two alleles: the C allele and the T allele. Remember an allele is an alternativeform of a gene. Almost all common SNPs have only two alleles. The effect of these SNPs on protein structure andfunction, and any effect on the resulting phenotype, is an extensive field of study.

You can watch a video about the Human Genome Project and how it cracked the "code of life" at this link: http://www.pbs.org/wgbh/nova/genome/program.html .

Our Molecular Selves video discusses the human genome, and is available at http://www.genome.gov/25520211 orhttp://www.youtube.com/watch?v=_EK3g6px7Ik . Genome, Unlocking Life’s Code is the Smithsonian’s National

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FIGURE 1.5(left) The Human Genome Project logo of the DOE. (right) A depiction of DNA sequence analysis. Note the 4colors utilized, each representing a separate base.

Museum of Natural History exhibit of the human genome. See http://unlockinglifescode.org to visit the exhibit.

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/69173

In DNA is only the beginning for understanding the human genome at http://www.dnaftb.org/41/animation.html ,Mario Capecchi discusses analyzing the functions of proteins to make sense of the information generated by theHuman Genome Project.

ENCODE: The Encyclopedia of DNA Elements

In September 2012, ENCODE, The Encyclopedia of DNA Elements, was announced. ENCODE was a colossalproject, involving over 440 scientists in 32 labs the world-over, whose goal was to understand the human genome. Ithad been thought that about 80% of the human genome was "junk" DNA. ENCODE has established that this is nottrue. Now it is thought that about 80% of the genome is active. In fact, much of the human genome is regulatorysequences, on/off switches that tell our genes what to do and when to do it. Dr. Eric Green, director of the NHGRI,which organized this project, states, "It’s this incredible choreography going on, of a modest number of genes andan immense number of ... switches that are choreographing how those genes are used."

It is now thought that at least three-quarters of the genome is involved in making RNA, and most of this RNA appearsto help regulate gene activity. Scientists have also identified about 4 million sites where proteins bind to DNA andact in a regulatory capacity. These new findings demonstrate that the human genome has remarkable and precise,and complex, controls over the expression of genetic information within a cell.

See ENCODE data describes function of human genome at http://www.genome.gov/27549810 for additional infor-mation.

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Vocabulary

• allele: An alternative form or different version of a gene.

• GenBank: The NIH genetic sequence database, a collection of all publicly available DNA sequences.

• human genome: All of the hereditary information encoded in the DNA of homo sapiens, including the genesand non-coding sequences.

• Human Genome Project (HGP): A project to understand the genetic make-up of the human species bydetermining the DNA sequence of the human genome and the genome of a few model organisms.

• National Center for Biotechnology Information (NCBI): U.S. government-funded national resource formolecular biology information; part of the United States National Library of Medicine on the NIH campus.

• National Human Genome Research Institute (NHGRI): One of 27 institutes and centers that make up theNIH; devoted to improving human health through human genetics and genomics basic and clinical research.

• National Institutes of Health (NIH): The United States’ medical research agency; supporting scientificstudies world-wide; composed of 27 institutes and centers.

• single nucleotide polymorphisms (SNP): A DNA sequence variation occurring when a single nucleotidediffers between members of a species or paired chromosomes in an individual.

Summary

• The goal of the Human Genome Project is to understand the genetic make-up of the human species bydetermining the DNA sequence of the human genome and the genome of a few model organisms.

• Understanding the human genome will allow research into genes and their corresponding proteins, and geneticdiseases.

Practice

Use this resource to answer the questions that follow.

• Human Genome Project at http://report.nih.gov/NIHfactsheets/ViewFactSheet.aspx?csid=45

1. What was the Human Genome Project? What were its goals?2. What role has the human genome project played in human genetics?3. What is The Cancer Genome Atlas?4. What is meant by individualized analysis based on each person’s genome? Why is this important?

Practice Answers

1. The Human Genome Project was the sequencing of the complete set of DNA in the human body. Its goal wasto provide researchers with powerful tools to understand the genetic factors in human disease, paving the wayfor new strategies for their diagnosis, treatment and prevention.

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2. The Human Genome Project has allowed for researchers to find a gene suspected of causing an inheriteddisease in a matter of days. Also, now there are more than 2,000 genetic tests for human conditions.

3. The Cancer Genome Atlas is the initiative to identify all the genetic abnormalities seen in 50 major types ofcancer.

4. Individualized analysis based on each person’s genome refers to a powerful form of preventive, personalizedand preemptive medicine. This will allow health care professionals to focus effects on specific strategies perindividual.

Review

1. What are the goals of the Human Genome Project?2. Is the DNA sequence information generated by the HGP available to anyone, and if so, how?3. What is maintained in the GenBank?4. What are single nucleotide polymorephisms?5. What is ENCODE?

Review Answers

1. The goal of the Human Genome Project is to understand the genetic make-up of the human species bydetermining the DNA sequence of the human genome and the genome of a few model organisms.

2. The sequence of the human DNA is stored in databases available to anyone on the Internet.3. The GenBank maintains the genomic sequences and protein sequences in the databases. The sequences in

these databases are combined sequences of anonymous donors.4. Single nucleotide polymorphisms are DNA sequence variations occurring at a single nucleotide in the genome.5. ENCODE was a colossal project whose goal was to understand the human genome and its incredible chore-

ography.

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1.5 Biotechnology and Medicine - Advanced

• Describe various applications of biotechnology as related to medicine.• How is DNA technology related to genetic testing and prenatal diagnosis?

Is this possible?

Of course DNA is much smaller than shown here and you cannot see it in a test tube. But you can manipulate andstudy DNA. Biotechnology and its associated techniques have made DNA-based biomedical research and analysisfairly routine.

Applications of DNA Technology: Medicine

Biotechnology and DNA technology could be considered synonymous. So much of biotechnology is DNA based.Remember that biotechnology is technology based on biological applications. Since the completion of the humangenome project, the data produced from the project and the tools and technologies associated with the project haveled to numerous biotechnology applications.

As discussed in the DNA Technology: The Human Genome Project (Advanced) concept, the Human Genome Projecthas opened up many avenues to take advantage of what we know about our genome in order to help us. Many ofthese possibilities are medically related. Others will be legally related. And yet still other uses of DNA technologyinclude those involving other genomes, especially in agriculture and the food sciences. However, it is the medicalpossibilities of biotechnology that most people associate with helping humans.

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Gene Therapy

Understanding and curing genetic diseases is the ultimate goal of human geneticists. As discussed in the HumanGenetics: Diagnosis and Treatment (Advanced) concept, gene therapy is the insertion of a new gene into anindividual’s cells and tissues to treat a disease, replacing a mutant disease-causing allele with a normal, non-mutantallele. Of course, the findings of the Human Genome Project are significant in determining the disease-causingalleles. Geneticists must know which are mutant alleles and which are non-mutant or "normal" alleles. They mustalso be able to identify alleles that are not just associated with a particular disease phenotype, but cause a diseasephenotype. And of course, scientists must develop and test the technology to replace mutant alleles.

Recombinant Insulin

In the 1920s, there was no known way to produce insulin, which was needed by people to remove excess sugarfrom the bloodstream. People with diabetes either lack insulin, produce low levels of insulin, or are resistant toinsulin, and thus they may need external insulin to control blood glucose levels. This problem was solved, at leasttemporarily, when it was found that insulin from a pig’s pancreas could be used in humans. This method was theprimary solution for diabetes until recently. The problem with insulin from pigs was that there were not enough pigsto provide the quantities of insulin needed. Scientists needed to devise another way to produce insulin. This led toone of the biggest breakthroughs in recombinant DNA technology: the cloning of the human insulin gene.

By methods discussed the DNA Technology: Gene Cloning (Advanced) concept, the specific gene sequence thatcodes for human insulin was introduced into the bacteria E. coli. The transformed gene altered the genetic makeupof the bacterial cells, such that in a 24 hour period, billions of E. coli containing the human insulin gene resulted.Once the recombinant human protein was isolated from the bacteria, enough insulin was available to be administeredto patients.

Though the production of human insulin by recombinant DNA procedures is an extremely significant event, manyother aspects of DNA technology are beginning to become reality. In medicine, modern biotechnology providessignificant applications in such areas as pharmacogenomics, genetic testing (and prenatal diagnosis), and genetherapy. These applications use our knowledge of biology to improve our health and our lives. Many of thesemedical applications are based on the findings of the Human Genome Project.

Pharmacogenomics

Currently, millions of individuals with high cholesterol take a similar type of drug, known as a statin. The drug,an inhibitor of HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase), the rate limiting enzyme incholesterol biosynthesis, decreases blood levels of cholesterol by induce the expression of low density lipoprotein(LDL) receptors in the liver. The increased levels of the LDL-receptors stimulate the catabolism of plasma LDL,removing cholesterol from plasma, which is an important determinant of atherosclerosis. You may know of peoplewho take a statin to help with their cholesterol levels. However, these drugs probably work slightly differently inmany of those people. In some, it lowers their cholesterol significantly; in others it may lower it only moderately;and in some, it may have no significant effect at all. (Luckily for those individuals, there are multiple versions ofthe statins, so different drugs can be tested to find the proper combination for that individual.) Why the difference?Because of the genetic background of all people; the different single nucleotide polymorphisms that make us alldifferent. Pharmacogenomics, a combination of pharmacology and genomics (the study of the genome) that refersto the study of the relationship between pharmaceuticals and genetics, may explain and simplify this problem.

Pharmacogenomics is the study of how the genetic inheritance of an individual affects his or her body’s responseto drugs. In other words, pharmacogenomics will lead to the design and production of drugs that are adapted to eachperson’s genetic makeup. Pharmacogenomics has applications in illnesses such as cancer, cardiovascular disorders,depression, attention deficit disorders, HIV, asthma, and diabetes, among others.

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FIGURE 1.6Genetically Engineering Bacteria to Pro-duce a Human Protein. Bacteria canbe genetically engineered to produce ahuman protein, such as a cytokine. Acytokine is a small protein that helps fightinfections.

Pharmacogenomics will result in the following benefits:

1. Development of tailor-made medicines. Using pharmacogenomics, pharmaceutical companies will be able tocreate drugs based on the proteins, enzymes and RNA molecules that are associated with specific genes anddiseases. These tailor-made drugs promise not only to maximize the beneficial effects of the medicine, butalso to decrease damage to nearby healthy cells.

2. More accurate methods of determining appropriate drug dosages. Knowing a patient’s genetics will enabledoctors to determine how well his or her body can process and metabolize a medicine. This will allow doctorsto prescribe the proper levels of the medicine, allowing the medicine to have optimal results.

3. Improvements in the drug discovery and approval process. Once the genes and proteins associated with adisease are known, the discovery of new medicines will be made easier using these genes and proteins astargets for the medicine. In addition to creating much more beneficial medicines, this could significantlyshorten the drug discovery process.

4. Better vaccines. Safer vaccines can be designed and produced by organisms transformed with DNA sequencesfrom an antigen. These vaccines will trigger the immune response without the risks of infection. They will becapable of being engineered to carry several strains of pathogen at once, combining several vaccines into one.

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Cytochrome P450 Genes

There are several known genes which are largely responsible for variances in drug metabolism and response amonghumans. The most common are the cytochrome P450 (CYP) genes, which encode enzymes that influence themetabolism of more than 75% of current prescription drugs. CYP450 proteins are hemoproteins, belonging to thesuperfamily of proteins containing a heme cofactor. Often, these proteins form part of multi-component electrontransfer chains, called P450-containing systems. The letter in P450 represents the word pigment, as these enzymesare red because of their heme group. The number 450 reflects the wavelength of the absorption maximum of theenzyme. Humans have 57 genes (and more than 59 pseudogenes) divided among 18 families of cytochrome P450genes and 43 subfamilies. Some examples are shown in the Table 1.2. CYP genes and enzymes are designated withthe abbreviation CYP, followed by a number indicating the gene family, a capital letter indicating the subfamily, andanother numeral for the individual gene.

TABLE 1.2: CYP Genes and Alleles

Family Function Members NamesCYP1 drug and steroid

(especially estrogen)metabolism

3 subfamilies, 3 genes, 1pseudogene

CYP1A1, CYP1A2,CYP1B1

CYP2 drug and steroidmetabolism

13 subfamilies, 16 genes,16 pseudogenes

CYP2A6, CYP2A7,CYP2A13, CYP2B6,CYP2C8, CYP2C9,CYP2C18, CYP2C19,CYP2D6, CYP2E1,CYP2F1, CYP2J2,CYP2R1, CYP2S1,CYP2U1, CYP2W1

CYP3 drug and steroid(including testosterone)metabolism

1 subfamily, 4 genes, 2pseudogenes

CYP3A4, CYP3A5,CYP3A7, CYP3A43

CYP4 arachidonic acid or fattyacid metabolism

6 subfamilies, 12 genes,10 pseudogenes

CYP4A11, CYP4A22,CYP4B1, CYP4F2,CYP4F3, CYP4F8,CYP4F11, CYP4F12,CYP4F22, CYP4V2,CYP4X1, CYP4Z1

CYP7 bile acid biosynthesis7-alpha hydroxylase ofsteroid nucleus

2 subfamilies, 2 genes CYP7A1, CYP7B1

CYP11 steroid biosynthesis 2 subfamilies, 3 genes CYP11A1, CYP11B1,CYP11B2

CYP21 steroid biosynthesis 2 subfamilies, 1 gene, 1pseudogene

CYP21A2

CYP24 vitamin D degradation 1 subfamily, 1 gene CYP24A1CYP46 cholesterol 24-

hydroxylase1 subfamily, 1 gene CYP46A1

How are these CYP genes important in individualized medicine? These genes, like all genes, have multiplealleles. As these enzymes are involved in drug metabolism, different alleles can have different effectiveness atthat metabolism.

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For example, the CYP1A1 gene has 13 known alleles, known as CYP1A1*1, CYP1A1*2A, CYP1A1*2B, CYP1A1*3,CYP1A1*4 and so on. CYP1A1*1 is the wild type allele, and the others are usually due to single nucleotidepolymorphisms (SNP). The CYP2D6 gene has over 100 known alleles. CYP2D6*1A is the wild type allele and theothers are variations on the *1A sequence. Some have a single SNP change, while other alleles have multiple basechanges. There are six separate CYP2D6*1 alleles within this gene family.

If a person receives one *1 allele each from his/her mother and his/her father to code for the CYP2D6 gene,then that person is considered to have an extensive metabolizer (EM) phenotype. An extensive metabolizer isconsidered the normal phenotype with average enzyme activity levels. Other CYP metabolism phenotypes include:intermediate, ultra-rapid, and poor. These phenotypes are due to allelic variation among the CYP genes. However,with myriad possibilities of allelic combinations, this developing individualized medicine guidelines is still wellunder development.

TABLE 1.3: CYP2D6 Allele and Enzyme Activity

Allele CYP2D6 Activity Metabolizer PhenotypeCYP2D6*1 normal extensiveCYP2D6*2 increased ultra-rapidCYP2D6*3 no activity poorCYP2D6*4 no activity poorCYP2D6*5 no activity poorCYP2D6*9 decreased intermediateCYP2D6*10 decreased intermediateCYP2D6*17 decreased intermediate

KQED: Pharmacogenomics

We know that, thanks to our genome, each of us is phenotypically unique. Some of those differences are obvious,like eye and hair color, but others are not obvious at all, such as how our bodies react to medication. As discussedabove, the CYP450 alleles have plenty to do with how each of us react to drugs. Researchers are beginning to look athow to individualize medical treatments, based on our genetic profiles. Some of the biggest breakthroughs have beenin cancer treatment. For additional information on this personalized medicine, listen to http://www.kqed.org/quest/radio/personalized-medicine and see http://www.kqed.org/quest/blog/2009/09/11/reporters-notes-personalized-medicine/ . Personalized medicine is a drug treatment regimen individually developed to an individual based on thatperson’s genetic profile.

Genetic Testing and Prenatal Diagnosis

Let’s propose a hypothetical situation: unfortunately, members of your family are predisposed to develop a debili-tating genetic disease. You and your spouse want to have a baby, but you want to know the likelihood of the childdeveloping the disease.

This scenario could happen to anyone. As we learn more and more about disease causing genes, it will becomeeasier to test for mutations in those genes. Currently, is there any way to determine if a baby will develop a diseasedue to a known mutation? Is it possible to screen for a mutation in a developing baby? The answer to both thosequestions is. . . yes.

Genetic testing involves the direct examination of DNA sequences. A scientist scans, by any number of methods, apatient’s DNA for mutated sequences. Genetic testing can be used to:

• Diagnose a disease.• Confirm a diagnosis.

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• Provide information about the course of a disease.• Confirm the existence of a disease.• Predict the risk of future development of a disease in otherwise healthy individuals or their children.• Identify carriers (unaffected individuals who are heterozygous for a recessive disease gene).• Perform prenatal diagnostic screening.• Perform newborn screening.

Consultations with human geneticists and genetic counselors are an important first step in genetic testing. They willmost likely prescribe some sort of prenatal screening (see the Human Genetics: Diagnosis and Treatment (Advanced)concept). Prenatal screening (also known as prenatal diagnosis or prenatal testing) is the testing for diseases orconditions in a fetus or embryo before it is born. Methods may involve amniocentesis or chorionic villus sampling toremove fetal cells. DNA can be isolated from these cells and analyzed. If the mutation that results in the phenotypeis known, that specific base can be analyzed, either through restriction fragment length polymorphism analysis or,more likely, through PCR and DNA sequence analysis. As it is the baby’s DNA that is being analyzed, the analysiswill determine if the developing baby will have the mutation and develop the phenotype, or not have the mutation.Parents can then be informed of the probability of the baby developing the disease.

In human genetics, preimplantation genetic diagnosis (PIGD) is genetic analysis performed on embryos prior toimplantation. PIGD is considered an alternative to prenatal diagnosis. Its main advantage is that it avoids selectivepregnancy termination, as the method makes it highly likely that the baby will be free of the disease in question. InPIGD, in vitro fertilization is used to obtain embryos for analysis. DNA is isolated from developing embryos priorto implantation, and specific genetic loci are screened for mutations, usually using PCR based analysis. Embryosthat lack the specific mutation can then be implanted into the mother, thereby guaranteeing that the developing babywill not have the specific mutation analyzed for (and thus not have the disease associated with that mutation).

KQED: Synthetic Biology

Can biotechnology be extended to develop new technologies based on biological systems? Imagine living cellsacting as memory devices, biofuels brewing from yeast, or a light receptor taken from algae that makes photographson a plate of bacteria. The new biotechnology field of Synthetic Biology is making biological systems easier toengineer, so that new functions can be derived and developed from living systems. Find out the tools that syntheticbiologists are using and the exciting things they are developing at http://www.kqed.org/quest/television/decoding-synthetic-biology and http://www.kqed.org/quest/television/web-extra-synthetic-biology-extended-interview .

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/523

Vocabulary

• biotechnology: Technology based on biological applications.

• cytochrome P450: A superfamily of a large and diverse group of enzymes that catalyze the oxidation oforganic substances.

• extensive metabolizer: Phenotype with normal level of organic substance metabolism.

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• gene therapy: Process to potentially cure genetic disorders; involves inserting normal genes into cells withmutant genes.

• genetic testing: The direct examination of DNA sequences for mutated sequence.

• individualized medicine: The concept that diagnosis and treatment can be tailored to a unique genetic code.

• personalized medicine: A medical model that customizes medical treatment to the individual patient by useof genetic or other information.

• pharmacogenomics: Field that is tailoring medical treatments to fit our genetic profiles.

• preimplantation genetic diagnosis: Genetic analysis performed on embryos prior to implantation.

• prenatal diagnosis: The diagnosis of a disease or condition before a baby is born.

• prenatal screening: A variety of tests can be used to detect birth defects and other genetic disorders in adeveloping baby prior to birth.

• recombinant DNA: DNA engineered through the combination of two or more DNA strands; combines DNAsequences which would not normally occur together.

• single nucleotide polymorphism: A DNA sequence variation occurring when a single nucleotide differsbetween members of a species or paired chromosomes in an individual.

• Synthetic Biology: Field of biology involved in engineering new functions from living systems.

Summary

• In medicine, modern biotechnology provides significant applications in such areas as pharmacogenomics,genetic testing (prenatal diagnosis), and gene therapy.

• Pharmacogenomics, the combination of pharmacology and genomics, is the study of the relationship betweenpharmaceuticals and genetics.

• Pharmacogenomics will result in the following benefits:

1. Development of tailor-made medicines.2. More accurate methods of determining appropriate drug dosages.3. Improvements in the drug discovery and approval process.4. Better vaccines.

• Genetic testing involves the direct examination of DNA sequences.• Genetic testing can be used to: diagnose a disease; confirm a diagnosis; provide prognostic information

about the course of a disease; confirm the existence of a disease; predict the risk of future development of adisease in otherwise healthy individuals or their children; screen for carriers (unaffected individuals who areheterozygous for a disease gene); perform prenatal diagnostic screening; and perform newborn screening.

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Practice I

Use these resources to answer the questions that follow.

• http://www.hippocampus.org/Biology → Biology for AP*→ Search: Medical Applications

1. What is the goal of biotechnology?2. What has PCR made possible?3. Describe how advances in biotechnology have helped medical applications.4. What is gene therapy?

• Genetic Variation, Disease Genes, and Risk Factors at http://biotech.about.com/od/DNA-Sequencing/a/Genetic-Variation-Disease-Genes-And-Risk-Factors.htm

1. What is a risk factor?2. What are the BRCA genes, and how are they related to breast cancer?3. What is meant by the genomics age?

Practice I Answers

Medical Applications

1. The goal of biotechnology is to enhance human existence in some way.2. PCR has made it possible to sequence all of an organism’s DNA.3. Advances in biotechnology have helped scientists develop new and better drugs and vaccines.4. Gene therapy is the treatment of a disorder by introducing specific engineered genes into a patient’s cells.

Genetic Variation, Disease Genes, and Risk Factors

1. A risk factor is the probability that a person with a specific set of gene variants will develop a certain trait, likea disease, when compared to other similar people without those same gene variants.

2. BRCA genes are genes that have been linked to breast cancer. Women with specific variations of these geneshave about a 60% chance of developing breast cancer, whereas the general rate of breast cancer is 12%.

3. The genomics age is the task of associating the tens of millions of genetic variations with the multitude ofdiseases, characteristics, and other traits they influence has a long way to go.

Practice II

• Craig Venter at http://www.youtube.com/watch?v=Ce8ZVyUqY-I

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/1753

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Review

1. What is gene therapy?2. What was the problem with insulin from pigs? How did scientists solve this problem?3. What are some of the benefits of pharmacogenomics?4. Describe how pharmacogenomics will result in specialty medicines.5. What are potential uses of genetic testing?

Review Answers

1. Gene therapy is the insertion of a new gene into an individual’s cells and tissues to treat a disease, replacing amutant disease-causing allele with a normal, non-mutant allele.

2. The problem with insulin from pigs was that there were not enough pigs to provide the quantities of insulinneeded. Scientists solved this problem through recombinant DNA technology: the cloning of the humaninsulin gene.

3. Some of the benefits of pharmacogenomics are (1) development of tailor-made medicines, (2) more accuratemethods of determining appropriate drug dosages, (3) improvements in the drug discovery and approvalprocess, and (4) better vaccines.

4. Pharmacogenomics will allow an individual to be prescribed specific dosages and/or drugs based upon theirallelic expression.

5. Genetic testing can be used to diagnose a disease, confirm a diagnosis, provide information about the courseof a disease, confirm the existence of a disease, predict the risk of future development of a disease, identifycarriers, perform prenatal diagnostic screening, and perform newborn screening.

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1.6 Biotechnology and Agriculture - Advanced

• Describe various applications of biotechnology as related to agriculture.• Why is biotechnology so important in agriculture?

Why would anyone grow plants like this?

Developing better crops is a significant aspect of biotechnology. But what is meant by "better?" Crops that areresistant to damage from insects or droughts? Crops that taste better? Crops that last longer? Crops that can growanywhere? How do you feed over 7,000,000,000 people? With biotechnology. Crops developed with biotechnologymust have a significant role in the world’s future. And it all starts in the lab.

Applications of DNA Technology: Agriculture

Biotechnology has many other useful applications besides those that are medically related. Many of these are inagriculture and food science. These include the development of transgenic crops - the placement of genes intoplants to give the crop a beneficial trait. Benefits include:

• Improved yield from crops.• Reduced vulnerability of crops to environmental stresses.• Increased nutritional qualities of food crops.• Improved taste, texture or appearance of food.• Reduced dependence on fertilizers, pesticides and other agrochemicals.

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FIGURE 1.7Creating a Transgenic Crop. A trans-genic crop is genetically modified to bemore useful to humans. The bacteriumtransfers the T-DNA (from the Ti plasmid)fragment with the desired gene into thehost plant’s nuclear genome.

• Production of vaccines.

Biotechnology in agriculture is discussed at http://www.youtube.com/watch?v=IY3mfgbe-0c (6:40).

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/164

Improved Yield from Crops

Using biotechnology techniques, one or two genes may be transferred into a crop to give a new trait to that crop.This is done in the hope of increasing its yield. However, these increases in yield have proved to be difficult toachieve. Current genetic engineering techniques work best for single gene effects - that is traits inherited in a simpleMendelian fashion. Many of the genetic characteristics associated with crop yield, such as enhanced growth, arecontrolled by a large number of genes, each of which just has a slight effect on the overall yield. There is, therefore,still much research, including plant genetic research, to be done in this area.

Reduced Vulnerability to Environmental Stresses

Crops are obviously dependent on environmental conditions. Drought can destroy crop yields, as can too much rainor floods. But what if crops could be developed to withstand these harsh conditions? Biotechnology will allow the

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development of crops containing genes that will enable them to withstand biotic and abiotic stresses. For example,drought and excessively salty soil are two significant factors affecting crop productivity. But there are crops that canwithstand these harsh conditions. Why? Probably because of that plant’s genetics. So biotechnologists and plantgeneticists are studying plants that can cope with these extreme conditions, trying to identify and isolate the genesthat control these beneficial traits. The genes could then be transferred into more desirable crops, with the hope ofproducing the same phenotypes in those crops.

Thale cress ( Figure 1.8), a species of Arabidopsis (A. thaliana), is a tiny weed that is often used for plant researchbecause it is very easy to grow and its genome has been extensively characterized. Scientists have identified a genefrom this plant, At-DBF2, that confers resistance to some environmental stresses. When this gene is inserted intotomato and tobacco cells, the cells were able to withstand environmental stresses like salt, drought, cold and heat farbetter than ordinary cells. If these preliminary results prove successful in larger trials, then At-DBF2 genes couldhelp in engineering crops that can better withstand harsh environments.

Researchers have also created transgenic rice plants that are resistant to rice yellow mottle virus (RYMV). In Africa,this virus destroys much of the rice crops and makes the surviving plants more susceptible to fungal infections.

FIGURE 1.8Thale cress.

Increased Nutritional Qualities of Crops

Maybe you’ve heard over and over that eating beans is good for you. True? Well, maybe. But what if it weregenetically possible to increase the nutritional qualities of food? One would think that would be beneficial to society.So, can biotechnology be used to do just that? Scientists are working on modifying proteins in foods to increasetheir nutritional qualities. Also, proteins in legumes and cereals may be transformed to provide all the amino acidsneeded by human beings for a balanced diet.

Improved Taste, Texture or Appearance of Food

Have you ever gone to the grocery store, bought some fruit and never gotten around to eating it? Maybe you haven’t,but maybe your parents have. Modern biotechnology can be used to slow down the process of spoilage so thatfruit can ripen longer on the plant and then be transported to the consumer with a still reasonable shelf life. Thisis extremely important in parts of the world where time from harvest to the consumer may be longer than in otherareas. In addition to improving the taste, texture and appearance of fruit, it will also extend the usable life of the fruit.As the world population grows and grows, this may become a fairly important issue. Extending the life of fruit canexpand the market for farmers in developing countries due to the reduction in spoilage. This has successfully been

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demonstrated with the tomato. The first genetically modified food product was a tomato which was transformed todelay its ripening. Researchers in Indonesia, Malaysia, Thailand, Philippines and Vietnam are currently working ondeveloping other delayed ripening fruits, such as the papaya.

Reduced Dependence on Fertilizers, Pesticides and Other Agrochemicals

There is growing concern regarding the use of pesticides in agriculture. Therefore, many of the current commercialapplications of modern biotechnology in agriculture are focused on reducing the dependence of farmers on thesechemicals. For example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein that can act as aninsecticide, known as the Bt toxin. But it is a naturally occurring protein, not a foreign chemical. Could this proteinbe used in crops instead of pesticides? Traditionally, an insecticidal spray has been produced from these bacteria. Asa spray, the Bt toxin is in an inactive state and requires digestion by an insect to become active and have any effect.Crop plants have now been engineered to contain and express the genes for the Bt toxin, which they produce in itsactive form. When an insect ingests the transgenic crop, it stops feeding and soon thereafter dies as a result of the Bttoxin binding to its gut wall. Bt corn is now commercially available in a number of countries to control corn borer(an insect like a moth or butterfly), which is otherwise controlled by insecticidal spraying.

FIGURE 1.9Kenyans examining genetically modifiedinsect resistant transgenic Bt corn.

In addition to insects, weeds have also been a menace to farmers - just ask anyone with a garden how much they hateweeds. They can quickly compete for water and nutrients needed by other plants. Sure, farmers can use herbicidesto kill weeds, but do these chemicals also harm the crops? Can biotechnology help with this issue? Some crops havealso been genetically engineered to acquire tolerance to the herbicides - allowing the crops to grow, but killing theweeds. But the lack of cost effective herbicides with a broad range of activity - that do not harm crops - is a problemin weed management. Multiple applications of numerous herbicides are routinely needed to control the wide rangeof weeds that are harmful to crops. And at times these herbicides are being used as a preventive measure – thatis, spraying to prevent weeds from developing rather than spraying after weeds form. So these chemicals are beingadded to crops. This practice is followed by mechanical and/or hand weeding to control weeds that are not controlledby the chemicals. Crops that are tolerant of herbicides would obviously be a tremendous benefit to farmers ( Figure1.9). The introduction of herbicide tolerant crops has the potential to reduce the number of chemicals needed duringa growing season, thereby increasing crop yield due to improved weed management and decreased harm to the crops.

In 2001, 626,000 square kilometers of transgenic crops were planted. Seventy-seven percent of the transgenic cropswere developed for herbicide tolerance in soybean, corn, and cotton, 15% were Bt crops for insect resistance, and

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8% were developed with genes for both insect resistance and herbicide tolerance in cotton and corn.

Production of Vaccines in Crop Plants

Most little children hate shots. And many children in parts of the world do not even have access to vaccines. Butwhat if these vaccines were available in an edible form? Modern biotechnology is increasingly being applied fornovel uses other than food. Banana trees and tomato plants have been genetically engineered to produce vaccines intheir fruit. If future clinical trials prove successful, the advantages of edible vaccines would be enormous, especiallyfor developing countries. The transgenic plants could be grown locally and cheaply. Edible vaccines would notrequire the use of syringes, which, in addition to being unpleasant, can be a source of infections if contaminated.

Scientists have created a transgenic purple tomato that contains a cancer-fighting compound and others that havehigh levels of antioxidants (see Figure 1.10). See http://extension.oregonstate.edu/gardening/purple-tomato-debuts-’indigo-rose’ for more information.

FIGURE 1.10Transgenic Purple Tomato. A purpletomato is genetically modified to containhigh levels of antioxidants. A gene for thecompound was transferred into normalred tomatoes.

Vocabulary

• abiotic: Nonliving, as in the non-living aspects of an ecosystem: soil, water, weather, climate, etc.

• biotic: Living, as in the living components of an ecosystem.

• Bt toxin: Toxin produced from Bacillus thuringiensis, a Gram-positive, soil-dwelling bacterium; commonlyused as a biological pesticide.

• thale cress: A small flowering plant native to Europe, Asia, and northwestern Africa; a popular modelorganism in plant biology and genetics.

• transgenic crop: The result of placement of genes into plants to give the crop a beneficial trait.

Summary

• Biotechnology in agriculture includes the development of transgenic crops - the placement of genes into plantsto give the crop a beneficial trait.

• Benefits of agriculture biotechnology include improved yield from crops, reduced vulnerability of crops toenvironmental stresses, increased nutritional qualities of food crops, improved taste, texture or appearance offood, reduced dependence on fertilizers, pesticides and other agrochemicals, and production of vaccines.

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Practice

Use this resource to answer the questions that follow.

• http://www.hippocampus.org/Biology → Biology for AP*→ Search: Agricultural Applications

1. How can biotechnology help with agricultural issues?2. What is test-tube cloning?3. Describe Golden rice.4. Describe a Ti plasmid and its process.

Practice Answers

1. Advances in agriculture and food biotechnology offer a way to help malnourished and hungry people aroundthe world. Biotechnology has the ability to genetically engineer plants for increased pest and disease resis-tance, and to create crops with increased nutritional value.

2. The test-tube cloning is the process of growing a plant from a single cell or fragment from an adult plant.3. Golden rice is a strain of rice that was engineered to contain extra iron and beta carotene, a precursor to vitamin

A.4. A Ti plasmid contains a stretch of DNA, called T DNA, which inserts into the plant DNA. Restriction enzymes

are used to place a gene of interest in the middle of the T DNA. When the Ti plasmid is introduced into theplant cell, the T DNA and the gene of interest are incorporated into the plant’s genome. A plant containingthe gene of interest is grown from the single cell.

Review

1. Describe uses of biotechnology in agriculture.2. Make a flow chart outlining the steps involved in creating a transgenic crop.3. Describe how DNA technology can improve yield from crops.4. Discuss how DNA technology can be used to reduce vulnerability to environmental stresses. Why is it

important? State an example.

Review Answers

1. Biotechnology is used in agriculture to improve yield from crops, reduce vulnerability of crops to environmen-tal stresses, increase nutritional qualities of food crops, improve taste, texture or appearance of food, reducedependence on fertilizers, pesticides and other agrochemicals, and production of vaccines.

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2.

FIGURE 1.11Creating a Transgenic Crop. A transgeniccrop is genetically modified to be moreuseful to humans.

3. Using biotechnology techniques, one or two genes may be transferred into a crop to give a new trait to thatcrop that has an effect on the overall yield, such as enhancing growth or size.

4. Biotechnology will allow the development of crops containing genes that will enable them to withstand bioticand abiotic stresses. An example is the thale cress, a tiny weed, that is easy to grow. It is important becauseit could help in engineering crops that can be better withstand harsh environments. Scientists have identifieda gene from this plant, At-DBF2, that confers resistance to some environmental stresses. When this geneis inserted into tomato and tobacco cells, the cells were able to withstand environmental stresses like salt,drought, cold and heat far better than ordinary cells.

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1.7 Cloning - Advanced

• Define and describe transgenic animals.• Define and describe animal cloning.

Are cows cloned?

They are, but so are many other animals like sheep and goats. Cloning allows large animals to produce drugs orproteins that are useful in medicine.

Applications of DNA Technology: Animal Cloning

DNA technology has proved very beneficial to humans. Transgenic animals are animals that have incorporated agene from another species into their genome. They are used as experimental models to perform phenotypic testswith genes whose function is unknown, or to generate animals that are susceptible to certain compounds or stressesfor testing purposes. Other applications include the production of human hormones, such as insulin. Many timesthese animals are rodents, such as mice, or fruit flies (Drosophila melanogaster). Fruit flies are extremely useful asgenetic models to study the effects of genetic changes on development. GloFish are the first genetically modifiedanimal to be sold as a pet and are transgenic zebrafish transfected with a natural fluorescence gene. Watch these fishat http://www.youtube.com/watch?v=6cQLGKH2ojY or in the video below.

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/143095

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But transgenic animals just have one novel gene. What about an animal with a whole new genome? Could a clone,a genetically exact copy of an organism, be developed using techniques associated with biotechnology? It couldbe argued that human cloning is one of the inevitable outcomes of modern biotechnology. It "simply" involves theremoval of the nucleus from a somatic cell and its placement into an unfertilized egg cell whose nucleus has eitherbeen deactivated or removed. This new cell would mimic the zygote, the first diploid cell of a new organism. Thisnew zygote is allowed to become established, and a few days later is placed into the uterus of a surrogate mother.Theoretically this would result in an individual genetically identical to the donor. Obviously, there are many ethicaland legal issues associated with human cloning, and of course, it is not a "simple" procedure. But animal cloning isarguably a different story.

Dolly

In February 1997, Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheepnamed Dolly from the mammary glands of an adult female ( Figure 1.12) (Nature 385, 810-13, 1997). Dolly wasthe first mammal to be cloned from an adult somatic cell. The cloning of Dolly made it apparent to many thatthe techniques used to produce her could someday be used to clone human beings. This resulted in tremendouscontroversy because of its ethical implications. After cloning was successfully demonstrated by Dolly’s creators,many other large mammals, including horses and bulls, were cloned. Dolly, however, was put down by lethalinjection in February 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis.Although most sheep like Dolly live to be 11 to 12 years of age, postmortem examination of Dolly seemed toindicate that, other than her cancer and arthritis, she appeared to be quite normal. Dolly was a mother to six lambs,bred through normal methods.

Cloning is now considered a promising tool for preserving endangered species.

In animal cloning, the nucleus from a somatic cell is inserted into an egg cell in which the nucleus has been removed.This process called somatic cell nuclear transfer results in essentially a fertilized egg, a zygote produced in anartificial manner. The resulting cell is cultivated and after a few divisions, the developing ball of cells is placed intoa surrogate mother’s uterus where it is allowed to develop into a fetus. The developing fetus will be geneticallyidentical to the donor of the original nucleus ( Figure 1.13).

For an animation of cloning, see this video: https://www.youtube.com/watch?v=q0B9Bn1WW_4

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/61285

See the Time magazine collection on cloning at http://www.time.com/time/archive/collections/0,21428,c_cloning,00.shtml .

Risks of Cloning

Producing a cloned animal is not an insignificant achievement. Most likely it has taken a significant effort bythe group of scientists attempting to make the clone. Rarely do scientists publish or discuss the many cloningexperiments that failed, or even in the successful clones, the issues that tend to arise later, during the animal’sdevelopment to adulthood. Cloning animals shows us what might happen if we try to clone humans.

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FIGURE 1.12Dolly the sheep. Dolly was the first largemammal to be cloned.

High Failure Rate

Producing a cone through somatic cell nuclear transfer is not every efficient. The success rate ranges from 0.1percent to 3 percent, which means that for every 1000 tries, only one to 30 clones are made. In other words, thereare 970 to 999 failures in every 1000 attempts. That’s a tremendous failure rate. This failure rate may be due to anumber of circumstances. Keep in mind that somatic cell nuclear transfer is an artificial process. It is not a naturalprocess, and there may still be components of the fertilization and development process that are not well understood.

1. The enucleated egg and the transferred nucleus may not be compatible.2. An egg with a newly transferred nucleus may not begin to divide or develop properly.3. Implantation of the embryo into the surrogate mother might fail.4. The pregnancy itself might fail.

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FIGURE 1.13Reproductive cloning: The nucleus is re-moved from a somatic cell and fused witha denucleated egg cell. The resulting cellmay develop into a colony of cloned cells,which is placed into a surrogate mother.In therapeutic cloning, the resulting cellsare grown in tissue culture; an animalis not produced, but genetically identicalcells are produced.

Large Offspring Syndrome

Cloned animals that do survive tend to be much bigger at birth than their natural conceived animals of the samespecies. This is known as "Large Offspring Syndrome". Cloned animals with this syndrome have abnormallylarge organs, which can lead to breathing, blood flow and other associated problems. However this syndrome isunpredictable; it does not always occur, so scientists cannot predict which clones will be affected.

Abnormal Gene Expression

Though surviving clones have identical genomes to their "parent," are they truly clones? Will they express thenecessary genes at the proper times? Gene expression is an extremely complicated and highly regulated cellularprocess (see the Regulation of Gene Expression (Advanced) concepts). One significant issue is to reprogram thetransferred nucleus so that it thinks it is in the zygote, mimicking the natural processes that must be initiated atfertilization, including the expression of the appropriate genes. The cell must be programmed so that the genes that

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must be expressed in that zygote are truly expressed. The nucleus cannot think it is in a differentiated cell, such asthe somatic cell it came from.

See Click and Clone at http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/ to test your knowledge ofsomatic cell nuclear transfer.

Telomeric Differences

As cells divide, their chromosomes get shorter. This is because the telomeres, the DNA sequences at both ends of achromosome, lose material every time the DNA is replicated. The older the animal is, the shorter its telomeres willbe, because of the number of cell cycles the cells have been through This is a natural part of aging. So, what happensto the clone if its transferred nucleus is already fairly old? Will the shortened telomeres affect its development orlifespan? The answer is still unclear. But starting a new organisms with "old" DNA with shortened telomeres isbound to have some effects, at least in some clones. Some cloned animals may be affected, others may not. Dollythe sheep’s chromosomes did have shorter telomere lengths than normal. This means that Dolly’s cells were agingfaster than the cells from a normal sheep.

Vocabulary

• clone: A genetically identical copy; may be a gene, a cell or an organism; an organism that is geneticallyidentical to its parent.

• gene expression: The process by which the information in a gene is "decoded" to produce a functional geneproduct, such as an RNA molecule or a polypeptide/protein molecule.

• somatic cell nuclear transfer: A technique for creating a embryo clone with a donor nucleus.

• telomere: A region of repetitive sequences at each end of a chromosome; protects the end of the chromosomefrom deterioration or from fusion with neighboring chromosomes.

• transgenic animal: An animal with a foreign gene that has been deliberately inserted into its genome.

• zygote: A fertilized egg; the first cell of a new organism.

Summary

• Transgenic animals are animals that have incorporated a gene from another species into their genome. Theyare used as experimental models to perform phenotypic tests with genes whose function is unknown, or togenerate animals that are susceptible to certain compounds or stresses for testing purposes. Other applicationsinclude the production of human hormones, such as insulin.

• Animal cloning is the generation of genetically identical animals using DNA from a donor animal, not agamete. Dolly, a sheep, was the first mammal to be cloned from an adult somatic cell.

Practice

Use this resource to answer the questions that follow.

• Why Clone? at http://learn.genetics.utah.edu/content/tech/cloning/whyclone/

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1. Why is cloning animal models of disease useful in research?2. Why is cloning stem cells for research beneficial?3. What does "Pharming for drug production" refer to?4. How can cloning be used to help endangered species?5. What are some issues associated with cloning?

Practice Answers

1. Cloning animal models of disease is useful in research because it is time efficient and gives scientists morecontrol over their experiments.

2. Cloning stem cells for research is beneficial because they are the body’s building blocks, responsible fordeveloping, maintaining and repairing the body, which can be utilized to repair damaged or diseased organsand tissues.

3. "Pharming for drug production" refers to genetically engineering animals to produce drugs or proteins that areuseful in medicine.

4. Cloning can be used to help to revive endangered species.5. There are a number of ethical, legal and social challenges that are associated with cloning. For example: How

sure can we be that cloned baby will be healthy long-term? What might go wrong?

Review

1. What is the difference between a transgenic animal and a cloned animal?2. Who was Dolly? Why was she important?3. What are the risks of cloning?

Review Answers

1. A transgenic animal is an animal with a foreign gene that has been deliberately inserted into its genome. Acloned animal is an animal genetically identically to its parent.

2. Dolly, the sheep, was the first mammal to be cloned form an adult somatic cell, specifically from the mammaryglands of an adult female. Cloning is now considered a promising tool for preserving endangered species.

3. Risks of cloning are high failure rate, large offspring syndrome, abnormal gene expression and telomericdifferences.

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1.8 Biotechnology and Forensic Science - Ad-vanced

• Describe various applications of biotechnology as related to forensic science.• Why is DNA analysis the most important tool of the forensic scientist?• Describe forensic STR analysis.

What’s a fingerprint made of DNA?

DNA fingerprinting creates a pattern based on an individual’s unique DNA. This can be used as an unique identifierfor a person. As you can imagine, this would be a key tool of the forensic scientist.

Applications of DNA Technology: Forensic DNA Analysis

You know that DNA can be used to distinguish individuals from each other. You may have heard that DNA canalso be used to match evidence and suspects and help solve crimes. This is demonstrated on shows like CSI: CrimeScene Investigation. But how is this done? How is a genetic fingerprint, or a DNA fingerprint, which is a DNApattern unique to each individual (except identical twins), created? Genetic fingerprinting, or DNA fingerprinting,distinguishes between individuals of the same species using only samples of their DNA. DNA fingerprinting has

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thus become one of the most powerful tools of the forensic scientist, enabling law enforcement personnel to matchbiological evidence from crime scenes to suspects. As any two humans have the majority of their DNA sequencein common, those sequences which demonstrate high variability must be analyzed. This DNA analysis was firstdeveloped using DNA hybridization techniques, but now is almost exclusively PCR-based.

DNA fingerprinting was developed by Sir Alec Jeffreys in 1985. Genetic fingerprinting exploits highly variablerepeating sequences. Two categories of these sequences are microsatellites and minisatellites. Microsatellites, alsoknown as short tandem repeats (STRs), consist of adjacent repeating units of 2 - 10 bases in length, for example(GATC)n, where GATC is a tetranucleotide (4 base) repeat and n refers to the number of repeats. It is the numberof repeating units at a given locus that is variable. An STR profile can be created for any individual by analyzinga series of STRs ( Figure 1.14). Two unrelated humans will be unlikely to have the same numbers of repeats at agiven locus.

In STR profiling, PCR is used to obtain enough DNA to then detect the number of repeats at 13 specific loci. PCRproducts are separated by gel or capillary electrophoresis. (Capillary electrophoresis is similar to gel electrophoresisbut uses a capillary tube filled with the gelatin material.) By examining enough STR loci and counting how manyrepeats of a specific STR sequence there are at a given locus, it is possible to create a unique genetic profile ofan individual. STR analysis has become the prevalent analysis method for determining genetic profiles in forensiccases. It is possible to establish a match that is extremely unlikely to have arisen by coincidence, except in thecase of identical twins, who will have identical genetic profiles. The polymorphisms (different in the number ofrepeats) displayed at each STR region will be shared by approximately 5 - 20% of individuals. When analyzingSTRs at multiple loci, such as the 13 STRs analyzed in forensic DNA analysis, it is the unique combinations of thesepolymorphisms in an individual that makes this method unmatched as an identification tool. The more STR regionsthat are analyzed in an individual the more discriminating the test becomes.

Genetic fingerprinting is used in forensic science to match suspects to samples of blood, hair, saliva or semen, orother sources of DNA. It has also led to several exonerations of formerly convicted suspects. Genetic fingerprintingis also used for identifying human remains, testing for paternity, matching organ donors, studying populations ofwild animals, and establishing the province or composition of foods. It has also been used to generate hypotheseson the pattern of human migration.

CODIS

In the United States, DNA fingerprint profiles generated from the 13 STR loci are stored in The National DNA Index(NDIS), part of CODIS, The Combined DNA Index System, maintained by the Federal Bureau of Investigation.As of June 2012, CODIS maintained over 9.7 million offender profiles, 1.1 million arrestee profiles and 436,000forensic profiles. Profiles maintained in CODIS are compiled from both suspects and evidence, and therefore areused to help solve criminal cases. Also as of June 2012, CODIS has produced over 182,200 "hits," assisting in morethan 174,600 investigations. See http://www.fbi.gov/about-us/lab/codis/ndis-statistics/ for additional information.

Profiles of missing persons are also maintained in CODIS. The true power of STR analysis is in its statistical powerof discrimination. Because the 13 loci are independently assorted, the laws of probabilities can be applied. Thismeans that if someone has the genotype of ABC at three independent loci, then the probability of having thatspecific genotype is the probability of having type A times the probability of having type B times the probability ofhaving type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1 with 18 zerosafter it) or more, that is, the chance of two samples matching by coincidence is greater than the number of people onthe planet, or the number of people that have ever lived.

The development of PCR has enabled STR analysis to become the method of choice for DNA identification. Priorto PCR, other methods were utilized. These include restriction fragment length polymorphism (RFLP) analysis andSouthern blot analysis.

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FIGURE 1.14The CODIS loci analyzed by STR anal-ysis. Notice they are spread over 14chromosomes, and that two are on the Xand Y chromosomes.

RFLP Analysis: Restriction Fragment Length Polymorphism

Prior to the development of PCR, restriction enzyme digestion of DNA followed by Southern blot analysis wasused for DNA fingerprinting. This analysis is based on the polymorphic nature of restriction enzyme sites amongdifferent individuals, hence restriction fragment length polymorphisms (RFLP) are formed after digestion ofDNA with these enzymes. A Southern blot, named after its inventor Edwin Southern, is a method used to check forthe presence of a specific DNA sequence in a DNA sample. Once an individual’s DNA is digested with a specificrestriction enzyme, the resulting fragments are analyzed by Southern blot analysis. These fragments will produce aspecific pattern for that individual. Southern blotting is also used for other molecular biology procedures, includinggene identification and isolation. Other blotting methods that employ similar principles have been developed. Theseinclude the western blot and northern blot. These procedures analyze proteins and RNA, respectively.

RFLP and Southern blot analysis involved several steps:

1. First, the DNA being analyzed is cut into different-sized pieces using restriction enzymes.2. The resulting DNA fragments are separated by gel electrophoresis.3. Next, an alkaline solution or heat is applied to the gel so that the DNA denatures and separates into single

strands.4. Nitrocellulose paper is pressed evenly against the gel and then baked so the DNA is permanently attached to

it. The DNA is now ready to be analyzed using a radioactive single-stranded DNA probe in a hybridizationreaction.

5. After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualizedon X-ray film by autoradiography ( Figure 1.15).

Hybridization is when two genetic sequences bind together because of the hydrogen bonds that form between thebase pairs. To make hybridization work, the radioactive probe has to be denatured so that it is single-stranded.The denatured probe and the Southern blot are incubated together, allowing the probe to bind to the correspondingfragment on the Southern blot. The probe will bond to the denatured DNA wherever it finds a fit. Hybridization ofa probe made to a variable segment of DNA will produce a DNA fingerprint pattern specific for an individual. Thisprocedure has a number of steps and is very labor intensive. PCR-based methods are much simpler.

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FIGURE 1.15Mutations can create or abolish restric-tion enzyme (RE) recognition sites, thusaffecting quantities and length of DNAfragments resulting from RE digestion.

Vocabulary

• CODIS: The Combined DNA Index System, is maintained by the Federal Bureau of Investigation and storesDNA profiles.

• DNA fingerprint: A unique DNA pattern that distinguishes between individuals of the same species usingonly samples of their DNA; also known as a genetic fingerprint.

• genetic fingerprint: A unique DNA pattern that distinguishes between individuals of the same species usingonly samples of their DNA; also known as a DNA fingerprint.

• microsatellite: Short sequences of 100-200 bp, usually due to repeats of 1-6 bp sequences; also known as aSTR (Short Tandem Repeat) polymorphism.

• restriction fragment length polymorphism: Genetic differences due to differences between restrictionenzyme sites; produces length variation of DNA segments upon analysis.

• short tandem repeat (STR): Short sequences of 100-200 bp, usually due to repeats of 1-6 bp sequences; alsoknown as a micro satellite.

• Southern blot: A method used to check for the presence of a specific DNA sequence in a DNA sample; namedafter its inventor Edwin Southern.

• STR profile: A genetic profile created through the analysis of 13 STR loci; often used in forensic analysis.

Summary

• Genetic fingerprinting, or DNA fingerprinting, distinguishes between individuals of the same species usingonly samples of their DNA. DNA fingerprinting has thus become one of the most powerful tools of the forensicscientist, enabling law enforcement personnel to match biological evidence from crime scenes to suspects.

Practice I

Use this resource to answer the questions that follow.

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• DNA Evidence: Basics of Analyzing at http://www.nij.gov/topics/forensics/evidence/dna/basics/analyzing.htm

1. What is the role of PCR in DNA typing?2. How does Short Tandem Repeat (STR) Analysis work?3. What is mitochondrial DNA analysis? Why is this useful?4. What is the purpose of DNA evidence?

Practice I Answers

1. The role of PCR in DNA typing is to reproduce millions of copies of DNA contained in a few skin cells. SincePCR analysis requires only a minute quantity of DNA, it can enable the laboratory to analyze highly degradedevidence for DNA.

2. Short tandem repeat (STR) technology is a forensic analysis that evaluates specific regions (loci) that are foundon nuclear DNA. The variable (polymorphic) nature of the STR regions that are analyzed for forensic testingintensifies the discrimination between one DNA profile and another.

3. Mitochondrial DNA (mtDNA) analysis allows forensic laboratories to develop DNA profiles from evidencethat may not be suitable for RFLP or STR analysis. While RFLP and PCR techniques analyze DNA extractedfrom the nucleus of a cell, mtDNA technology analyzes DNA found in a different part of the cell, themitochondrion. Old remains and evidence lacking nucleated cells —such as hair shafts, bones, and teeth—that are unamenable to STR and RFLP testing may yield results if mtDNA analysis is performed.

4. The purpose of DNA evidence is to identify or to exculpate an individual under investigation.

Practice II

• Create a DNA Fingerprint at http://www.pbs.org/wgbh/nova/education/body/create-dna-fingerprint.html

Review

1. What is a DNA fingerprint and how is it used?2. What is STR profiling?3. What is CODIS? What is it used for?4. What are the five steps in RFLP and Southern blot analysis?5. What is hybridization in regards to RFLP?

Review Answers

1. DNA fingerprinting is a unique DNA pattern that distinguishes between individuals of the same species usingonly samples of their DNA. It is used in forensic analysis, enabling law enforcement personnel to matchbiological evidence from crime scenes to suspects.

2. STR profiling is a genetic profile created through the analysis of 13 STR loci.3. CODIS is the Combined DNA Index System which maintained over 9.7 million offender profiles, 1.1 million

arrestee profiles and 436,000 forensic profiles. Profiles maintained are compiled from both suspects andevidence, and therefore are used to help solve criminal cases.

4. The five steps are:

a. First, the DNA being analyzed is cut into different-sized pieces using restriction enzymes.b. The resulting DNA fragments are separated by gel electrophoresis.c. Next, an alkaline solution or heat is applied to the gel so that the DNA denatures and separates into single

strands.

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d. Nitrocellulose paper is pressed evenly against the gel and then baked so the DNA is permanently attachedto it. The DNA is now ready to be analyzed using a radioactive single-stranded DNA probe in ahybridization reaction.

e. After hybridization, excess probe is washed from the membrane, and the pattern of hybridization isvisualized on X-ray film by autoradiography.

5. Hybridization is when two genetic sequences bind together because of the hydrogen bonds that form betweenthe base pairs.

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1.9. Ethical, Legal, and Social Issues of Biotechnology - Advanced www.ck12.org

1.9 Ethical, Legal, and Social Issues ofBiotechnology - Advanced

• Discuss some of the ELSI associated with biotechnology and the Human Genome Project.

Right or wrong? Good or bad? Legal or illegal?

The completion of The Human Genome Project is one of the most important scientific events of the past 50 years.However, is knowing all of our DNA a good thing? Could this tell a person if they are predisposed to develop acertain disease? Could this lead to types of discrimination? The advancement of biotechnology has raised manyinteresting "ethical, legal and social" questions.

Ethical, Legal, and Social Issues

Biotechnology will have a tremendous impact on our future - of this there is no doubt. Is society entering somedangerous areas? Well, many of these issues have never been analyzed until recently. With the discovery of countlessamounts of genetic information and the development of its applications, many ethical, legal and social issues ( ELSI)need to be addressed. The ability to sequence and analyze one’s genome is both of tremendous value and tremendousconcern.

Some ELSI issues (or questions) associated with the Human Genome Project are:

• Who should have access to personal genetic information and how will it be used?• Who owns and controls genetic information?• How does personal genetic information affect an individual and society’s perceptions of that individual?

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• How does genomic information affect members of minority communities?• How reliable and useful is fetal genetic testing?• How will genetic tests be evaluated and regulated for accuracy, reliability, and utility?• How do we prepare the public to make informed choices?• Should testing be performed when no treatment is available?• Should parents have the right to have their minor children tested for adult-onset diseases?• Are genetic tests reliable and interpretable by the medical community?• Where is the line between medical treatment and enhancement?• Are genetically modified foods and other products safe for humans and the environment?• How will these technologies affect developing nations’ dependence on the West?• Who owns genes and other pieces of DNA?• Will patenting DNA sequences limit their accessibility and development into useful products?

Many of these questions will be argued for many years. Many of these questions can only be answered by theindividual. Many of these questions need to be and are being addressed by governments.

Are scientific fantasies, such as those depicted on TV shows such as Star Trek or in the movie GATTACA http://www.youtube.com/watch?v=ZppWok6SX88 , a possibility? Who can really say? How, really, will biotechnologyaffect our future? It seems as if the possibilities are endless.

MEDIAClick image to the left or use the URL below.URL: http://www.ck12.org/flx/render/embeddedobject/165

See the ELSI Research Program of the NIH for further information (http://www.genome.gov/10001618 ).

Vocabulary

• ELSI: Ethical, Legal, and Social Issues; this term is associated with the Human Genome Project.

Summary

• ELSI stands for Ethical, Legal and Social Issues. This is a term associated with the Human Genome project.Rapid advances in DNA-based research, human genetics, and their applications have resulted in new andcomplex ethical and legal issues for society. ELSI programs that identify and address these implications havebeen an integral part of the Human Genome Project since its inception. These programs have resulted in abody of work that promotes education and helps guide the conduct of genetic research and the developmentof related medical and public policies.

Practice

Use this resource to answer the questions that follow.

• http://www.hippocampus.org/Biology → Biology for AP*→ Search: Practical and Ethical Concerns

1. What are two concerns associated with biotechnology?

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2. Why could genetically engineered plants replace naturally grown plants?3. What is cloning? What was the first cloned large mammal?4. What are two ethical considerations associated with the human genome sequence?

Practice Answers

1. The concerns associated with biotechnology are the long-term repercussions of genetic manipulation of or-ganisms in our environment, the effects of genetically engineered organisms escaping into the wild and ethicalimplications of cloning.

2. Genetically engineered plants could replace naturally grown plants because they have advantages, such as pestresistance.

3. Cloning is where a genetic duplicate of an animal is made from a single cell of the animal. The first mammalto be cloned was Dolly, the sheep.

4. Two ethical considerations associated with the human genome sequence are health insurances reactions or theconception of a "perfect" person.

Review

1. Describe why ELSI programs are important.2. List five significant ELSI issues.

Review Answers

1. ELSI programs are important because they identify and address the implications of the Human Genome Projectsince its inception. These programs have resulted in a body of work that promotes education and helps guidethe conduct of genetic research and the development of related medical and public policies.

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1.10 References

1. Left: Sam McCabe; Right: Zachary Wilson. CK-12 Foundation . CC BY-NC 3.02. Left: Image copyright phloxii, 2014; Right: Image copyright Jarrod Erbe, 2014. http://www.shutterstock.com

. Used under licenses from Shutterstock.com3. Jodi So. CK-12 Foundation . CC BY-NC 3.04. User:Plociam/Wikimedia Commons. http://commons.wikimedia.org/wiki/File:Human_genome_to_genes.png

. CC BY 2.05. Left: U.S. Department of Energy Genome Programs; Right: Redrawn based on image by Chris Dixon. Left: ht

tp://commons.wikimedia.org/wiki/File:Logo_HGP.jpg; Right: http://commons.wikimedia.org/wiki/File:DNA_sequence.png . Public Domain

6. Mariana Ruiz Villarreal (LadyofHats) for CK-12 Foundation. CK-12 Foundation . CC BY-NC 3.07. Mariana Ruiz Villarreal (LadyofHats) for CK-12 Foundation. CK-12 Foundation . CC BY-NC 3.08. Marie-Lan Nguyen. http://commons.wikimedia.org/wiki/File:Arabidopsis_thaliana_JdP_2013-04-28.jpg .

CC BY 2.59. Dave Hoisington/CIMMYT. http://commons.wikimedia.org/wiki/File:Btcornafrica.jpg . CC BY 2.5

10. Purple tomato: F Delventhal; Red tomato; Flickr:photo_de. Purple tomato: http://www.flickr.com/photos/krossbow/7826368470/; Red tomato; http://www.flickr.com/photos/photon_de/2700323949/ . CC BY 2.0

11. Jodi So. CK-12 Foundation . CC BY-NC 3.012. Colin and Sarah Northway. www.flickr.com/photos/46174988@N00/4822043093/ . CC BY 2.013. Zachary Wilson. CK-12 Foundation . CC BY-NC 3.014. Courtesy of Chemical Science Technology Laboratory, National Institute of Standards and Technology. http

://commons.wikimedia.org/wiki/File:Codis_profile.jpg . Public Domain15. NCBI Probe Database. http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml . Public

Domain

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