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Genetic Technologies: Amplifying, Modifying, and Monitoring DNA

Genetic Technologies: Amplifying, Modifying, and Monitoring DNA

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Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



© The McGraw−Hill

Companies, 2010



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



19.1 Patenting DNA

Biotechnology is the use or alteration of cells or biological molecules for specific applications. It is an ancient art as well as a

modern science. Using yeast to ferment fruit or produce wine are

biotechnologies, as is extracting biochemicals from organisms.

The popular terms “genetic engineering” and “genetic

modification” refer broadly to any biotechnology that manipulates DNA. This includes altering the DNA of an organism to

suppress or enhance the activities of its own genes, as well as

combining the genetic material of different species. Organisms

that harbor DNA from other species are termed transgenic and

their DNA is called recombinant DNA.

Creating transgenic organisms is possible because all life

uses the same genetic code (figure 19.1). Mixing DNA from

different species may seem unnatural, but in fact DNA moves

and mixes between species in nature—bacteria do it, and it is

why we have viral DNA sequences in our chromosomes. But

human-directed genetic modification usually endows organisms with traits they would probably not acquire naturally, such

as fish that can tolerate very cold water, tomatoes that grow in

salt water, and bacteria that synthesize human insulin.



381



DNA sequence from the human genome might be used to diagnose a specific disease, but could inhibit research unless exceptions to use are made for researchers. DNA is also patentable as

a research tool, as are algorithms used to extract information

from DNA sequences, and databases built of DNA sequences.

The Technology Timeline highlights some of the events and

controversy surrounding patenting of genetic material.

Patent law has had to evolve to keep up with modern

biotechnology. In the 1980s, when sequencing a gene was

painstakingly slow, only a few genes were patented. Then, in

the mid-1990s, with faster sequencing technology and shortcuts

to finding the protein-encoding parts of the genome, the U.S.

National Institutes of Health and biotech companies began seeking patent protection for thousands of short DNA sequences,

even if their functions weren’t known. Because of the flood of

applications, the U.S. Patent and Trademark Office tightened

requirements for usefulness. Today, with entire genomes being



Technology Timeline

PATE NTI N G LI FE AN D G E N E S



1790 U.S. patent act is enacted. An invention must be new,

useful, and not obvious to earn a patent.



What Is Patentable?

Creating transgenic organisms raises legal questions, because

the design of novel combinations of DNA may be considered

intellectual property, and therefore patentable. To qualify for

patent protection, a transgenic organism, as any other invention,

must be new, useful, and not obvious to an expert in the field. A

corn plant that manufactures a protein naturally found in green

beans but not in corn, thereby making the corn more nutritious,

is an example of a patentable transgenic organism. A patent for a



1873 Louis Pasteur is awarded first patent on a life form, for

yeast used in industrial processes.



1930 New plant variants can be patented.

1980 First patent is awarded on a genetically modified

organism, a bacterium given four plasmids (DNA rings)

that enable it to metabolize components of crude oil.

The plasmids are naturally occurring, but do not all occur

naturally in a single type of bacterium.



1988 First patent is awarded for a transgenic organism, a

mouse that manufactures human protein in its milk.

Harvard University granted patent for “OncoMouse”

transgenic for human cancer.



1992 Biotechnology company is awarded a broad patent

covering all forms of transgenic cotton. Groups

concerned that this will limit the rights of subsistence

farmers contest the patent several times.



1996–1999 Companies patent partial gene sequences and certain

disease-causing genes as the basis for developing specific

medical tests.



2000 With gene and genome discoveries pouring into the

Patent and Trademark Office, requirements tightened for

showing utility of a DNA sequence.



2003 Attempts to enforce patents on nonprotein-encoding

parts of the human genome anger researchers who

support open access to the information.



Figure 19.1 The universality of the genetic code makes

biotechnology possible. The greenish mice contain the gene

encoding a jellyfish’s green fluorescent protein (GFP). Researchers

use GFP to mark genes of interest. The GFP mice glow less greenly

as they mature and more hair covers the skin. The non-green mice

are not genetically modified. The Nobel prize was awarded in 2008

for uses of GFP to mark molecules in live organisms.



2007 Patent requirements must embrace new, more complex

definition of a gene.



2010 Direct-to-consumer genetic testing companies

struggle to license DNA patents for multi-gene and SNP

association tests.



Chapter 19 Genetic Technologies: Amplifying, Modifying, and Monitoring DNA



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Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



sequenced much faster (sometimes in only a matter of days)

than it once took to decipher a single gene, a DNA sequence

alone does not warrant patent protection. It must be useful as

a tool for research or as a novel or improved product, such as

a diagnostic test or a drug. In the United States, more than

48,000 patents have been filed on DNA, with 63,000 pending.

However, since 1987, only thirty-one lawsuits involving DNA

patents have actually gone to court.

Despite the increasing stringency of patent requirements,

problems still arise concerning the status of DNA sequences.

A biotechnology company in the United States, for example,

holds a patent on the BRCA1 breast cancer gene that includes

any diagnostic tests based on the DNA sequence. The American Civil Liberties Union is challenging the BRCA1 patent,

because tests that use the gene are expensive and available

only from this one company. The Bioethics: Choices for the

Future box in chapter 20 explores another patent-related

issue: families helping to discover genes by donating their

children’s DNA, then having to pay for the tests developed

from the research.

Analysis of human genome information continues

to complicate patenting. One problem is redundancy. For

the same gene, it is possible to patent the entire sequence

(termed genomic DNA), or just the protein-encoding exons. A

researcher can also patent a gene variant, such as a sequence

containing a SNP or mutation. A company or researcher developing a tool or test based on a particular gene or its encoded

protein might infringe upon several patents that are based on

essentially the same information. For example, it is unclear

how patents would cover exons common to different genes.

Now, as genetics shifts from a gene-by-gene focus to analyzing expression patterns of suites of interacting genes, patent

law will have to once again adjust to keep up with scientific

developments.



© The McGraw−Hill

Companies, 2010



government has stepped in. A committee has suggested ways

around the patent thicket:









Ban the patenting of associations between DNA

sequence variants and disease.

Allow DNA to be patented only for use in a diagnostic test.

Exempt physicians and researchers from litigation if

they use patented DNA sequences.



A broader action is the Genomic Research and Accessibility Act,

which would ban patenting any DNA or its encoded proteins.

While the laws are being worked out, companies can

navigate the patent thicket by moving the parts of their operations that use the patented DNA sequences to countries where

the restrictions on use do not apply. They can also tweak the

recipes for a patented procedure, such as substituting a different type of cell in culture that produces a particular protein, or

altering chemical protocols.

The direct-to-consumer companies are finding themselves

in an identity crisis. When these companies began to spring up

a few years ago, they circumvented regulations on genetic tests

for disease by claiming that they provided only information as

an educational service. If the law disallows patents for use of

DNA sequences in diagnostic tests, these companies would not

be included because of how they identify themselves. But if the

companies change their tune, claiming to offer tests for diseases

so that they have access to patented sequences, they will be under

scrutiny of the federal agencies that regulate genetic testing and

products for health-related purposes.



Key Concepts

1. Biotechnology is the use or modification of cells or

biological molecules for a specific application.

2. DNA patenting is evolving to embrace genome-wide

applications.



The Patent Thicket

A new problem with patenting DNA stems from the shift in

focus of the entire field from a single-gene to a genome-wide

approach. A company seeking to use part of a gene sequence

in a test must license the use of that sequence from the patent holder. Many companies, however, offer “panels” of tests,

such as for several heart-related disorders or for conditions

that are more prevalent among Ashkenazi Jews. The companies must license many patented DNA sequences. The challenge is amplified for the many direct-to-consumer genetic

testing companies that scan clients’ DNA for many thousands

of SNPs, looking for “associations” rather than diagnoses. If

each SNP is patented, and requires payment of 1 to 5 percent

of the profit, such tests cannot be developed unless a company

owns the patents.

People in the business of using DNA sequences in tests

and products term the need to license patents for every SNP or

snippet of DNA the “patent thicket.” Because such companies

are forming at a rate that is faster than the Patent and Trademark office can handle the issuing of DNA patents, the U.S.

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19.2 Amplifying DNA

Some forensic and medical tests require many copies of a specific

DNA sequence from a small sample. Mass-producing a DNA

sequence, called nucleic acid amplification, was invented in the

1970s and 1980s. The first and best known technique is the polymerase chain reaction (PCR), which works on DNA molecules

outside cells. Another approach, recombinant DNA technology,

amplifies DNA from one type of organism placed in the cell of

another. Recombinant DNA technology is addressed in section 19.3.

PCR is based on the natural process of DNA replication.

Recall from chapter 9 that every time a cell divides, it replicates

all of its DNA. PCR uses DNA polymerase to rapidly replicate

a specific DNA sequence millions of times.

Applications of PCR are eclectic (table 19.1). In forensics,

PCR is used routinely to amplify DNA sequences that are profiled

to establish blood relationships, to identify remains, and to help

convict criminals or exonerate the falsely accused. In agriculture,

veterinary medicine, environmental science, and human health



Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



Table 19.1



VI. Genetic Technology



© The McGraw−Hill

Companies, 2010



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



Uses of PCR



PCR has been used to amplify DNA from:

■ a cremated man, from skin cells left in his electric shaver, to



diagnose an inherited disease in his children.

■ a preserved quagga (a relative of the zebra) and a marsupial wolf,



both extinct.

■ microorganisms that cannot be cultured for study.

■ the brain of a 7,000-year-old human mummy.

■ the digestive tracts of carnivores, to reveal food web interactions.

■ roadkills and carcasses washed ashore, to identify locally



threatened species.

■ products illegally made from endangered species.

■ genetically altered bacteria that are released in field tests, to



follow their dispersion.

■ one cell of an 8-celled human embryo to detect a disease-related



genotype.

■ poached moose meat in hamburger.

■ remains in Jesse James’s grave, to make a positive identification.

■ the guts of genital crab lice on a rape victim, which matched the



DNA of the suspect.

■ fur from Snowball, a cat that linked a murder suspect to a



crime.



383



care, PCR is used to amplify the DNA or RNA of pathogens to

detectable levels. In genetics, PCR is both a crucial laboratory tool

to identify genes and it is a component of many diagnostic tests.

PCR was born in the mind of Kary Mullis on a moonlit night in northern California in 1983. As he drove the hills,

Mullis was thinking about the precision of DNA replication,

and a way to tap into it popped into his mind. He excitedly

explained his idea to his girlfriend and then went home to think

it through. “It was difficult for me to sleep with deoxyribonuclear bombs exploding in my brain,” he wrote much later.

The idea behind PCR was so simple that Mullis had

trouble convincing his superiors at Cetus Corporation that he

was onto something. Over the next year, he used the technique

to amplify a well-studied gene. Mullis published a landmark

1985 paper and filed patent applications, launching the field of

nucleic acid amplification. He received a $10,000 bonus for his

invention, which the company sold to another for $300 million.

Mullis did, however, win a Nobel prize.

PCR rapidly replicates a selected sequence of DNA in a

test tube (figure 19.2). The requirements include:

1. Knowing parts of a target DNA sequence to be amplified.

2. Two types of lab-made, single-stranded, short pieces

of DNA called primers. These are complementary in

sequence to opposite ends of the target sequence.

3. A large supply of the four types of DNA nucleotide

building blocks.

4. Taq1, a DNA polymerase produced by Thermus

aquaticus, a microbe that inhabits hot springs. This

enzyme is adapted to its host’s hot surroundings and

makes PCR easy because it does not fall apart when

DNA is heated, as most proteins do.



Primers

Free

nucleotides



Polymerase



Raise

temperature



Target

sequence



Target

sequence



Lower

temperature



Heat

separates

strands



Primers hybridize

due to base

complementarity



Polymerase finishes

replicating DNA

Repeat process

many times



Figure 19.2



Amplifying a specific DNA sequence. In the polymerase chain reaction, specific primers, a thermostable DNA

polymerase, and free nucleotides replicate a DNA sequence of interest. The reaction rapidly builds up millions of copies of the target

sequence. Figure 14.9 shows an application of PCR.

Chapter 19 Genetic Technologies: Amplifying, Modifying, and Monitoring DNA



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Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



In the first step of PCR, heat is used to separate the two

strands of the target DNA. Next, the two short DNA primers and

Taq1 DNA polymerase are added. The temperature is lowered.

Primers bind by complementary base pairing to the separated

target strands. In the third step, the Taq1 DNA polymerase adds

bases to the primers and builds a sequence complementary to

the target sequence. The newly synthesized strands then act as

templates in the next round of replication, which is initiated

immediately by raising the temperature. All of this is done in an

automated device called a thermal cycler, or in a device that uses

microscopic layers of heated and cooled silicon, to control the

key temperature changes. The heat-resistant DNA polymerase is

crucial to the process.

The pieces of identical DNA accumulate exponentially. The

number of amplified pieces of DNA equals 2n, where n equals the

number of temperature cycles. After just 20 cycles, 1 million copies of the original sequence have accumulated in the test tube.

PCR’s greatest strength is that it works on crude samples

of rare, old, and minute sequences. PCR’s greatest weakness,

ironically, is its exquisite sensitivity. A blood sample submitted

for diagnosis of an infection, if contaminated by leftover DNA

from a previous test, or a stray eyelash from the person running

the reaction, can yield a false result.

Using layered silicon instead of a thermal cycler to

amplify DNA greatly speeds PCR. Thirty cycles using the

thermal cycler take ninety minutes; with the silicon layers, it

takes a little over 4 minutes. The speed is valuable in situations

where rapid diagnosis is important, such as the case of a person

with a life-threatening infection who requires a certain antibiotic, or on a battlefield to detect biological weapons.

The invention of PCR inspired other nucleic acid amplification techniques. One is transcription-mediated amplification,

which copies target DNA into RNA and then uses RNA polymerase to amplify the RNA. This procedure doesn’t require

temperature shifts, and it generates 100 to 1,000 copies per

cycle, compared to PCR’s doubling, and can yield 10 billion

copies of a selected sequence in a half hour.



Key Concepts

1. PCR rapidly replicates a short DNA sequence.

2. PCR is based on DNA replication and has many uses.

3. Other nucleic acid amplification technologies

followed PCR.



“Cloning” in this context refers to making many copies of a

specific DNA sequence.



Recombinant DNA

Researchers first began pondering the potential uses and risks

of mixing DNA from different species in the 1970s. It started

in February 1975, when 140 molecular biologists convened at

Asilomar, on California’s Monterey Peninsula, to discuss the

safety and implications of a new type of experiment: combining genes of two species. Would planned experiments that use

a cancer-causing virus to deliver DNA be safe? The researchers discussed restricting the types of organisms used in recombinant DNA research and brainstormed ways to prevent escape

of a resulting organism from the laboratory. The guidelines

drawn up at Asilomar outlined measures of “physical containment,” such as using specialized hoods and airflow systems

that would keep recombinant microorganisms inside the laboratory, and “biological containment,” such as weakening organisms so that they could not survive outside the laboratory.

Recombinant DNA technology turned out to be safer

than expected, and it spread to industry faster and in more

diverse ways than anyone had imagined. However, recombinant

DNA-based products have been slow to reach the marketplace

because of the high cost of the research and the long time it

takes to develop any new drug. Today, several dozen such drugs

are available, and more are in the pipeline. Recombinant DNA

research initially focused on providing direct gene products

such as peptides and proteins. These included insulin, growth

hormone, and clotting factors. However, the technology can target carbohydrates and lipids by affecting the genes that encode

enzymes required to synthesize them.



Constructing Recombinant DNA Molecules—

An Overview

Manufacturing recombinant DNA molecules requires restriction

enzymes that cut donor and recipient DNA at the same sequence;

DNA to carry the donor DNA (called cloning vectors); and recipient cells (bacteria or other cultured single cells).

After inserting donor DNA into vectors, the procedure

requires several steps to get the desired modified cell type:











19.3 Modifying DNA

Recombinant DNA technology adds genes from one type of

organism to the genome of another. It was the first gene modification biotechnology, and was initially done in bacteria to

produce peptides and proteins useful as drugs. When bacteria

bearing recombinant DNA divide, they yield many copies of

the “foreign” DNA, and under proper conditions they produce

many copies of the protein that the foreign DNA specifies.

Recombinant DNA technology is also known as gene cloning.

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Companies, 2010



Selecting cells where the genetic material includes any

foreign DNA

Selecting cells that received the gene of interest

Stimulating transcription of the foreign gene and

translation of its protein product

Collecting and purifying the desired protein



The natural function of restriction enzymes is to protect

bacteria by cutting DNA of infecting viruses. Methyl (CH3) groups

shield the bacterium’s own DNA from its restriction enzymes.

Bacteria have hundreds of types of restriction enzymes. Some of

them cut DNA at particular sequences of four, five, or six bases

that are symmetrical in a specific way—the recognized sequence

reads the same, from the 5′ to 3′ direction, on both strands of

the DNA. For example, the restriction enzyme EcoR1, shown

in figure 19.3, cuts at the sequence GAATTC. The complementary sequence on the other strand is CTTAAG, which, read



Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



backwards, is GAATTC. (You can try this with other sequences

to see that it rarely works this way.) In the English language, this

type of symmetry is called a palindrome, referring to a sequence

of letters that reads the same in both directions, such as “Madam,

I’m Adam.” Unlike the language comparison, however, palindromic sequences in DNA are on complementary strands.

The cutting action of some restriction enzymes on

double-stranded DNA creates single-stranded extensions. They

are called “sticky ends” because they are complementary to

each other, forming hydrogen bonds as their bases pair. Restriction enzymes work as molecular scissors in creating recombinant DNA molecules because they cut at the same sequence in

any DNA source. That is, the same sticky ends result from the

same restriction enzyme, whether the DNA is from a mockingbird or a maple.

Another natural “tool” used in recombinant DNA technology is a cloning vector. This structure carries DNA from

the cells of one species into the cells of another. A vector can

be any piece of DNA into which other DNA can insert. A commonly used type of vector is a plasmid, which is a small circle

of double-stranded DNA that occurs naturally in some bacteria, yeasts, plant cells, and other types of organisms. Viruses

that infect bacteria, called bacteriophages, are another type of



G A A T T C

C T T A A G



a.

Restriction enzymes cut

DNA at specific sequences.

Sticky end



A A T T C

G

G

C T T A A



A A T T C

G

G

C T T A A



b.

Sticky end

Donor DNA



A A T T C

G



G

C T T A A



DNAs from two sources cut with the

same restriction enzyme have

“sticky” ends. Two pieces of

DNA hydrogen bond, and ligase seals

the sugar-phosphate backbone.

G A A T T C

C T T A A G

Host DNA



G A A T T C

C T T A A G

Donor DNA



385



vector. Bacteriophages are manipulated to transport DNA but

not cause disease. Disabled retroviruses are used as vectors too,

as are DNA sequences from bacteria and yeast called artificial

chromosomes.

When choosing a cloning vector, size matters. The desired

gene must be short enough to insert into the vector. Gene size

is typically measured in kilobases (kb), which are thousands

of bases. Various types of cloning vectors can hold up to about

2 million DNA bases.

To create a recombinant DNA molecule, a restriction

enzyme cuts DNA from a donor cell at sequences known to

bracket the gene of interest (figure 19.4). The enzyme leaves

single-stranded ends on the cut DNA, each bearing a characteristic base sequence. Next, a plasmid is isolated and cut with the

same restriction enzyme used to cut the donor DNA. Because

the same restriction enzyme cuts both the donor DNA and the

plasmid DNA, the same complementary single-stranded base

sequences extend from the cut ends of each. When the cut plasmid and the donor DNA are mixed, the single-stranded sticky

ends of some plasmids base pair with the sticky ends of the

donor DNA. The result is a recombinant DNA molecule, such

as a plasmid carrying the human insulin gene. The plasmid and

its human gene can now be transferred into a cell, such as a

bacterium or a white blood cell.



Isolating the Gene of Interest



Restriction enzyme

recognition sequence

G A A T T C

C T T A A G



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Host DNA



c.



Figure 19.3 Recombining DNA. A restriction enzyme makes

“sticky ends” in DNA by cutting it at specific sequences. (a) The

enzyme EcoR1 cuts the sequence GAATTC between G and A.

(b) This staggered cutting pattern produces “sticky ends” of

sequence AATT. The ends attract through complementary base

pairing. (c) DNA from two sources is cut with the same restriction

enzyme. Pieces join, forming recombinant DNA molecules.



Constructing recombinant DNA molecules usually begins by

cutting all of the DNA in the donor cell. This DNA, which

includes non-protein-encoding sequences, is termed genomic

DNA. Researchers assemble collections of recombinant bacteria (or other single cells) that harbor pieces of a genome. By

using several copies of a genome, the pieces overlap where

sequences align. Such a collection is called a genomic library.

For each application, such as using a human protein as a drug,

a particular piece of DNA must be identified and isolated from

a genomic library. There are several ways to do this “needle in

a haystack” type of search.

A piece of DNA that is complementary to part of the

template strand of the gene in question can be linked to a label,

such as a radioactive or fluorescent molecule. This labeled

gene fragment is called a DNA probe. It emits a signal when

it binds to its complement in a cell that contains a recombinant

plasmid. DNA probes can also be made using genes of similar

sequence from other species—they will bind the human version of the gene. Using such a probe is a little like mistakenly

typing “hipropotamus” to google “hippopotamus.” You’d probably still come up with a hippo.

A genomic library contains too much information for a

researcher seeking a particular protein-encoding gene—it may

also contain introns, the genes that encode rRNAs and tRNAs,

and many repeated sequences. A shortcut is to use another type

of library, called a complementary DNA, or cDNA library,

that represents only protein-encoding genes. A cDNA library is

made from the mRNAs in a differentiated cell, which represent

the proteins manufactured there. For example, a muscle cell has

abundant mRNAs that encode contractile proteins, whereas a



Chapter 19 Genetic Technologies: Amplifying, Modifying, and Monitoring DNA



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Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



© The McGraw−Hill

Companies, 2010



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



Bioreactor



DNA isolated

A specific restriction

from donor cell enzyme fragments

(animal or plant) donor DNA

Scale

up



Plasmid

isolated

from

bacterium



The same restriction

enzyme that fragmented

donor DNA is also used

to open plasmid DNA



Donor and plasmid Modified plasmids

DNA are mixed;

(recombinant DNA)

“sticky ends” of

are introduced into

donor DNA

bacteria

hydrogen bond

with sticky ends

of plasmid DNA

fragment; recombinant

molecule is sealed

with ligase



Bacteria divide and

clone the gene spliced

into the plasmids



Drug is produced



Figure 19.4 Recombinant DNA. DNA isolated from a donor cell and a plasmid are cut with the same restriction enzyme and mixed.

Sticky ends from the donor DNA hydrogen bond with sticky ends of the plasmid DNA, forming recombinant DNA molecules. When such a

modified plasmid is introduced into a bacterium, it is mass produced as the bacterium divides.



fibroblast has many mRNAs that represent connective tissue

proteins.

To make a cDNA library, researchers first extract the

mRNAs from cells. Then, these RNAs are used to construct

complementary or “c” DNA strands using reverse transcriptase, DNA nucleotide triphosphates, and DNA polymerase

(figure 19.5). Reverse transcriptase synthesizes DNA complementary to RNA. DNA polymerase and the nucleotides then

can synthesize the complementary strand to the single-stranded

cDNA to form a double-stranded DNA. Different cell types

yield different cDNA collections, or libraries, that reflect which

genes are expressed. They do not, however, reveal protein abundance because in a cell mRNA molecules are transcribed and

degraded at different rates.

A specific cDNA can be taken from a cDNA library and

used as a probe to isolate the original gene of interest from the

genomic library. If the goal is to harness the gene and eventually

collect its protein product, then the genomic version is useful,

because it includes control regions such as promoters. Once a gene

of interest is transferred to a cell where it can be transcribed into

mRNA and that RNA can be translated, the protein is collected.

Such cells are typically grown in containers called bioreactors,

with nutrients sent in and wastes removed. The desired product is

collected from the medium in which the cells are growing.

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Mature mRNA

transcript

mRNA isolated;

reverse transcriptase

added

mRNA-cDNA

hybrid

mRNA-degrading

enzymes added

Single-stranded

cDNA

DNA polymerase

added

Double-stranded

cDNA



Figure 19.5



Copying DNA from RNA. Researchers make cDNA

from mRNA using reverse transcriptase, an enzyme from a retrovirus.

A cDNA version of a gene includes the codons for a mature mRNA,

but not sequences for promoters and introns. Control sequences

from bacteria may be added so that the eukaryotic gene can be

transcribed and translated in a prokaryote (the bacterium).



Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



Selecting Recombinant DNA Molecules

Much of the effort in recombinant DNA technology is in identifying and separating cells that contain the gene of interest,

once the foreign DNA is inserted into the vector. Three types

of recipient cells can result:

1. Cells that lack plasmids

2. Cells that contain plasmids that do not contain a foreign gene

3. Cells that contain plasmids that have picked up a foreign

gene (the goal)

The procedure is set up to distinguish bacteria that have

taken up recombinant plasmids from those that have not taken up

plasmids or that have admitted plasmids that do not carry foreign

DNA. One type of strategy has two steps: using an antibiotic resistance gene and a color change reaction to highlight the plasmids

that have picked up the gene of interest. First, human and plasmid

DNA are cut with the same restriction enzymes and mixed. The

plasmids are closed with ligase (the enzyme that glues the sugarphosphate backbone when DNA replicates), and transferred to

bacterial cells. When the antibiotic is applied, only cells harboring

plasmids survive. The plasmids also include a gene that encodes

an enzyme that catalyzes a reaction that produces a blue color. If

a human gene inserts and interrupts the gene for the enzyme, the

bacterial colony that grows is not blue, and is therefore easily distinguished from the blue bacterial cells that have not incorporated

the human gene.

When cells containing the recombinant plasmid divide,

so does the plasmid. Within hours, the original cell gives rise to

many cells harboring the recombinant plasmid. The enzymes,

ribosomes, energy molecules, and factors necessary for protein

synthesis transcribe and translate the plasmid DNA and its foreign gene, producing the desired protein.



Products from Recombinant DNA Technology

In basic research, recombinant DNA technology provides a way

to isolate individual genes from complex organisms and observe

their functions on the molecular level. Recombinant DNA has

many practical uses, too. The first was to mass-produce proteinbased drugs.

Drugs manufactured using recombinant DNA technology are pure, and are the human version of the protein. Before

recombinant DNA technology was invented, human growth

hormone came from cadavers, follicle-stimulating hormone

came from the urine of post-menopausal women, and clotting

factors were pooled from hundreds or thousands of donors—

introducing great risk of infection, especially after HIV and

hepatitis C became more widespread.

The first drug manufactured using recombinant DNA

technology was insulin. Before 1982, people with type 1 diabetes mellitus obtained the insulin that they injected daily from

pancreases removed from cattle in slaughterhouses. Cattle

insulin is so similar to the human peptide, different in only

two of its fifty-one amino acids, that most people with diabetes could use it. However, about one in twenty patients is allergic to cow insulin because of the slight chemical difference.



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387



Until recombinant DNA technology was developed, the allergic patients had to use expensive combinations of insulin from

other animals or human cadavers. Table 19.2 lists some drugs

produced using recombinant DNA technology.

Insulin is a simple peptide and is therefore straightforward to mass-produce in bacteria. Some drugs, however,

require that sugars be attached, or must fold in specific, intricate ways to function. These molecules must be produced in

eukaryotic cells.

Drugs developed using recombinant DNA technology must

compete with conventional products. Deciding whether a recombinant drug is preferable to an existing similar drug is often a

matter of economics. For example, interferon β-1b treats a type

of multiple sclerosis, but this recombinant drug costs more than

$20,000 per year. British researchers calculated that more people

would be served if funds were spent on improved supportive care

for many rather than on this costly treatment for a few.

Tissue plasminogen activator (tPA), a recombinant clotbusting drug, also has cheaper alternatives. If injected within 4

hours of a heart attack, tPA dramatically limits damage to the

heart muscle by restoring blood flow. It costs $2,200 a shot. An

older drug, streptokinase, is extracted from unaltered bacteria

and is nearly as effective, at $300 per injection. tPA is very

valuable for patients who have already had streptokinase and

could have an allergic reaction if they were to use it again. Bioethics: Choices for the Future on page 387 considers another

drug derived from recombinant DNA technology, erythropoietin (EPO).

An application of recombinant DNA technology in the

textile industry is a novel source of indigo—the dye used to

make blue jeans blue. The dye originally came from mollusks

and fermented leaves of the European woad plant or Asian

indigo plant. The 1883 discovery of indigo’s chemical structure

led to the invention of a synthetic process to produce the dye

using coal-tar. That method has dominated the industry, but it

releases toxic by-products.

In 1983, microbiologists discovered that E. coli, with a little help, can produce indigo. The bacterium converts glucose to

the amino acid tryptophan, which then forms indole, a precursor

to indigo. Another type of bacterium takes the indole to indigo.

Researchers altered E. coli to suppress alternative pathways for

metabolizing glucose, coaxing the cells to synthesize excess

tryptophan. They then added genes from the other bacterial species, extending the biochemical pathway all the way to produce

indigo. The result: common bacteria that manufacture the blue

dye of denim jeans from glucose, a simple sugar.



Transgenic Animals

Eukaryotic cells growing in culture are generally better at

producing human proteins than are prokaryotic cells such as

bacteria. An even more efficient way to express some recombinant genes is in a body fluid of a transgenic animal, such as

milk. The fact that the cells secreting the human protein are

part of an animal more closely mimics the environment in the

human body.



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Concepts and Applications,

Ninth Edition



Table 19.2



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



Drugs Produced Using Recombinant DNA Technology



Drug



Use



Atrial natriuretic peptide



Dilates blood vessels, promotes urination



Colony stimulating factors



Help restore bone marrow after marrow transplant; restore blood cells following cancer chemotherapy



Deoxyribonuclease (DNase)



Thins secretions in lungs of people with cystic fibrosis



Epidermal growth factor



Accelerates healing of wounds and burns; treats gastric ulcers



Erythropoietin (EPO)



Stimulates production of red blood cells in cancer patients



Factor VIII



Promotes blood clotting in treatment of hemophilia



Glucocerebrosidase



Corrects enzyme deficiency in Gaucher disease



Human growth hormone



Promotes growth of muscle and bone in people with very short stature due to hormone deficiency



Insulin



Allows cells to take up glucose in treatment of type 1 diabetes



Interferons



Treats genital warts, hairy cell leukemia, hepatitis C and B, Kaposi sarcoma, multiple sclerosis



Interleukin-2



Treats kidney cancer recurrence



Lung surfactant protein



Helps lung alveoli to inflate in infants with respiratory distress syndrome



Renin inhibitor



Lowers blood pressure



Somatostatin



Decreases growth in muscle and bone in pituitary giants



Superoxide dismutase



Prevents further damage to heart muscle after heart attack



Tissue plasminogen activator



Dissolves blood clots in treatment of heart attacks, stroke, and pulmonary embolism



Transgenic sheep, cows, and goats have all expressed

human genes in their milk, including those that encode clotting factors, clot busters, and collagen. Production of human

antibodies in rabbit and cow milk illustrates the potential value

of transgenic animals. Recall from figure 17.9 that antibodies

are assembled from the products of several genes. Researchers

attach the appropriate human antibody genes to promoters for

milk proteins. (Recall from chapter 10 that a promoter is a short

sequence at the start of a gene that controls the rate of transcription.) These promoters normally oversee production of abundant

milk proteins. The mammary gland cells of transgenic animals

can assemble antibody parts to secrete the final molecules—just

as if they were being produced in an activated B cell in the human

immune system. Such antibodies can be used to treat cancer.

Several techniques are used to insert DNA into animal

cells to create transgenic animals. They include:













386



© The McGraw−Hill

Companies, 2010



chemicals that open transient holes in plasma

membranes

liposomes (fatty bubbles) that carry DNA into cells as

plasma membranes envelop them

brief jolts of electricity (electroporation) that open

transient holes in plasma membranes

microscopic needles that inject DNA into cells

(microinjection)

metal particles coated with foreign DNA shot into cells

(particle bombardment)

Part 6



Genetic Technology



As in plant cells, once foreign DNA is introduced into

an animal cell, it must enter the nucleus, replicate along with

the cell’s own DNA, and be transmitted when the cell divides.

Finally, an organism must be regenerated from the altered cell.

If the trait is dominant, the transgenic organism must express it

in the appropriate tissues at the right time in development. If the

trait is recessive, crosses between heterozygotes may be necessary to yield homozygotes that express the trait. Then the organisms must pass the characteristic on to the next generation.



Animal Models

Herds of transgenic farm animals supplying drugs in their milk

have not become important sources of pharmaceuticals—they

are too difficult to maintain. Transgenic animals are far more

useful as models of human disease. Inserting the mutant human

beta globin gene that causes sickle cell disease into mice, for

example, results in a mouse model of the disorder. Drug candidates can be tested on these animal models and abandoned if

they cause significant side effects before testing in humans.

Transgenic animal models, however, have limitations.

Researchers cannot control where a transgene inserts in a

genome, and how many copies do so. The level of gene expression necessary for a phenotype to emerge may also differ in the

model and humans. This was the case for a mouse model of

familial Alzheimer disease (MIM 104760). The transgene has

the exact same DNA sequence that disrupts amyloid precursor

protein in a Swedish family with the condition, but apparently



Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



© The McGraw−Hill

Companies, 2010



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



389



Bioethics: Choices for the Future



EPO: Built-in Blood Cell Booster or Performance-Enhancing Drug?

Athletes are chastised for using performance-enhancing substances.

Should this happen if an athlete’s body naturally produces greater-



patients and is given with cancer chemotherapy to avoid the need for

transfusions.



than-average amounts of a substance that gives a competitive

advantage? This is the situation for EPO (erythropoietin), a glycoprotein

hormone that the kidneys produce in response to low levels of oxygen



EPO’s ability to increase the oxygen-carrying capacity of

blood under low oxygen conditions is the reason why athletic

training at high altitudes increases endurance. Since the early



in the blood. EPO travels to the bone marrow and binds receptors on

cells that give rise to red blood cell progenitors. Soon, more red blood

cells enter the circulation, carrying more oxygen to the tissues



1990s, athletes have abused EPO to reproduce this effect, at great

risk. EPO thickens the blood, raising the risk of a blockage that can

cause a heart attack or stroke, especially when intense, grueling



(see photo).

The value of EPO as a drug became evident after the invention

of hemodialysis to treat kidney failure in 1961. Dialysis removes EPO

from the blood, causing severe anemia. But boosting EPO levels proved

difficult because levels in human plasma are too low to pool from



exercise removes water from the bloodstream. Excess EPO caused

sudden death during sleep for at least eighteen cyclists abusing the

hormone. Olympic athletes are now routinely given urine tests to

screen for EPO abuse.

People with familial erythrocytosis get extra EPO naturally. Type



donors. Instead, in the 1970s, the U.S. government obtained EPO from

South American farmers with hookworm infections and Japanese

aplastic anemia patients, who secrete abundant EPO into urine. But

when the AIDS epidemic came, biochemicals from human body fluids

were no longer safe.

Recombinant DNA technology solved the EPO problem. It

is sold under various names to treat anemia in dialysis and AIDS



1 (MIM 133100) is autosomal dominant, and is caused by mutation in

the EPO receptor. It causes large and abundant red blood cells, but low

blood serum levels of EPO. A member of a family from Finland with this

condition won several Olympic medals for skiing thanks to his inborn

ability. An autosomal recessive form of the condition, type 2 (MIM

263400), increases the level of EPO in the bloodstream. Both forms

of erythrocytosis usually have no symptoms, but increase the risk of

circulation blocked by the sluggish, oxygen-laden blood.



Questions for Discussion



At least two genes control EPO secretion. Certain variants of these genes

increase the number of red blood cells, increasing endurance but also

raising risk of heart attack and stroke.



did nothing to the mice—until researchers increased transcription rate tenfold. Only then did the telltale plaques and tangles,

and neuron cell death, appear in the mouse brains.

Animal models might not mimic the human condition

exactly because of differences in their rates of development, or different symptoms. Difficulty in relating the abnormal movements

and behavior of a mouse model of Huntington disease, for example,

led researchers to create transgenic macaques (figure 19.6), which,

as primates, are much closer to humans in lifespan, metabolism,

reproduction, behavior, and cognition. Recall from chapter 12

that HD is caused by an expansion of a triplet nucleotide repeat.

Researchers created macaques with varying numbers of triplet

repeats. As in humans, fewer than thirty or so repeats did not

affect locomotion or behavior, but longer repeats, or insertion of

more than one transgene, produced symptoms.



1.



Was it ethical in the 1970s to obtain EPO from sick, poor

people in South America and Japan to treat relatively well-off

Americans?



2.



Should taking a substance made naturally in the body be

considered performance enhancement?



3.



Do you think that tests should be developed to identify athletes

whose genes, anatomy, or physiology give them a competitive

advantage in a particular sport or event? What should be done

with such information?



4.



When developing drugs that use recombinant DNA

technology, should researchers consider how the product

could be abused?



Sometimes it isn’t clear why a transgenic mouse model

yields results that do not apply to people who have the exact

same mutation. Consider a mouse model of the form of familial amyotrophic lateral sclerosis in which the enzyme SOD1 is

not produced (MIM 105400). Ten drugs were found to extend

the 2-year lifespan of the mice; none of the drugs helped people, and one actually worsened symptoms. The dog model of

ALS in figure 12.1b is not transgenic; it has a dog version of

the condition.



Bioremediation

Recombinant DNA technology and transgenic organisms provide

processes as well as products. In bioremediation, bacteria or

plants with the ability to detoxify certain pollutants are released



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Concepts and Applications,

Ninth Edition



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



© The McGraw−Hill

Companies, 2010



Bioremediation cleans up munitions dumps from wars.

One application uses bacteria that normally break down

trinitrotoluene—better known as TNT, the major ingredient in

dynamite and land mines. The enzyme that provides this capability is linked to the GFP gene. When the bacteria are spread in a

contaminated area, they glow near land mines, revealing the locations much more specifically than a metal detector could. Once the

land mines are removed, the bacteria die as their food vanishes.



Key Concepts

1. In recombinant DNA technology, a cell receives a cloning

vector that contains foreign DNA encoding a protein of

interest.

2. Genes are isolated from genomic DNA libraries or cDNA

libraries.

3. Antibiotic resistance genes and gene variants that

change the color of growth media are used to select cells

bearing recombinant plasmids.

4. Recombinant DNA technology is used to manufacture

large amounts of a pure protein in single cells and to

create multicellular transgenic organisms.

5. Some transgenic plants use Ti plasmids to obtain foreign

DNA. Transgenic animals receive foreign DNA naked,

in liposomes, or by electroporation, microinjection, or

particle bombardment. The gene must be transcribed

and translated and its product collected and purified.

For multicellular organisms, crosses may be necessary to

obtain homozygous recessives.

6. Transgenic animals model human disease.

7. Bioremediation uses natural abilities to detoxify

environmental contaminants.



Figure 19.6



Monkeys stand in for humans. Transgenic

macaques are models for Huntington disease, an autosomal

dominant neurodegenerative disorder.



19.4 Monitoring Gene Function

or grown in a particular area. Natural selection has sculpted such

organisms, perhaps as adaptations that render them unpalatable

to predators. Bioremediation uses genes that enable an organism

to metabolize a substance that, to another species, is a toxin. The

technology uses unaltered organisms, and also transfers “detox”

genes to other species so that the protein products can more easily penetrate a polluted area.

Nature offers many organisms with interesting tastes. A

type of tree that grows in a tropical rainforest on an island near

Australia, for example, accumulates so much nickel from soil

that slashing its bark releases a bright green latex ooze. This

tree can be used to clean up nickel-contaminated soil.

Bioremediation can tap the metabolisms of transgenic

microorganisms, sending them into plants whose roots then distribute the detox proteins in the soil. For example, transgenic yellow poplar trees can thrive in mercury-tainted soil if they have a

bacterial gene that encodes an enzyme, mercuric reductase, that

converts a highly toxic form of mercury in soil to a less toxic gas.

The tree’s leaves then release the gas.

388



Part 6



Genetic Technology



Gene expression DNA microarrays (gene chips) are devices

that detect and display the mRNAs in a cell. The creativity of

the technique lies in choosing the types of cells to interrogate.

Evaluating a spinal cord injury illustrates the basic steps in

creating a DNA microarray to assess gene expression. Researchers knew that in the hours after such a devastating injury, immune

system cells and inflammatory biochemicals flood the injured

area, but it took gene expression profiling to reveal just how fast

healing begins.

A microarray is a piece of glass or plastic that is about

1.5 centimeters square—smaller than a postage stamp. Many small

pieces of DNA (oligonucleotides) of known sequence are attached

to one surface, in a grid pattern. The researcher records the position

of each DNA piece in the grid. In many applications, a sample from

an abnormal situation (such as disease, injury, or environmental

exposure) is compared to a normal control. Figure 19.7 compares

cerebrospinal fluid (CSF; the liquid that bathes the spinal cord)

from an injured person (sample A) to fluid from a healthy person (sample B). Messenger RNAs are extracted from the samples



Lewis: Human Genetics:

Concepts and Applications,

Ninth Edition



VI. Genetic Technology



19. Genetic Technologies:

Amplifying, Modifying, and

Monitoring DNA



Sample A:

Spinal cord injury



© The McGraw−Hill

Companies, 2010



391



Sample B:

Control



1 Isolate RNA



Reverse

transcriptase



2 Generate cDNAs



3 Label probes with

fluorescent tags



Fluorescent

tags



+



Apply DNA probes



4 Incubate labeled

cDNAs with DNA

microarray



5 Laser scanner

detects bound,

fluorescent

DNA probes

DNA microarray with target DNA from

genes whose protein products could

function in the spinal cord



6 Computer

analyzes

data



Figure 19.7



Sample A > B



Neither binds



Sample B > A



Sample A = B



A DNA microarray experiment reveals gene expression in response to spinal cord injury.

Chapter 19 Genetic Technologies: Amplifying, Modifying, and Monitoring DNA



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